INTRODUCTION
Schistosomiasis is a water-borne parasitic disease,caused by blood-dwelling flukes of the genus Schistosoma, which continues to cause widespread affliction in many developing countries in tropical areas. An estimated 200 million people are infected worldwide, and more than 600 million live in endemic areas (World Health Organization, 2005). While the main species responsible for human infections are Schistosoma mansoni, S. haematobium and S. japonicum, the species S. mekongi is found in communities along the Mekong River in Cambodia and Lao People's Democratic Republic (Lao PDR) with high mortality rates (Muth et al. Reference Muth, Sayasone, Odermatt-Biays, Phompida, Duong and Odermatt2010). Recently, increasing numbers of travellers, migrants and foreign workers have resulted in the importation of schistosomiasis (Leshem et al. Reference Leshem, Meltzer, Marva and Schwartz2009; Clerinx and Van Gompel, Reference Clerinx and Van Gompel2011). The S. mekongi life cycle involves the snail intermediate host, Neotricula aperta, and the definitive hosts, i.e. humans, dogs and pigs (Strandgaard et al. Reference Strandgaard, Johansen, Pholsena, Teixayavong and Christensen2001; Attwood et al. Reference Attwood, Fatih, Campbell and Upatham2008; Ishikawa and Ohmae, Reference Ishikawa and Ohmae2009).
Microscopic methods for detection of eggs from infected stool and cercarial shedding from infected snails are tedious and time-consuming. Stool examination has certain limitations: it is not easy to differentiate between S. japonicum and S. mekongi eggs, it is not feasible during the pre-patent period, and it is insensitive in the case of light infections. In addition, it is difficult to positively identify species of Schistosoma cercariae. Parasite-specific antibody detection, such as an enzyme-linked immunosorbent assay, is a potential alternative diagnostic method for a schistosomiasis mekongi control program (Ohmae et al. Reference Ohmae, Sinuon, Kirinoki, Matsumoto, Chigusa, Socheat and Matsuda2004; Kirinoki et al. Reference Kirinoki, Chigusa, Ohmae, Sinuon, Socheat, Matsumoto, Kitikoon and Matsuda2011). However, serological diagnosis is problematic for the detection of parasites in infected stool or snail samples. Recently, molecular-based approaches, such as conventional polymerase chain reaction (c-PCR), have been developed for identification and differentiation of the major human schistosomes including S. mansoni, S. haematobium, S. japonicum and S. mekongi (Kato-Hayashi et al. Reference Kato-Hayashi, Kirinoki, Iwamura, Kanazawa, Kitikoon, Matsuda and Chigusa2010), but these procedures demand analysis by agarose gel electrophoresis. This process, however, requires a long time, has a limited throughput and a tendency toward carry-over contamination. The effective real-time PCR technique has increasingly replaced c-PCR due to its greatly improved molecular detection and differential diagnosis of microorganisms.
As this method has not yet been applied for the detection of S. mekongi in fecal specimens and infected snails, our study was designed to use real-time fluorescence resonance energy transfer (FRET) PCR, another assay arrangement of real-time based PCR, combined with melting-curve analysis for the detection of this parasite's DNA in infected rat stool and snail samples. In addition, a method for detection of S. mekongi spiked in water in laboratory setting conditions was developed.
MATERIALS AND METHODS
Parasite and DNA materials
Schistosoma mekongi (Loatian strain)-experimentally infected Neotricula aperta (beta race) snails and infected rats as well as the S. mekongi adults and cercariae were obtained from the Applied Malacology Center, Department of Social and Environmental Medicine, Faculty of Tropical Medicine, Mahidol University, Thailand. All animal experiments were performed according the Guidelines for Animal Experimentation of the National Research Council of Thailand. For specificity evaluations of real-time FRET PCR, S. japonicum (Japanese Yamanashi strain) adults were provided by the Faculty of Tropical Medicine, Mahidol University, Thailand. Other parasite DNAs were extracted from adult worms of Opisthorchis viverrini, Haplorchis taichui, Centrocestus spp., Clonorchis sinensis, Echinostoma malayanum, Fasciola gigantica, Paragonimus heterotremus, Stellantchasmus spp., metacercariae of Haplorchoides spp., cercariae of animal schistosomes, human leukocytes, S. japonicum-infected mouse feces and negative healthy human feces, as well as from human feces infected with Strongyloides stercoralis, Capillaria philippinensis, hookworm, Trichostrongylus spp., Trichuris trichiura, Ascaris lumbricoides, Taenia spp., Echinostoma spp., minute intestinal flukes, Giardia lamblia and Isospora belli. The DNA samples were kept at −70°C in the DNA bank at the Department of Parasitology, Faculty of Medicine, Khon Kaen University. This study was approved by the Khon Kaen University Ethics Committee for Human Research (reference numbers HE511023 and HE531201). Informed consent was obtained from all human adult participants and from parents or legal guardians of minors.
