Medicine
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This repository contains the published and unpublished research of the Faculty of Medicine by the staff members of the faculty
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Item Introduction of Recombinase Polymerase Amplification assay based mobile suitcase laboratory as a point of need tool to diagnose cutaneous leishmaniasis in Sri Lanka(Sri Lanka Medical Association, 2018) Gunaratna, G.P.S.; Ranasinghe, P. H. K. I. S.; Manamperi, A.; Pathirana, N.; Pathirana, H.; Wickremasinghe, R.; de Silva, N.R.; Sooriyarachchi, M.; Ahmed, A.E.W.INTRODUCTION AND OBJECTIVES: Cutaneous leishmaniasis (CL) caused by the vector-borne protozoan parasite is now endemic in Sri Lanka. Microscopy of Giemsa stained slit skin smears (SSS), lesion aspirates or scrapings for the presence of amastigotes, is widely used for laboratory confirmation of CL, although the reported sensitivity is low. Facilities for more sensitive culture and molecular techniques are available only in reference laboratories. A newly developed, Recombinase Polymerase Amplification (RPA) assay based Mobile Suitcase Laboratory (MSL) is a promising, molecular point of care test with high sensitivity and specificity for diagnosis of both post-kala• azar dermal leishmaniasis and visceral leishmaniasis. Objective was to assess RPA based MSL as a point of need tool to diagnose CL in Sri Lanka.METHODS: Twenty seven army personnel at Mullaitivu Army camp clinically suspected of having CL were recruited for this pilot study. Two slit skin smears and two punch biopsy specimens were obtained from each of them. Slit skin smears were stained with Giemsa in the field and examined for the presence of amastigotes and RPA was carried out at the point of collection. PCR was performed at the Parasitology Department, Sri Jayewardenepura University. RESULTS: Fifteen patients were confirmed by PCR as having CL and 14 of them were also positive by RPA based MSL conducted in the field (93.33% sensitivity). Only 3/15 were positive with microscopy of SSS (20% sensitivity). CONCLUSION: This pilot study shows RPA based MSL as a promising tool to diagnose CL at point of need.Item Evaluation of rapid extraction and isothermal amplification techniques for the detection of Leishmania donovani DNA from skin lesions of suspected cases at the point of need in Sri Lanka(BioMed Central, 2018) Gunaratna, G.; Manamperi, A.; Bohiken-Fascher, S.; Wickremasinghe, R.; Gunawardena, K.; Yapa, B.; Pathiana, N.; Pathirana, H.; de Silva, N.; Sooriyaarachchi, M.; Deerasinghe, T.; Mondal, D.; Ranasinghe, S.; Abd EI Wahed, A.BACKGROUND: Leishmaniasis is a disease caused by vector-borne protozoans. In Sri Lanka, the cutaneous form of the disease is predominant, which is usually diagnosed using Giemsa-stained slit skin smear examination and by histology. However, the sensitivity of slit skin smears and histology are reportedly low. Moreover, facilities for the highly sensitive polymerase chain reaction (PCR) are available only in a few highly-equipped parasitology laboratories. Therefore, there is a need for low cost, sensitive and specific screening tests for diagnosis of leishmaniasis at the point of need. RESULTS: In this study, a mobile suitcase laboratory applying novel extraction (SpeedXtract) and isothermal amplification and detection (recombinase polymerase amplification assay, RPA) methods were evaluated for the diagnosis of cutaneous leishmaniasis in Sri Lanka. First, the developed assay was applied to three different sample types (punch biopsy, slit skin smears and fine needle aspirates) at a local hospital. The results showed that the 2 mm punch biopsy sample produced the best exponential amplification curve and early fluorescence signal in the RPA assay. Secondly, punch biopsies were collected from 150 suspected cutaneous leishmaniasis cases and screened with SpeedXtract/RPA, RNAlater/PCR and ATL buffer/PCR, in addition to Giemsa-stained slit skin smears. Fifty-seven samples were negative in all detection methods. In total 93 samples were positive with assay sensitivities of 65.5% (SpeedXtract/RPA), 63.4% (RNAlater/PCR) and 92.4% (ATL buffer/PCR). The Giemsa-stained slit skin smear delivered the worst clinical sensitivity (32.2%). CONCLUSIONS: The SpeedXtract/RPA method under field conditions took 35 min, while almost 8 h were needed to finalize the extraction and detection by PCR in the laboratory. The SpeedXtract/RPA method produced similar sensitivity to samples preserved in RNAlater and subjected to PCR amplification, but both were less sensitive than ATL-preserved samples subjected to PCR amplification. There is a need for a standardization of sample collection and nucleic acid extraction methods.