Multiplex Real-Time PCR For The Detection Of Pathogenic Intestinal Parasites And Comparison With Parasitological Techniques
| dc.contributor.author | Basuni, Madihah | |
| dc.date.accessioned | 2020-01-29T02:56:16Z | |
| dc.date.available | 2020-01-29T02:56:16Z | |
| dc.date.issued | 2012-01 | |
| dc.description.abstract | Intestinal parasitic infections by helminths and protozoa are among the most prevalent infections and remain a major public health burden in underdeveloped countries. Most intestinal helminth infections cause abdominal pain accompanied by aneroxia, nausea and diarrhea while intestinal protozoa cause diarrheal diseases. Routine diagnostic methods for intestinal parasitic infections which rely heavily on microscopic detection are insensitive, and require well-trained microscopists to avoid misdiagnosis or overdiagnosis of the infection. These limitations have led to the development of highly sensitive DNA-based assays. PCR has been proven to be sensitive and specific for detection of enteric pathogens. Since conventional PCR is time consuming and prone to cross-contamination, it is desirable to develop a realtime PCR assay which is rapid and can provide quantitative and real-time information on the amplified products. In this study, a real-time multiplex PCR assay for the detection of closely related intestinal helminths namely Ascaris lumbricoides, Strongyloides stercoralis, Necator americanus and Ancylostoma duodenale and an assay for the detection of three closely related intestinal protozoa which comprised Entamoeba histolytica, Cryptosporidium parvum and Giardia lamblia were optimized and evaluated.total of 302 stool samples were collected from patients with gastrointestinal problems from Hospital Serian, Hospital Lundu and Hospital Universiti Sains Malaysia. Parasitological techniques were performed by direct wet smear, zinc sulphate floatation/sedimentation, trichrome staining for E. histolytica and G. lamblia, acid-fast staining for C. parvum and Kato Katz technique for helminth ova. The primers were tested at five concentrations and two PCR thermal profiles. The PCR products were cloned into TOPO® cloning vector and the DNA plasmids were used as positive control for standard curve construction. The detection limit for each organism was as follows: one DNA copy for G. lamblia; 10 copies for each Ancylostoma, A. lumbrocoides and S. stercoralis; and 102 copies for each N. americanus, E. histolytica and C. parvum. For real-time multiplex PCR assay optimizations, primer limitation steps were performed in order to avoid any inhibition due to high abundant template. Phocine herpesvirus 1 (PhHV-1) with an optimum dilution of 10-2 PFU/ml was included at the template preparation stage in order to detect false negative results due to the presence of any inhibitor compounds or PCR failure. Each assay was tested with one or multiple DNA template with or without the addition of PhHV-1 DNA in each reaction. Two real-time multiplex PCR assays i.e. for detection of intestinal helminths and intestinal protozoa were performed on all DNA samples. The real-time PCR results were compared with those obtained by parasitological techniques based on sensitivity, specificity and Chisquare tests. For the detection of intestinal helminths, out of 302 samples, microscopic examination detected 13 (4.3%) positive samples while real-time PCR assay detected 94 (31.1%). For the detection of intestinal protozoa, microscopic examination detected five (1.7%) positive cases and real-time PCR detected 23 (7.6%) with p<0.05. Multiple infections by two and more organisms (either helminths or protozoa) were recorded in 29 out of 110 positive cases (26%) by real-time multiplex PCR, while no cases of multiple infections were reported by microscopic examination. The real-time multiplex PCR assays optimized in this study successfully detected all the target organisms. They were also successfully evaluated on patients’ samples for the detection of four important intestinal helminths and three common intestinal protozoa. The PCR assay detected 7.2 times more positive samples for intestinal helminths and 4.6 times for intestinal protozoa as compared to parasitological techniques. In conclusion, the real-time PCR assays described in this study provide an alternative laboratory diagnostic method for intestinal parasitic infections and would be useful for treatment monitoring and epidemiological studies. | en_US |
| dc.identifier.uri | http://hdl.handle.net/123456789/9480 | |
| dc.language.iso | en | en_US |
| dc.subject | Intestines -- Diseases | en_US |
| dc.title | Multiplex Real-Time PCR For The Detection Of Pathogenic Intestinal Parasites And Comparison With Parasitological Techniques | en_US |
| dc.type | Thesis | en_US |
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