Capability of detection
To determine the capability of real-time FRET PCR, tissues from non-infected N. aperta snails were ground separately. Then, individual aliquots of 1, 5 and 10 non-infected snail samples were separately inoculated with 1, 2, 5, 10, and 40 S. mekongi cercariae. For detection of cercariae in water, deionized water samples spiked with live cercariae were prepared for filtration. Each 1 L water sample was spiked with 1, 2, 5, and 10 S. mekongi cercariae. Subsequently, samples were filtered through a Whatman Nuclepore membrane filter (Fisher Scientific, Pittsburgh, PA, USA) as previously described (Hung and Remais, Reference Hung and Remais2008). For detection of parasite eggs in fecal samples, 100 mg fecal aliquots of non-infected rats were separately inoculated with 1, 2, 5, 8, and 10 S. mekongi eggs. These samples were also used for genomic DNA extraction (see below). The resulting DNA samples were then used in real-time FRET PCR.
For a specificity evaluation of the method, genomic DNA from parasites other than S. mekongi and the control samples were used.
Preparation of specimens for real-time FRET PCR
DNA samples were extracted from all non-infected (n=30) and experimentally infected (n=30) N. aperta snails, including their shells, as well as from snail tissue samples artificially inoculated with S. mekongi cercariae. Each specimen was homogenized with disposable polypropylene pestles (Bellco Glass Inc., Vineland, NJ, USA) and extracted using a NucleoSpin Tissue kit (Macherey-Nagel GmbH & Co., KG, Duren, Germany). For DNA extraction from cercariae attached to filtered membranes, the protocol was performed as described above. For DNA extraction from rat feces, 100 mg samples of S. mekongi-infected rat feces, non-infected rat feces, and non-infected rat feces artificially inoculated with S. mekongi eggs were mixed thoroughly with 200 μl of normal saline solution (0·85% NaCl in distilled water), followed by centrifugation at 8000 g for 5 min. The supernatant was discarded and then the fecal pellet was frozen at −20°C for 30 min. The frozen pellets were homogenized with disposable polypropylene pestles (Bellco Glass Inc.) and extracted using a QIAamp® DNA Stool Mini Kit (Qiagen, Hilden, Germany). The intensity of S. mekongi eggs in the feces of infected rats (n=10) was presented as eggs per gramme (e.p.g.) of feces (ranging from 100 to 2200 e.p.g.; geometric mean=255·4 e.p.g.). DNA was eluted in 100 μl of distilled water for both extraction kits, of which 5 μl was used in the real-time PCR reaction.
Real-time FRET PCR assay
LightCycler PCR detection and analysis systems (LightCycler 2.0, Roche Applied Science, Mannheim, Germany) were used for amplification and quantification. The reaction was performed in glass capillaries. Specific primers, i.e. SM-F (5′-GTT CTT GAG TAG TGT AGT C-3′) and SM-R (5′-ATA ACC TTA ACA AAC TCA TAA AC-3′) (Proligo, Singapore), were designed to bind to the S. mekongi mitochondrion complete genome sequence (GenBank Accession number AF217449) (Le et al. Reference Le, Blair, Agatsuma, Humair, Campbell, Iwagami, Littlewood, Peacock, Johnston, Bartley, Rollinson, Herniou, Zarlenga and McManus2000). For amplification detection, the LightCycler FastStart DNA Master HybProbe Kit (Roche Applied Science) was used as recommended by the manufacturer. Briefly, a pair of adjacent oligoprobes was hybridized with the S. mekongi mitochondrion. One probe had been labelled at the 5′ end with the LightCycler Red 705 fluorophore (5′ Red 705-ATT AAC CTT TGG TTG TTA TAC TAC GCG TAT G-Phosphate 3′) (SMLC705 probe) and the other had been labelled at the 3′ end with 530 fluorescein (5′-CTT ATA GTG TGC GAT TAT TTA TTA TAT TTT TTG GT-Flou 530-3′) (SMFL530 probe) (Tib Molbiol, Berlin, Germany). The probes and primers were designed by LC probe design software (Roche Applied Science). When the probes hybridized to the same DNA strand internal to the PCR primers, the probes came in close proximity and produced a FRET (Lyon and Wittwer, Reference Lyon and Wittwer2009). The PCR mixture contained LightCycler FastStart DNA Master HybProbe (Roche Applied Science), 3 mM MgCl2, 0·5 μM SM-F primer, 0·5 μM