Sri Lanka

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Browsing Sri Lanka by Author "ABAYASEKARA, SNW"

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    A Polymerase chain reaction (PCR) based assay for the detection of DIROFILARIA REPENS
    (University of Colombo: UC(MED)., 1997) ABAYASEKARA, SNW
    Filariasis is a group of infectious diseases caused by parasitic nematodes. Of the many species of filarial worms eight are truly specific for man, but there are also zoonotic forms which can infect humans. Dirofilaria repens is one such parasitic nematode of dogs in Sri Lanka. According to current estimates, 410 cases of human dirofilariasis caused by Dirofilaria repens has been reported exclusively in the old world including Sri Lanka. Given the low sensitivity of current diagnostic methods (blood smears) and carying expertise between laboratories and technicians, a rapid and sensitive assay would greatly assist the early detection of microfilariae in dogs and developing infective larvae in vectors. The capability to detect genetic elements (DNA or RNA) of a particular parasite or organism as a means of identifying the causative agent has been the traditional function of nucleic acid hybridization assays. However the low copy number of genetic material and the need for using radiolabelling fostered the development of PCR based assays. A previously cloned and characterized tandemly repeated DNA sequence of D.repens was selected for designing primers for the development of a PCR based assay. The primer pair (DRF4 \& DRR2) of three pairs of primers was chosen for the assay. The PCR assay was optimized in relation to the concentration of deoxyribonucleotides (dNTP), MgCl2, primers, Taq polymerase and annealing temperature. The assay selectively amplified D.repens DNA only and was sensitive enough to detect 100 fg of parasite DNA and a single microfilaria in infected blood samples. Analysis of blood samples from dogs (n=50) using the PCR assay was compared with that of microscopy. The results obtained by PCR were in total agreement with that of microscopy, except for one sample which was negative. It is likely that this sample may have had a very low level of microfilariaemia and hence not detected by microscopy. The high sensitivity of the assay could perhaps also aid in the identification of immature adults found as lumps or nodules in human clinical specimens obtained from biopsies