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Myeloproliferative neoplasms include a heterogeneous group of stem cell disorders with overproduction of myeloid cells. They have very different clinical courses and prognosis and are amenable to specific targeted therapy. A prompt and accurate diagnosis is therefore very important. Genetic characterisation plays an important role in diagnosis and classification of these disorders. BCR-ABL1fusion gene and JAK2V617F mutation are the particular major molecular markers to be detected because of availability of targeted therapy. In this study, a new molecular testing system was developed for the differential diagnosis of myeloproliferative neoplasms. A multiplex reverse-transcriptase polymerase chain reaction was developed for fast detection of JAK2 V617F mutation and BCR-ABL1fusion simultaneously. It was demonstrated to be fast and highly sensitive and specific for the mutations as validated by analysis of clinical samples. The sensitivity limit was well suited for clinical diagnosis. There was great potential saving in consumables and manpower with a much shortened turn-around-time in most cases when compared to conventional diagnostic protocol. / published_or_final_version / Pathology / Master / Master of Medical Sciences

Background Tuberculosis (TB) infection is a contagious disease due to infection by Mycobacterium tuberculosis(MTB) causing global health burden. There is increasing effort to develop early case detection methods and also to address the issue of multidrug resistance TB (MDR-TB). Molecular methods have been applied to provide rapid and accurate diagnosis. In addition to commercial kits being available for the detection of MTB from clinical specimens, In-house PCR assays have also been developed for the detection of MTB, and can be adjusted according to the laboratories’ own demand. Several molecular techniques like TaqMan probes and Hybridization probes may be applied to target for markers of MTB, e.g. 16s rRNA and IS6110.Detection of the mutation genes, for example, katGfor isoniazid (INH), enables determination of susceptibility of the antibiotic more rapidly than traditional culture methods, and is especially useful due to the increasing emergence of MDR-TB. A wide range of genes have been reported to be related to the resistance of INH, katG315 mutation is the most common gene among them. Therefore, genotyping katG315 allows determination of the susceptibility of INH. Objective The first objective is to evaluate the diagnostic performance of IS6110 One-tube Nested Real-Time PCR for the detection of MTB. Clinical pulmonary specimens collected from Tuen Mun Hospital were retrieved for investigation. All the specimens have already been tested for COBAS TaqMan MTB test and culture results have been obtained for all the samples. During the first stage of the study, all the specimens were tested with IS6110 One-tube Nested Real-Time PCR. Sensitivity, specificity, positive predictive value, negative predictive value and diagnostic odds ratio were obtained from the comparison with the gold standard of MTB detection, i.e., culture. During the second stage of the study, samples were selected to undergo katG315 HybProbe Real-Time PCR assay to determine the genotype of katG. The performance of the assay was evaluated statistically. Result In the first stage of the study, 200 samples were tested with IS6110 One-tube Nested Real-Time PCR. The assay was found to have a sensitivity of 76.92%, specificity of 98.52%, positive predictive value of 96.15%, negative predictive value of 89.86% and the diagnostic odds ratio of 221.667. In the second stage of the study, 66 samples were selected and tested for katG315 HybProbe Real-Time PCR assay, 36 samples were successfully genotyped while 30 samples failed to be genotyped. The only culture proven INH resistance specimen was not amplified at first, and culture isolate was extracted for genotyping again. The repeated test confirmed the genotype of the resistance strain to be a mutant. Conclusion katG315 HybProbe Real-Time PCR assay is a valid approach for genotyping katG. However, the sensitivity and efficiency has to be improved before application for clinical use. From the statistics obtained, COBAS TaqMan PCR assay, which is routinely used in Tuen Mun Hospital, is statistically proven to have comparatively better performance than the IS6110 One-tube Nested Real-Time PCR. Improvement on the assay is required for IS6110 One-tube Nested Real-Time PCR. However, there is great potential of applying both IS6110 One-tube Nested Real-Time PCR and katG315 HybProbe Real-Time PCR assay in clinical use with the same platform available. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Master / Master of Medical Sciences

Thesis (M. Med. Sc.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 70-79).