SM-R primer, 0·2 μM SMLC705 probe and 0·2 μM SMFL530 probe. The total reaction volume was 20 μl. The samples were run through 45 cycles of repeated denaturation (10 s at 95°C), annealing (30 s at 45°C), and extension (15 s at 72 °C). The temperature transition rate was 20°C/s. After amplification, a melting curve was produced by heating the product at 20°C/s to 95°C, cooling it to 60°C, keeping it at 60°C for 15 s, and then slowly heating it at 0·1°C/s to 80°C. The fluorescence intensity change was measured throughout the slow heating phase. In order to determine the specificity of the oligonucleotide hybridization based on the FRET technique, DNA extracted from control samples (those not containing S. mekongi) were analysed separately. Each run contained at least 1 negative control consisting of 5 μl of distilled water. For improved visualization of the melting temperatures (Tm), melting curves were derived as previously described (Thanchomnang et al. Reference Thanchomnang, Intapan, Lulitanond, Choochote, Manjai, Prasongdee and Maleewong2008). Melting curves were applied to determine hybridization between the fluorophore-labelled probes and the specific amplified products. The specific amplified products were shown by conventional agarose gel electrophoresis. The cycle number (Cn) indicating the target sequence copy number was presented as the number of PCR cycles needed for the fluorescence signal of the amplicons to exceed the detected threshold value.
S. mekongi-positive control plasmid
A positive control plasmid was constructed by cloning a PCR product of the S. mekongi mitochondrion into the pGEM-T Easy Vector (Promega, Madison, WI, USA), according to the manufacturer's instructions. The PCR products were obtained by c-PCR using primers SM-F and SM-R. The plasmid was propagated in Escherichia coli, and the nucleotide sequence of the inserted gene was sequenced in both directions. The nucleotide sequence of the cloned mitochondrion revealed an identical structure to the S. mekongi genome (GenBank Accession no. AF217449).
Data analysis
The diagnostic sensitivity and specificity were calculated and expressed using the standard method (Galen, Reference Galen1980). The relationship between the worm loads and the Cn was analysed by Pearson Correlation Test.
RESULTS
Standardization of real-time PCR
The sensitivity of real-time FRET PCR was determined using 5 μl of 10-fold serial dilutions (4·3×109–4·3×102 copies) of S. mekongi-positive control plasmid in distilled water. The detection limit of the mitochondrion gene target DNA sequence was 4·3×104 copies of positive control plasmid (Fig. 1L), when considering 35 cycles as the cutoff detection limit. In addition, 10-fold serial dilutions (40–4×10−5 ng) of S. mekongi genomic DNA were spiked to individual non-infected N. aperta snail and genomic DNAs were separately extracted from each sample as described above. Then sensitivity of the real-time FRET PCR was determined using 5 μl of each extracted sample. The detection limit was as little as 4×10−3 ng S. mekongi genomic DNA/snail (Fig. 1R). No fluorescence signal was obtained when testing purified DNA from: non-infected N. aperta snails, non-infected rat feces, S. japonicum-infected mouse feces, and panel of other control samples (see Materials and Methods section).
Amplification plot of fluorescence (y-axis) versus cycle numbers (x-axis) showing the analytical sensitivity of real-time PCR for detecting serial dilutions of Schistosoma mekongi plasmid DNA copies (L) and genomic DNA spiked Neotricula aperta snail (R). (A–H): 10-fold dilution concentrations of S. mekongi plasmids from 4·3×109 to 4·3×102 copies/reaction, (I and Z) distilled water and 10-fold dilution concentrations of S. mekongi genomic DNA spiked snail from 40 to 4×10−5 ng/snail (S–Y).
In terms of the capability of detection, as little as 2 eggs could be clearly detected in 100 mg of non-infected rat feces; and a DNA sample from 1 S. mekongi cercaria in filtered paper could be measured. DNA samples from each aliquot of 1, 5 and 10 non-infected N. aperta snail tissues artificially inoculated with 1 S. mekongi cercaria were positive (data not shown).
