Improvement of a PCR-based method for the detection of Opisthorchis viverrini eggs in human stool samples by targeting internal transcribed spacer-2 (ITS-2), cytochrome oxidase subunit 1 (cox1), and cytochrome b (cyb)

Abstract

Opisthorchis viverrini infection causes various complications in patients, ranging from asymptomatic to severe chronic disease including cholangiocarcinoma (CCA). O. viverrini is endemic in Southeast Asia and acting as a risk for CCA. Early diagnosis of O. viverrini infection can reduce the number of CCA cases. The routine diagnosis for opisthorchiasis is direct wet smear, sometimes coupled with concentration techniques, which has limitations when investigating light infection or if done by laboratorians with lack of experiences. PCR-based methods have been established for the detection of O. viverrini egg DNA from stool samples, but have never fully succeeded for light infections when compared to wet smear concentration techniques. This study aims to improve the PCR-based method for detection of O. viverrini eggs in stool samples by targeting the genes ITS-2, cox1, and cyb. The results reveal higher sensitivity than conventional concentration techniques, with all newly designed primers. ITS-2 has an overall sensitivity of 76.9% with 66.7% in the samples with < 50 EPG, while cox1 has shown 96.2% overall sensitivity and 94.1% in the same EPG intervals. Interestingly, the new pointing target, cyb, has shown 100% sensitivity in all egg intervals in this study, particularly for light infections (EPG less than 100). No cross-reactivity was found in Taenia spp., Trichuris trichiura, Ascaris lumbricoides, Capillaria philippinensis, and hookworm. The procedure is convenient, with shorter steps compared to previous reports, and it appears appropriate for use in the diagnosis of light infection with O. viverrini. These three genes are good candidates for use in PCR-based detection of the parasite eggs. Further testing with a larger cluster of samples is however necessary.

Introduction

Opisthorchis viverrini infection remains a major contagious problem in the Mekong sub-region including Cambodia, Laos, Vietnam, and Thailand (Sripa et al. 2010; Sithithaworn et al. 2012). The highest recent incidence has been reported from Laos in 2016, with a prevalence of more than 50% in some provinces (Saiyachak et al. 2016; Suwannatria et al. 2018). In Thailand, the infection rate has been decreasing due to several managing strategies with an overall prevalence of approximately 5% in 2014 (Suwannatria et al. 2018). Humans acquire O. viverrini metacercaria (an infective stage) by ingesting cyprinoid fish, the parasite’s secondary intermediate host. Infections develop mostly asymptomatic for many years. Long-term infection can ultimately lead to cholangiocarcinoma (CCA) - bile duct cancer - linked to chronic inflammation of the bile canaliculi where adult parasites settle and release offending molecules (Sripa et al. 2007; Surapaitoon et al. 2017). An effective diagnostic procedure is required for detecting early stages of the infection, and thus definitely reducing the opportunity for CCA to develop. A simple direct wet smear is routinely used for diagnosis of O. viverrini infection, yet it shows low sensitivity, especially for low numbers of eggs and when applied by laboratorians with lack of experiences. The better method is the concentration technique which increases sensitivity up to 100% in > 1000 eggs-per-gram (EPG) samples, and exceeding 91% in cases of light infection (Charoensuk et al. 2019). However, the limitation of the concentration technique is that the morphology of O. viverrini and minute intestinal fluke (MIF) eggs are sufficiently similar to make discrimination difficult, causing false positive detection (Kaewkes et al. 1991). Molecular methods have been developed for diagnosis of opisthorchiasis including antigen and antibody detection. Copro-antigen detection using a capture ELISA is a promising method with sufficient sensitivity (Sirisinha et al. 1992, 1995). Antigen detection in urine samples has also been reported with 81% sensivitity and 70% specificity (Worasith et al. 2015). Immunodiagnostic method detecting antibody in human serum showed commonly higher sensitivity and specific comparing with antigen detection but with suspicious present infection and invasive sample collection (Poopyruchpong et al. 1990; Wongsaroj et al. 2001; Ruangsittichai et al. 2006).

