Comparative detection of Mycobacterium Ulcerans dna by PCR from FTA cards, cell Lysis solution and RNA protect solution

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OCTOBER, 2016.
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Buruli ulcer is a necrotizing skin disease caused by Mycobacterium ulcerans. It occurs in more than 33 countries with tropical and subtropical conditions. Samples for diagnosis and confirmation are usually transported in liquid media including cell lysis solution (CLS), PANTA, and RNA protect solution from the disease centers to reference laboratories. FTA cards have been developed to store, preserve and protect nucleic acids stored on it. It serves as a transport medium for samples requiring the use of nucleic acids and reduces storage space. Diagnosis of Buruli ulcer requires transporting samples from the remote communities to the laboratories as well as for storage and sample collection. The usage of FTA cards has however not been explored in the field of Buruli ulcer and in Ghana. This study therefore sought to evaluate the use of FTA for obtaining DNA samples and transporting of M. ulcerans to the laboratories and to also establish its detection limit. Methods: Known concentrations of M. ulcerans suspensions were spotted on the FTA cards and allowed to air dry. Two millimeters of discs were cut out and DNA was extracted using the in-house extraction procedure to optimize the cards and determine the detection limit of the cards. Swab samples from ulcerative lesions and FNA samples from pre-ulcerative lesions were taken from 53 suspected Buruli ulcer patients and distributed into CLS, RNA protect solutions and spotted on the FTA cards. DNA was extracted from the samples and amplified using the Dry Reagent Based PCR for the CLS and FTA card samples and qPCR for RNA protect solution samples. Amplicons were viewed on agarose gel and results recorded as positive, negative or inhibited. Samples were also smeared on glass slides for the Zeihl Neelson stain for acid fast bacilli. Samples stored on the FTA cards were kept over a period of six months and extraction done on monthly basis to detect the preservative ability of the card. Comparisons were made and results analyzed using GraphPad prism version 5.0 and Stata version12.0. From the optimization test, it was observed that in absolute numbers, 1 bacterium in a concentration of 10 bacteria/ml spotted on the card can be detected. The sensitivity of the FTA cards using patient samples was recorded as 58% and a specificity of 70% whiles that of qPCR was 94% sensitivity and 67% for specificity when compared with the gold standard, the DRB PCR. When compared with the smear for microscopy, the sensitivity for FTA cards was 80% and a specificity of 48%. Samples stored over a period of two months had the highest positivity ratio of 100% followed by those stored for five months with 58.8% and 55%, 50%, 44%, and 27% for four months, one month, three months and six months respectively. inter-assay agreement between FTA PCR and standard CLS PCR was 50% and a p-value of 0.58 for swab samples and 67.7%, p value of 0.22 for FNA samples. FTA cards serve as a good storage and transport medium However because of low sensitivity and low inter-assay agreement rate, it cannot be used as a replacement for the CLS. It may however be used in addition to the smear for microscopy in areas where access to the reference laboratories is difficult.
A thesis submitted to the Department of Molecular Medicine, in fulfilment of the requirements for the degree of Master of Philosophy, Immunology,