Efficiency of the Stool-PCR Test Targeting NADH Dehydrogenase (Nad) Subunits for Detection of Opisthorchis viverrini Eggs

Abstract

Opisthorchis viverrini infection is the major parasitic infection problem in Southeast Asian countries, and long-term infection will lead to cholangiocarcinoma (CCA), the bile duct cancer. The early diagnosis of O. viverrini infection may interrupt the progression of the opisthorchiasis and other related illnesses, especially CCA. The current diagnostic procedure is stool examination by microscope-based methods such as direct smear and concentration techniques but it is limited by low parasite egg numbers. The molecular diagnosis prompts the chance to evaluate the light infection with low number of parasite eggs but is currently inconvenient for routine use due to special equipment requirement and unstable sensitivities. Our present study aims to establish the efficiency of OvNad subunits, the mitochondrial gene, for introducing as a potential diagnostic target by conventional PCR, the cheapest and easiest molecular procedure. A total of 166 stool samples were investigated microscopically by the PBS-ethyl acetate concentration technique (PECT); 75 samples were O. viverrini positive with 28 samples that were positive with single parasite (hookworm, A. lumbricoides, S. stercoralis, Taenia spp., and T. trichiura), 11 samples were with mixed infection, and 52 samples were without parasite detection. The detection limits of OvNad subunits were evaluated in artificially spiked samples containing 0, 1, 5, 10, 20, 50, and 100 Ov-eggs. The result suggested that the best detection efficacy was of OvNad5 that had exact detection limits at only 5 eggs. In the PCR amplification of OvNad subunits, there exist 100% specificities with varied sensitivities from 64%, 88%, 80%, and 100% of OvNad1, OvNad2, OvNad4, and OvNad5, respectively. OvNad subunits were amplified specifically without cross reactivity with the other collected parasites. Our study established that OvNad subunits, especially OvNad5, are the potent candidates for PCR amplification of stool containing Ov-eggs with high confidential sensitivity, specificity, PPV, and NPV even in the light infection that would be a benefit for developing as a routine diagnosis of O. viverrini infection.

Production and Immunological Characterization of scFv Specific to Epitope of Opisthorchis viverrini Rhophilin-Associated Tail Protein 1-like (OvROPN1L)

“…The detection of specific proteins or target molecules with little cross-reactivity is ideal. For O. viverrini infections, several monoclonal antibodies have been developed along with other procedures [ 9 , 14 , 45 , 46 , 47 ]. Most researchers consider secreted antigens in fecal specimens, coproantigens, as the best specimens containing parasite proteins.…”

“…However, these methods require the professional experience of the investigator to differentiate O. viverrini eggs from other parasite eggs, and they do not enable the diagnosis of light infections with a low level of infection [ 7 , 8 ]. Molecular diagnosis is another possibility; several targets have been reported, such as NADH dehydrogenase (NAD) subunits [ 9 , 10 , 11 ], internal transcribed spacer (ITS-1 and ITS-2) [ 12 , 13 ], and cytochrome c oxidase 1 (cox1) [ 13 , 14 ]. Various methods have been introduced, such as conventional PCR, qPCR, and LAMP, but the variation in sensitivity and specificity of the target genes is still questionable.…”

Development of a protocol for soil transmitted helminths DNA extraction from feces by combining commercially available solutions

“…The use of commercially available parasite concentrators for feces is a fairly common practice in parasitological studies using microscopy methods [44][45][46][47][48][49]. At the same time, we found a small number of works where protocols for isolating helminth DNA from feces used flotationsedimentation for concentrating parasite eggs [50][51][52]. At the same time, in most of these studies, the eggs were washed off the coverslips for further DNA isolation after flotation, which greatly complicates the DNA isolation procedure and increases the risk of losing some of the parasite eggs.…”

Current prevalence and geographic distribution of helminth infections in the parasitic endemic areas of rural Northeastern Thailand

“…The positive samples from modified Kato-Katz thick smear were subjected to sedimented by PBS-ethyl acetate concentration technique (PECT) as previously described [29][30][31] with a few modifications. In brief, 0.5-1 g of stool specimen was mixed with 10 mL of 0.01 M PBS, pH 7.4, and filtered through three layers of sterile gauze pad.…”

“…The morphological characteristics of O. viverrini (Ov) and minute intestinal flukes (MIFs) eggs are similar. So, the Ov egg was confirmed by specific PCR amplification from genomic DNA using OvNad5 primer as previously described [30]. PCR amplifications were done by using GoTaq ® Colorless Master Mix (Promega, USA) containing 3 µL of genomic DNA, and 25 pmol of each forward and reverse primer (OvNad5-F: TTT GCG GAG GTT TGT TAC CT and OvNad5-R: CAC CTC ACC AAT TCA ACA CG) in a thermal cycler (Mastercycler nexus Eppendorf flexlid, Germany).…”