Polymerase chain reaction (PCR) based methods have been established for detecting O. viverrini eggs in stool samples by validating various target genes such as internal transcribed spacer (ITS), cytochrome c oxidase (cox), and nicotinamide adenine dinucleotide dehydrogenase (nad) (Duenngai et al. 2008; Umesha et al. 2008; Sato et al. 2009; Buathong et al. 2015). However, the sensitivity in the case of light infection has been limited, and the PCR inhibitors present in stool are a major problem (Umesha et al. 2008; Sato et al. 2009). Our aim was to develop a highly sensitive PCR-based method for the detection of O. viverrini eggs in stool samples by introducing new primer sets for the previously reported targets ITS-2, cox1, as well as by establishing a new mitochondrial target, cytochrome b (cyb).

Materials and methods

The protocol was ethically approved by the Human Research Ethic Committee of Thammasat University (No. 019/2561). Written informed consents were obtained from the patients for publication of this study and the copy of the written consents is available upon request. Ninety-two human stool samples were collected from Nakhon-Phanom province, Northeastern Thailand. All samples were screened for parasitic infection by direct wet smear in 0.85% NaCl, and simultaneously in 2% Lugol’s iodine. We also took into account reports of suspected parasitic infection by trained non-professional village health volunteers. All samples were then re-investigated by using the PBS ethyl acetate concentration technique (PECT) as previously described (Buathong et al. 2015). The parasite eggs per gram (EPG) were counted and calculated from PECT. Initially, the sediments were autoclaved for 5 minutes at 121 °C, to open the operculum, and DNA was extracted by using QIAamp® PowerFecal® DNA kit (QIAGEN, Germany) according to the manufacturer’s instructions. The final filtrates contained 50 µL of eluted DNA, of which 2 µL each was used as a template for PCR amplification.

The adapted primers for internal transcribed spacer 2, ITS-2 (accession number AY584735.1), with a 245 bp amplicon, was newly designed as follows: OvITS2-F 5’- AAC AAT TGA GCC ACG ACT CC -3’ and OvITS2-R 5’- CAT CGA CAT CTT GAA CGC ATA-3’. Two mitochondrial target genes were also verified from a partial sequence of O. viverrini mitochondrial DNA available in GenBank (accession number JF739555.1), including cytochrome c oxidase subunit 1 (cox1, nt 6934–8484, 244 bp) and cytochrome b (cyb, nt 753–1862, 266 bp). The newly designed primers for cox1 (244 bp) and cyb (266 bp) were OvCOX1-F 5’-TTG CCA GGA TTC GGT ATG AT-3’; OvCOX1-R 5’-CCG CAA GCA TAT ACA ACC AA-3’; OvCYB-F 5’- TTG CTG ATG TAG GCA AGA GG -3’, and OvCYB-R 5’- GGT TGC CGC TCA ATA AGA CA-3’, respectively. PCR amplification was performed in a total volume of 25 µL, containing 2 µL of DNA template, 1X Taq buffer with (NH₄)₂SO₄, 1.5 mM MgCl₂, 200 µM of each dNTP, 25 pmol of each primer, and 1.25 units of Taq DNA polymerase (Thermo Scientific, Lithuania). PCR products were amplified in a thermal cycler (Mastercycler Nexus Eppendorf Flexlid, Germany), with initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 50°C for 30 sec, extension at 72°C for 30 sec, and a final extension at 72°C for 10 min to complete the amplification. The PCR products were sizeseparated in 2% agarose gels containing ViSafe Red Gel Stain (Vivantis, USA), using 1X TBE buffer at 100 V for 45 min. The positive control was DNA extracted from O. viverrini eggs of adult parasites obtained from the livers of infected hamsters (the animal ethics was approved by the Thammasat animal care and use committee [TU-ACUC] no. 024/2559, following the care and use of laboratory animals’ guideline (NRC 2011). The PCR products of ITS-2, cox1, and cyb from positive samples were evaluated by DNA sequencing (Macrogen, South Korea), and BLAST-searched in the GenBank database to confirm the correct species.