Real-time FRET PCR for the detection of S. mekongi in infected N. aperta snails and rats
Real-time FRET PCR combined with melting curve analysis of the amplicons was applied to detect S. mekongi DNA in infected N. aperta snails and rats. A total of 30 S. mekongi-infected and 30 non-infected N. aperta snails, as well as 10 S. mekongi-infected and 10 non-infected rat fecal samples, were separately analysed. The melting curve analyses are shown in Fig. 2. When using S. mekongi-specific primers and probes, the mean±s.d., range and median of the Tm values of the S. mekongi-infected N. aperta snails were 62·85±0·09, 62·74–63·00 and 62·85, respectively, and those of the S. mekongi-infected rat fecal samples were 62·81±0·05, 62·73–62·9 and 62·8, respectively. A total of 30 S. mekongi-infected N. aperta snails (Cn range 29·49–32·04; mean±s.d.=31·43±1·09; median=31·86) and 10 S. mekongi-infected rat fecal samples (Cn range 31·08–35·34; mean±s.d.=31·98±1·11; median=31·74) tested positive by real-time FRET PCR with melting-curve analysis, whereas all of the specific control DNA was negative. However, no significant correlation was reported between the cycle numbers and the intensity of S. mekongi eggs in fecal samples (P> 0·05) (data not shown). The sensitivity and specificity were all 100%. The validity of the real-time FRET PCR method used in this study was verified by the presence of the prominent 260 bp product amplified from S. mekongi genomic DNA, and from DNA from S. mekongi-infected N. aperta snails, S. mekongi-infected rat feces, and the positive control plasmids (Fig. 3, lanes 1–3 and P). In contrast, genomic DNAs from other control materials were not amplified. Although the genomic DNAs from parasites other than S. mekongi, or from fecal samples infected with other parasites, gave various amplified bands, but no specific fluorescence signal was detected by melting-curve analysis.
Representative melting-curve analysis of 2 fluorophore-labelled probes hybridized to the amplification products from mitochondrion DNA of Schistosoma mekongi. The melting temperature (Tm) of the double-stranded fragment is visualized by plotting the negative derivative of the change in fluorescence divided by the change in temperature in relation to the temperature [−(d/dT) Fluorescence (705/530)]. The turning point of this converted melting curve results in a peak and permits easy identification of the fragment's specific Tm. Melting curves of genomic DNA from (A) S. mekongi adults; (B) positive control plasmid; (C, D) S. mekongi-infected rat feces; and (E, F) S. mekongi-infected Neotricula aperta snails; as well as (G) genomic DNA from non-infected N. aperta snails, non-infected rat feces, a panel of other control samples (see Materials and Methods section), and a negative control (distilled water).
Ethidium bromide staining patterns of the PCR products on a 1·5% agarose gel. The arrows indicate the 260 bp Schistosoma mekongi-specific bands. (Lane N): negative control containing no DNA. (Lane P): the PCR products obtained from the positive control plasmid. Genomic DNA from: S. mekongi adults (lane 1); S. mekongi-infected Neotricula aperta snails (lane 2); S. mekongi-infected rat feces (lane 3); non-infected N. aperta snails (lane 4); non-infected rat feces (lane 5); Opisthorchis viverrini (lane 6); Centrocestus spp. (lane 7); Clonorchis sinensis (lane 8); Haplorchis taichui (lane 9); Fasciola gigantica (lane 10); Echinostoma malayanum (lane 11); Paragonimus heterotremus (lane 12); Haplorchoides spp. (lane 13); Stellantchasmus spp. (lane 14); animal schistosomes (lane 15); individual human feces infected with Strongyloides stercoralis (lane 16), Taenia spp. (lane 17), minute intestinal flukes (lane 18), Capillaria philippinensis (lane 19), Giardia lamblia (lane 20), hookworm (lane 21), Isospora belli (lane 22), Trichostrongylus spp. (lane 23), Trichuris trichiura (lane 24), and Ascaris lumbricoides (lane 25); S. japonicum-infected mouse feces (lane 26); and negative healthy human feces (lane 27). (Lane M): DNA size markers (1 kb plus DNA ladder from Invitrogen, Carlsbad, CA, USA).