published_or_final_version / Microbiology / Master / Master of Medical Sciences

Introduction: Mycoplasma pneumoniae(M. pneumoniae) causes 10% to 30% of community-acquired pneumonia (CAP). The commonly used first-line antibiotic macrolide (ML) against respiratory tract infection may lead to the increase of ML-resistant M. pneumoniaeinfection. To resolve the problem, a rapid and accurate method for detection of ML-resistant M. pneumoniaeis necessary for treatment adjustment. Aims: The study aims to (1) develop a rapid method for diagnosis of ML-resistance of M. pneumoniaedirectly from clinical specimens; and (2) investigate the prevalence of M. pneumoniaeand ML-resistant M. pneumoniae. Methods: The M. pneumoniaeqPCR results of 689 respiratory tract samples from Queen Mary Hospital collected during April 2010 to May of 2012 were analyzed. Positive nucleic acid from M. pneumoniaeqPCR samples were tested with SimpleProbe real-time PCR coupled to melting curve analysis (SimpleProbe PCR), pyrosequencing and 23S rRNA gene sequencing(23S sequencing) for detection of ML-resistance. Results: A total of 111 samples (16.11%) in 689respiratory tract samples were found M. pneumoniaepositive by qPCR. Of 111, 96 positive nucleic acids were available for this study. Overall, 29 (30.21%, n=96) of ML-resistant M. pneumoniaewere found. 23S sequencing identified 28 mutants (29.17%) and 62 wild–type (64.58%), while 6 (6.25%) of them are failed to be identified. Pyrosequencing identified 28 mutants (29.17%) and 63 wild–type (65.63%), while 5 (5.21%) of them are failed to be identified. The SimpleProbe PCR identified 29 mutants (30.21%) and 65 wild–type (67.71%), while 2 (2.08%) of them are failed to be identified. All ML-resistant M. pneumoniaepositives were found to have A2063G mutation either by 23S sequencing or pyrosequencing. Conclusion: From this study, SimpleProbe PCR is the most sensitive and simple to perform. Therefore, it is highly recommended to be included in the routine testing with positive M. pneumoniaesamples for diagnosis of ML-resistant strain. 23S sequencing or pyrosequencing is recommended to use as a confirmatory test if necessary. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

Introduction: Mycoplasma pneumoniae (M. pneumoniae) has been a major cause of community-acquired pneumonia (CAP), accounting for about 10-30% of the cases. Previously, a local study revealed that more than 60% of clinical isolates of M. pneumoniae exerted A2063G mutation, which confers a high level of macrolide drug resistance and results in treatment failure. While A2063G is the only mutation identified locally, a rapid diagnostic assay for detection of this single point mutation is urgently needed for switching the drug of choice. Aims: This study aims to develop a rapid PCR assay for detection of A2063G mutation of M. pneumoniae isolates for our locality, to compare with other commercially available assays and to further confirm the prevalence of A2063G mutation in macrolide-resistance M. pneumoniae (MRMP) in Hong Kong. Methods: A total of 110 respiratory tract samples were collected from 102 patients in Hong Kong Sanatorium and Hospital during April 2013 to April 2014. They were analyzed by an in-house hybridization-probe real-time PCR assay coupled with melting curve analysis to detect the presence of M. pneumoniae and the target A2063G point mutation. Results were compared with a commercial real-time PCR assay and the A2063G point mutation was further confirmed by 23S rRNA gene sequencing. The limit of detection (LOD), mutation threshold determination and cross reactivity of the in-house assay were also evaluated. Results: Over 40% (47/110) of the respiratory tract samples were tested positive for M. pneumoniae by the in-house assay and 36.2% (17/47) of the positive samples exerted A2063G mutation. The limit of detection was 500 copies/ml as evaluated using external quality control samples. Twenty well-characterized clinical isolates of M. pneumoniae were used to evaluate the A2063G mutation threshold. The mutation threshold for A2063G mutant detection was above 60%. This assay did not show any cross-reactivity with common clinical isolates from the respiratory tract samples. Conclusion: In this study, an in-house real-time PCR assay was evaluated and demonstrated its great potential as a rapid clinical diagnostic tool. The assay was highly sensitive and specific in detecting M. pneumoniae and its A2063G mutation from clinical samples in Hong Kong. The results were almost concordant to the current routine testing, with the advantage of lower cost and shorter turnaround time for rapid detection. / published_or_final_version / Microbiology / Master / Master of Medical Sciences