Results

Direct wet smears coupled with PECT of 92 human stool specimens, revealed that 20 of 92 (21.7%) samples were O. viverrini-positive. Other parasite eggs found were from Ascaris lubricoides (2/92; 2.2%), Taenia spp. (7/92; 7.6%), Trichuris trichiura (3/92; 3.3%), Capillaria philippinensis (1/92; 1.1%), and hookworm (7/92; 7.6%). Fifty-two samples were parasite-free (52/92; 56.5%) and no mixed infections were found, as shown in Table 1. All samples were subjected to amplification by PCR as mentioned above, for evaluation of the efficiency of the new primers (ITS-2, cox1, and cyb), and for cross-reactivity assessment. Representative PCR products of ITS-2 (245 bp), cox1 (244 bp) and cyb (266 bp) were size-separated by 2% agarose gel electrophoresis (Fig. 1). All PECT-O. viverrini-positive samples (n = 20) were positive for all three genes, while the other parasitic samples (n = 20) did not yield any product. Interestingly, the PECT-parasite-undetected group (n = 52) included two samples positive with ITS2, five samples positive with cox1, and six samples positive with cyb, as shown in Table 1. The sequencing results confirmed that the PCR products had 100% identity with O. viverrini DNA and the sequences of the PCR products were deposited in GenBank with accession numbers MW131224 (ITS-2), MW134725 (cox1), MW131879 (cyb). This means that PCR with our primer sets could detect a lower concentration of parasite eggs than was possible by PECT. Sensitivities of adapted primers for ITS-2, cox1 and cyb are shown in Table 2. The overall sensitivities for ITS-2, cox1 and cyb are 76.9%, 96.2% and 100%, respectively. Moreover, 100% sensitivity was found in samples containing > 50 EPG for all three sets of primers and the primers for cyb showed still 100% sensitivity for samples containing < 50 EPG.

Table 1.

Stool samples collected from Nakhon-Phanom province, Northeastern Thailand, tested for O. viverrini by PECT and PCR

Parasites	Positive samples by PECT	Positive samples by PCR with O. viverrini-specific primers
		ITS-2	cox1	cyb
Opisthrochis viverrini	20	22	25	26
Taenia spp.	7	0	0	0
Hookworm	7	0	0	0
Trichuris trichiura	3	0	0	0
Ascaris lumbricoides	2	0	0	0
Capillaria philippinensis	1	0	0	0
No parasite detection	52	50	47	46
Total	92	92	92	92

Fig. 1.

Agarose gels showing the resolved PCR products obtained from O. viverrini and other parasite DNA in human fecal samples with primers targeting ITS-2 (a), cox1 (b), and cyb (c). Lane M, 100-bp plus DNA marker (Thermo Fisher Scientific, Lithuania); lane P, positive control; lane N, negative control; Opisthorchis viverrini (Ov), Taenia spp. (Te), Hook worm (Hw), Trichuris trichiura (Tt), Ascaris lumbricoides (Al), Capillaria philippinensis (Cp), and parasite-negative sample (Pn)

Table 2.

