Development Of Loop Mediated Isothermal Amplification Assay For Rapid Diagnosis Of Tuberculosis

Development of Loop-mediated Isothermal Amplification Assay for Rapid Diagnosis of Tuberculosis

Development of Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of Tuberculosis

This dissertation, "Development of Loop-mediated Isothermal Amplification Assay for Rapid Diagnosis of Tuberculosis" by Ka-lun, Wong, 王嘉倫, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Tuberculosis (TB), a severe disease caused by Mycobacteria tuberculosis (MTb), remains a globally severe health problem. In modern days, emergence of drug resistant TB is a new threat to public health since it can lead to treatment failure, increased transmission of TB to other hosts and further development of drug resistant complications. The traditional diagnostic method for TB by Acid Fast Bacilli (AFB) smear and Lowenstein-Jensen medium (LJ) culture are poor in sensitivity and time consuming respectively. There is a need for rapid diagnosis and identification of MTb. Nucleic acid amplification like PCR and real-time PCR are options for rapid diagnosis. However, such techniques require sophisticated technique and complex equipment. The high cost would constitute a barrier for countries with a high demand but only limited resources. Loop-mediated isothermal amplification (LAMP) assay is a novel technique, proven by many studies as to its high sensitivity and highly specificity to MTb. Most importantly, LAMP assay is economical and affordable by developing countries. The first objective of this study was to evaluate the analytical sensitivity and specificity of LAMP assay. The specificity of LAMP assay was determined by performing LAMP assay on 19 clinical isolates, which had already been identified previously. The clinical isolates included 14 mycobacteria tuberculosis complex (MTb), and five mycobacteria other than tuberculosis (MOTT) strains that were positive for AFB smear and LJ culture but negative for IS6110 single-tube nested real time PCR. The specificity was 100%. The analytical sensitivity as well as the limit of detection (LOD) were determined by testing on a duplicate set of serial DNA dilution, where each duplicate consisted of dilution of 100,000, 10,000, 1000, 300, 100, 10 and 1 colony forming unit/milliliter (CFU/ml). The LOD of LAMP assay was about 3 CFU per reaction. The second objective of this study evaluated the diagnostic performance of LAMP assay against AFB culture and IS6110 single tube nested real time PCR for identification of MTb in 200 respiratory specimens from 123 patients. All the specimens have already been tested for IS6110 single tube nested real time PCR, and culture results and AFB smears results have been obtained for all the specimens. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of three diagnostic methods (AFB smear microscopy, LAMP assay amplification and IS6110 single-tube nested real time PCR) were calculated with 95% confidence interval using LJ culture as gold standard. The LAMP assay had a sensitivity of 80%, specificity of 92.6%, PPV of 60% and NPV of 97.1%. However, MTb might fail to grow on the LJ culture medium for various reasons, for instance, MTb might already be dead after antibiotic treatment of the patient, or there might be poor laboratory practices during the processing of the specimens. Since the LJ culture method could produce false negatives in the situations described above, an alternative to the LJ culture method, ''Hybrid Method'' was used as the gold standard. Under this method, a specimen was regarded as positive if the LJ culture result was positive. On the other hand, if a specimen generated a negative result using the LJ culture method, the results from the LAMP assay and IS6110 nested real time PCR would be considered, i.e., if both the LAMP assay

Evolving Landscape of Molecular Diagnostics

Evolving Landscape of Molecular Diagnostics: Applications and Techniques presents cutting-edge reviews on current and emerging technologies in the diagnosis of microbial infections. The book discusses the fundamentals of Molecular Diagnostics for bacteria, fungi, viruses and a variety of state-of-the-art diagnostics tools and techniques for obtaining qualitative and quantitative results. It includes topics such as Next generation sequencing and application of "OMICS" for early disease diagnosis. Sections cover the entire spectrum of emerging diagnostic tools and techniques, as well as the principles, pros and cons of each method and applications for diagnosis of infectious disease in plants, humans and veterinary. This is a resource for both researchers and students working in clinical microbiology, infectious biology, applied life sciences and scientists working in the clinical diagnostic industry. - Provides a wide range of diagnostics tools and technologies in the field of infectious biology - Presents discussions on emerging technologies for early disease diagnosis and novel diagnostics techniques for detection of COVID, Urea Breath Tests for Detection of Helicobacter pylori, Loop-Mediated Isothermal Amplification as Point-of-Care Diagnosis, Molecular imaging, Lab-on-a-chip Technologies, and several others - Analyzes literature reviews, underpinning methodologies, and opportunities and limitations - Includes case studies and examples that highlight applications using the underpinning techniques