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

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.


Introduction
Opisthorchis viverrini (O. viverrini; Ov), a digenean liver fluke, is a major cause of human opisthorchiasis and Ovinduced cholangiocarcinoma (CCA), the biliary ductal cancer, in enduring infection [1]. It is a food-borne parasite that spreads throughout the Southeast Asian subregional countries, especially in the Mekong Basin including Laos PDR, Cambodia, Vietnam, and ailand [2][3][4]. Huge incidence of O. viverrini is highly related to the prevalence of CCA in those areas according to the consumption of raw Cyprinid fish, a secondary intermediate host of O. viverrini [5]. CCA is a highly progressive cancer originating from the transformed intra-or extrahepatic cholangiocytes which lack early diagnostic markers [6]. e diagnosis of O. viverrini infection is possible, but it has a lot of limitations. e gold standard for diagnosis of O. viverrini infection is microscope-based methods such as direct smear and concentration techniques including the formalin-ethyl acetate concentration technique (FECT) and related procedures [7]. e conventional methods are convenient but have low sensitivity; moreover, the observation could be erroneous and need professional laboratorians for identification by their naked eyes [8].
e molecular detection of Ov-eggs has been introduced for several years by many methods such as conventional PCR, LAMP, and quantitative real-time PCR [9][10][11][12][13][14]. e target genes are varied but mostly concentrated on the mitochondrial gene due to the presence of high conservation such as internal transcribed spacer (ITS), cytochrome c oxidase 1 (cox1), and NADH dehydrogenase 1 (nad1) [15][16][17][18]. e detection limit of those genes is not stable among the specimen types, collection procedures, preservation methods, extraction protocols, and the number of eggs [19].
NADH dehydrogenase (Nad) is the enzyme which converts nicotinamide adenine dinucleotide (NAD) from NADH (reduced form) to NAD + (oxidized form) encoded by the mitochondrial genome [20]. e NADH dehydrogenase complex spans inside the inner membrane of the mitochondria. e complex provides energy from the oxidation of NADH to NAD + and proton transfer from the mitochondrial matrix to the intermembrane space. Moreover, the complex has numerous metabolic functions such as fatty acid oxidation, TCA cycle, amino acid catabolism, and cytosolic reactions given the crucial central position of metabolic pathways responsible for carbohydrate, fatty acid, and amino acid catabolism [21]. As it has various vital functions for the cells, Nad is abundantly expressed in every cell which is a good compelling target for molecular detection.
e established mitochondrial genome of O. viverrini permits the possibility to encounter the Nad subunits for development as the diagnostic target other than Nad1 that was previously tested [17]. e schematic drawing of the O. viverrini mitochondrial genome is illustrated in Figure 1. Hence, this study aimed to establish the new target for the molecular identification of Ov-egg directly in the stool specimen by comparison of the efficiency of Nad subunits using conventional PCR which is the easiest and cheapest molecular procedure for developing a potential diagnostic target in the future.

Study Areas and Stool Collection.
A total of 166 stool samples were collected from several provinces in the north, northeast, and central ailand where O. viverrini is endemic in Chiang Mai, Phrae, Phetchabun, Nakhon Phanom, Pathumthani, and Chachoengsao provinces during 2018 to 2020. e participants living in the risk areas of these provinces were included in this study. All participants were educated and consented for a noninvasive stool collection procedure.
e collected samples were transferred to the laboratory and kept in 4-10°C until processed by the concentration method.

PBS-Ethyl Acetate Concentration Technique (PECT).
All samples were subjected to the PBS-ethyl acetate concentration technique (PECT) as previously described [15] with a few modifications. In brief, one gram of each stool specimen was mixed with 10 mL of PBS, pH 7.4, and filtered through three layers of gauze pad. 3 mL of ethyl acetate was added to the filtrate and mixed thoroughly by inverting. e tube was centrifuged at 2,000×g, for 10 min, and the upper phases were removed. e sediment was investigated under the light microscope at 100 and 400 magnifications.

Genomic DNA Isolation.
e genomic DNA of parasitic eggs was isolated from PECT sediments of stool. Before extraction, the sediments were autoclaved at 121°C, for 5 min to open the operculum of parasite eggs. e genomic DNAs were then extracted by using the QIAamp® PowerFecal® DNA kit (QIAGEN, Germany) according to the manufacturer's instructions with the final filtrates containing 50 µL of eluted DNAs.  e amplification steps included initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min, and one cycle of a final extension at 72°C for 10 min. e PCR products were size separated on 2% agarose gel containing ViSafe Red Gel Stain (Vivantis, USA) using 1X TBE buffer at 100 V for 2 h. e PCR products were confirmed for their correct sequences by DNA sequencing service (Macrogen, Republic of Korea).

Cross-Reactivity Evaluation.
e cross reactivities of OvNad subunit specific primers were simultaneously investigated in the collected stools. e PECT sediments with other parasites were monitored for the cross reactivity. e samples included both single and mixed infections with either flatworms or roundworms.

Statistical Analysis.
e sensitivity, specificity, positive predicted value (PPV), and negative predicted value (NVP) of OvNad subunit detection by PCR in the stool were calculated as previously described [22] comparing with PECT as a gold standard method.  Table 2.