Sensitivities of PCR-based detection of O. viverrini eggs in stool samples, using ITS-2, cox1, and cyb gene-specific primers

EPG	Number of samples	ITS-2		cox1		cyb
		Positive samples	Sensitivity (%)	Positive samples	Sensitivity (%)	Positive samples	Sensitivity (%)
< 50	17	11	66.7 (11/17)	16	94.1 (16/17)	17	100.0 (17/17)
51–100	6	6	100.0 (6/6)	6	100.0 (6/6)	6	100.0 (6/6)
> 100	3	3	100.0 (3/3)	3	100.0 (3/3)	3	100.0 (3/3)
Total	26	20	76.9 (20/26)	25	96.2 (25/26)	26	100.0 (26/26)

Discussion

This study aimed to evaluate the sensitivity of the new PCR gene-targets for O. viverrini eggs. Twenty-six samples positive detected by PECT and by PCR, were included for the validation. For the adapted ITS-2 primer, the overall sensitivity was 76.9% (20/26) with 100% in samples containing > 50 EPG (9/9), and with 66.7% (11/17) in < 50 EPG samples. The overall sensitivity was not higher than in the previous study (Duenngai et al. 2008), however, light infection samples (> 50 EPG) were detected at 100% sensitivity which is higher than reported in a study that used a higher egg concentration (Sato et al. 2009). The newly designed ITS-2 primer illustrated higher sensitivity in the low EPG range, but overall it had a similar percentage (71.0-92.4%) with the PCR-RFLP of ITS-2 that is currently used as a reference method for PCR detection of O. viverrini eggs (Traub et al. 2009; Buathong et al. 2017). However, our new primer sets make possible a quicker one-step procedure, by eliminating the need for restriction endonucleases digestion after PCR. For the mitochondrial genes, the new cox1 primers showed a sensitivity of 96.2% (25/26) overall, 94.1% (16/17) in samples containing < 50 EPG, 100% (6/6) in 51–100 EPG, and 100% (3/3) in > 100 EPG. Lamaningao’s study (Lamaningao et al. 2017) using combined conventional PCR and qPCR to detect O. viverrini cox1 gene, showed 100% sensitivity but did only specify egg concentration for a mock-infected sample. However, the present study provided 100% sensitivity (at > 50 EPG) without qPCR. Moreover, overall cox1 sensitivity is higher than the 66.7% sensitivity mentioned by Buathong and colleagues (Buathong et al. 2015). Remarkably, the cyb primers gave 100% overall sensitivity, meaning 100% for samples with < 50, 51–100, as well as > 100 EPG as shown in Table 2. Therefore, the newly designed primers for cyb should be further evaluated for PCR-based screening of O. viverrini-contaminated fecal samples. In addition, all three primer sets showed no cross reactivity with other helminth infections including Taenia spp, hookworms, Trichuris trichiura, Ascaris lubricoides and Capillaria philippinensis. Minute intestinal fluke infections could not be investigated in this study due to a lack of samples. This is unfortunate as the eggs of minute intestinal flukes are difficult to discriminate by microscopy. Finally, PCR inhibitors in fecal samples are a major obstacle and must be carefully removed during DNA extraction. Immunodiagnosis could replace PCR in case of bile duct obstruction or early infection (McCarthy et al. 2012; Saijuntha et al. 2018).

Conclusions

The sensitivities of PCR-based methods depend on factors such as gene copy number, the number of eggs, extracted DNA quality, and PCR inhibitors (Lovis et al. 2009). Therefore, recent infections harboring only immature parasites and infections with obstructed bile ducts cannot be detected. Our present study has reduced the limitations of DNA cleanup and PCR interference by using a new generation DNA isolation kit (QIAamp® PowerFecal® DNA kit, QIAGEN, Germany) which allowed us to obtain a high quality of extracted DNA. Highly purified DNA makes PCR more reliable, with only one amplicon per primer. Moreover, the selected ITS-2 target gene has an abundant copy number in the genomic DNA of O. viverrini eggs, making it suitable for routine amplification. Conclusively, our present study establishes not only a convenient PCR-based method but also reports a new target gene, cyb, which is useful in the detection of light O. viverrini infections without cross-reactivity (Ascaris lubricoides, Taenia spp, Trichuris trichiura, Capillaria philippinensis, and hookworms). However, the small sample size is the limitation of this study, and more samples are needed to evaluate the method more thoroughly.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.