Multiplex Real-Time PCR For The Detection Of Pathogenic Intestinal Parasites And Comparison With Parasitological Techniques
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Date
2012-01
Authors
Basuni, Madihah
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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.
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Keywords
Intestines -- Diseases