DISCUSSION
Identification and differentiation of major human schistosomes by real-time PCR has been developed for supplemental detection of S. japonicum (Lier et al. Reference Lier., Simonsen, Wang, Lu, Haukland, Vennervald, Hegstad and Johansen2009; Hung and Remais, Reference Hung and Remais2008), S. mansoni (Gomes et al. Reference Gomes, Melo, Werkhauser and Abath2006; ten Hove et al. Reference ten Hove, Verweij, Vereecken, Polman, Dieye and van Lieshout2008) and S. haematobium (ten Hove et al. Reference ten Hove, Verweij, Vereecken, Polman, Dieye and van Lieshout2008; Kjetland et al. Reference Kjetland, Hove, Gomo, Midzi, Gwanzura, Mason, Friis, Verweij, Gundersen, Ndhlovu, Mduluza and Van Lieshout2009). To our knowledge, this is the first successful study to use 2 individually fluorophore-labelled hybridization probes based on real-time FRET PCR combined with melting-curve analysis for the detection of S. mekongi infection. This method can detect parasite DNA not only in infected snails and fecal samples, but can also be used for detection of S. mekongi in water samples. This method was able to detect as little as 1 cercaria implanted in a pool of 10 non-infected N. aperta snails, and 2 eggs inoculated in 100 mg of non-infected rat feces. The results were positive for all S. mekongi-infected snails and rats, which revealed 100% sensitivity. Moreover, the procedure can detect 1 cercaria per litre of spiked laboratory water.
No fluorescence appeared, and the primers did not amplify the 260 bp band when DNAs belonging to parasites other than S. mekongi were tested, indicating 100% specificity. However, a non-specific band at approximately 380 bp was seen when S. mekongi-infected rat feces were observed. This result did not possibly cause a problem because it presented in 1 of 10 S. mekongi-infected rat fecal samples (data not shown). Hence, real-time FRET PCR could be useful for distinguishing S. mekongi cercariae from a panel of other flukes, i.e. S. japonicum, O. viverrini, Centrocestus spp., H. taichui, F. gigantica, E. malayanum, C.sinensis, P. heterotremus, Haplorchoides spp., Stellantchasmus spp., and animal schistosomes as well as non-infected N. aperta snails. The method was also able to distinguish S. mekongi eggs in rat fecal samples from extracted DNA of human leukocytes, S. japonicum-infected mouse feces, and negative human feces, as well as from human feces infected with other intestinal parasites (see Materials and Methods section). On the other hand, no significant correlation between cycle numbers and intensity of S. mekongi eggs in fecal samples was observed. This may be due to the degradation of larval DNA in S. mekongi eggs in fecal samples. Further studies need to improve the quantitative efficacy of this method.
The real-time FRET PCR protocol presented here provides an alternative choice to the available microscopic, serological or molecular methods for the detection of S. mekongi in snails, the first intermediate host, and in infected fecal samples of the final hosts. Two fluorophore hybridization probes and an assay system can be used to detect S. mekongi, but do not react with S. japonicum. Due to other schistosome DNA samples (i.e. S. mansoni and S. haematobium) not being available in our laboratory, this procedure did not specifically test these DNA samples. Further bioinformatic analysis demonstrated that our probes are highly specific, showing no identical complementarity with other parasite nucleotide sequences in the databases (NCBI, GenBank Flat File Release 183.0, April 15, 2011; S. mansoni genome, http://www.sanger.ac.uk/cgi-bin/blast; S. japonicum genome, http://www.genedb.org/blast), except for the SMFL530 probe that shows partial complementary with certain sequences of S. japonicum (Sjp_0125110.1..mRNA and Sjp_0123690.1..mRNA) and S. japonicum mitochondrion gene (GenBank Accession no. AF215860). However, no positive result was documented when S. japonicum genomic DNA was investigated. Notwithstanding, further studies need to test for specificity using DNA samples from S. mansoni and S. haematobium as well as other animal schistosomes.
This detection system can provide standardized information on S. mekongi epidemiology. Presently, countries in the lower area of the Mekong River basin, including Laos PDR, Cambodia and Thailand, are popular areas for travellers from developed, non-endemic countries. The developed procedure could be useful as a detection method for screening patients returning from high-risk areas. Moreover, the tool can be used for surveillance of Mekong schistosomiasis in people returning from military operations, and among the influx of immigrants from endemic countries.
In conclusion, a specific, sensitive and fast real-time FRET PCR for the detection of S. mekongi in snail intermediate hosts and in infected rat fecal samples was developed. In addition, the method has the potential to be applied to testing of environmental water samples, serving as a fast and reliable tool for cercarial location in endemic areas. The test is suitable for epidemiological studies and eradication programs for intermediate hosts.
FINANCIAL SUPPORT
This research was supported by grants from: the National Science and Technology Development Agency (Discovery Based Development Grant); the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission; and the Faculty of Medicine, Khon Kaen University.