Detection Limits of OvNad Subunits.
e PCR amplification of OvNad subunits by conventional PCR with 0, 1, 5, 10, 20, 50, and 100 Ov-egg reviewed the different detection limits among the OvNad subunits. OvNad1 has the lowest detection limit at 50 eggs followed by OvNad4 at 20 eggs and OvNad2 at 10 eggs. Interestingly, OvNad5 had unyielding activities in the low egg number in only 5 eggs. e amplification of OvNad subunits is illustrated in Figure 2.

Sensitivity, Specificity, and Cross Reactivity of OvNad
Subunits.
e PCR amplification of 166 samples with OvNad subunit specific primers suggested that OvNad subunits had different sensitivities. Only 48/75 of PECTpositive samples can be amplified by OvNad1, while 66/75 and 60/75 were successfully amplified by OvNad2 and OvNad4, respectively. e OvNad5 showed luminous activity with 75/75 of PECT-positive samples and another 2 from PECT-negative samples (the sequences had been correctly confirmed). e sensitivities of OvNad1, OvNad2, OvNad4, and OvNad5 were 64.0%, 88.0%, 80.0%, and 100%, respectively, and specificities were 100% in all subunits. e positive predicted values (PPV) of OvNad1, OvNad2, OvNad4, and OvNad5 were 100% in all subunits, whereas the negative predicted values (NVP) were 77.12%, 91.00%, 85.85%, and 100%, respectively. e sensitivities, specificities, PPV, and NPV are shown in Table 3. In the testing of cross reactivities, the OvNad subunits were used to amplify in all simultaneously collected parasites of both single and mixed infections. All OvNad subunits showed excellency without amplification of all other kinds of parasites as mentioned in Table 2 and

Discussion
e molecular technology allows many researchers to develop several diagnostic procedures for detecting of any diseases, especially infectious diseases with perilous 2500   COX3   CYTB  ND4  ND2  ND1  COX1  ND5  rRNA  ND4L  ATP6  ND3  rRNA COX2 ND6   5000  7500 10,000 12,500 complexity. e O. viverrini infection is also in the same pipeline with other diseases, which has some of established molecular diagnostic procedures, but the sensitivity has been limited. e ITS-based PCR is the first and most famously used target for molecular identification, but later, it showed varied sensitivity comparable with the newly identified targets such as Cox1, Cytochrome B (CytB), and Nad1 [17]. Even though these targets showed better or equal sensitivities to ITS, they have many drawbacks, especially the detection in the low number of parasite eggs [10]. Nad1 is a mitochondrial gene which has been previously introduced but never verified with the other Nad subunits. e detection limits of OvNad subunits were verified in our study. e result revealed that the OvNad5 subunit had better activities than the other subunits.     PECT, the stool concentration technique, is a routinely used method for investigating parasitic infection. It has shown the highest sensitivity than the other microscope-based methods up to 100% in >1000 eggs-per-gram (EPG) samples and exceeding 91% in cases of light infection [8]. As a result, we used PECT as a reference method for sensitivity, specificity, PPV, and NPV calculations. e best sensitivity from our study is of OvNad5 which showed 100% sensitivity followed by OvNad2, OvNad4, and OvNad1, respectively. Surprisingly, the OvNad2 and OvNad5 revealed better sensitivities than OvNad1 that was formerly established. OvNad5 showed the best efficiency in all investigated parameters including sensitivity, specificity, PPV, and NPV with 100%. In comparison with the other target genes, OvNad5 also has better capacity than ITS-2 and Cox1 mentioned in previous reports by Lamaningao [25] and Buathong [15], with 83.1% and 89.1%, respectively, especially in the light infection (less than 100 EPG) where the sensitivities of ITS-2 and Cox1 were up to 73.9% and 89.1% [10,15,23,24]. DNA sequencing of the PCR products amplified by the introduced primers confirmed the sequences of OvNad that referred to the Oveggs with less identity to the minute intestinal fluke. e introduced stool PCR, especially OvNad5, showed a relevant result to natural Ov-positive samples investigated by PECT. All of the PECT-positive samples were also positive with OvNad5 PCR; interestingly, two PECT-negative samples were positive with OvNad5 PCR, emphasizing the limitation of the conventional method to detect the egg in low-intensity samples. For the cross reactivities to other parasites that could be found together with Ov infection including Hookworm, A. lumbricoides, Taenia spp., S. stercoralis, E. vermicularis, and T. trichiura, the result suggested that OvNad has no cross reactivity with these parasite eggs. It is confidentially confirmed that OvNad subunits could be the exact targets for PCR amplification of Ov-egg.
In conclusion, our study established that OvNad subunits can be the potential targets for Ov-egg DNA detection in the stool specimen. Moreover, OvNad5 could be the best candidate due to the potent detection limit, highest sensitivity, specificity, PPV, and NPV. e introduced primers can be applied for using in routine diagnosis because conventional PCR is easier and cheaper than real-time PCR. It is also useful for other researchers to develop more tests on OvNad subunits in the future.

Data Availability
All data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare no conflicts of interest regarding the publication of this paper. wrote and finalized the manuscript. All authors reviewed and approved the final manuscript for submission.