Detection of Schistosoma japonicum and Oncomelania hupensis quadrasi environmental DNA and its potential utility to schistosomiasis japonica surveillance in the Philippines

Autoři: Raffy Jay C. Fornillos aff001;  Marcello Otake Sato aff003;  Ian Kim B. Tabios aff004;  Megumi Sato aff005;  Lydia R. Leonardo aff001;  Yuichi Chigusa aff003;  Toshifumi Minamoto aff007;  Mihoko Kikuchi aff008;  Emelda R. Legaspi aff009;  Ian Kendrich C. Fontanilla aff001
Působiště autorů: DNA Barcoding Laboratory, Institute of Biology, College of Science, University of the Philippines Diliman, Quezon City, Philippines aff001;  Natural Sciences Research Institute, University of the Philippines Diliman, P. Velasquez St. Diliman, Quezon City, Philippines aff002;  Department of Tropical Medicine and Parasitology, Dokkyo Medical University, Tochigi, Japan aff003;  College of Medicine, University of the Philippines Manila, Ermita Manilla, Philippines aff004;  Graduate School of Health Sciences, Niigata, Japan aff005;  Graduate School, University of the East Ramon Magsaysay Memorial Medical Center, Quezon City, Philippines aff006;  Graduate School of Human Development and Environment, Kobe University, Tsurukabuto, Nada-ku, Kobe, Japan aff007;  Department of Immunogenetics, Institute of Tropical Medicine, Nagasaki University, Sakamoto, Nagasaki, Japan aff008;  Medical Zoology Laboratory, Schistosomiasis Research and Training Center, Palo Leyte, Philippines aff009
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0224617


In recent years, the prevalence and infection intensity of Schistosoma japonicum in endemic areas of the Philippines have significantly decreased due to yearly population-based treatment strategies, yet transmission rates remain high and uninterrupted. An important indicator of active disease transmission is the presence of Schistosoma japonicum and its snail intermediate host Oncomelania hupensis quadrasi in freshwater habitats. In this study, we sought to apply a species-specific real-time PCR (qPCR) assay for the detection of S. japonicum and O. hupensis quadrasi in freshwater samples using environmental DNA approach that can complement the commonly utilized malacological survey in determining potential transmission foci in order to have a more effective snail surveillance strategy for schistosomiasis japonica in endemic areas. The newly developed assay was specific to S. japonicum and O. hupensis quadrasi with no amplification detected against non-target trematode Fasciola spp. and snails such as Lymnaea spp., Pomacea canaliculata, and Melanoides spp. that typically co-exist in the same environment. The assay effectiveness was determined using 19 environmental water samples collected from Northern Samar (N = 5 sites), Leyte (N = 11 sites) and Compostela Valley (N = 3 sites) and compared to malacological survey for determining O. hupensis quadrasi snail colonies and snail crushing to visualize S. japonicum cercariae. TaqMan qPCR targeting a short fragment of the cytochrome c oxidase subunit 1 (cox1) gene was positive for S. japonicum in 9 sites, for O. hupensis quadrasi in 9 sites, and for both S. japonicum and O. hupensis quadrasi in 5 sampling sites. Moreover, it was able to detect O. hupensis quadrasi in 3 out of 12 sites found negative and 6 out of 7 sites found positive through malacological survey, and in 4 of the 5 snail sites positive for snails with cercariae. Overall, this method can complement malacological surveys for monitoring of schistosomes in endemic areas of the Philippines, especially those with high risk of human infection.

Klíčová slova:

Fresh water – Malacology – Philippines – Polymerase chain reaction – Schistosomiasis – Snails – Schistosoma japonicum – Schistosomiasis japonica


1. Blas BL. Handbook for the control of Schistosomiasis japonica. A monograph on Schistosoma japonicum infection in the Philippines. Schistosomiasis Control Service Department of Health. 1991.

2. Olveda DU, Li Y, Olveda RM, Lam AK, McManus DP, Chau TNP, et al. Bilharzia in the Philippines: past, present, and future. Int J Infect Dis. 2014;18: 52–56. doi: 10.1016/j.ijid.2013.09.011 24211228

3. Leonardo LR, Acosta LP, Olveda RM, Aligui GDL. Difficulties and strategies in the control of schistosomiasis in the Philippines. Acta Trop. 2002;82: 295–299. doi: 10.1016/s0001-706x(02)00022-0 12020904

4. Conlan J V, Sripa B, Attwood S, Newton PN. A review of parasitic zoonoses in a changing Southeast Asia. Vet Parasitol. 2011;182: 22–40. doi: 10.1016/j.vetpar.2011.07.013 21846580

5. Tubangui MA, others. The molluscan intermediate host in the Philippines of the oriental blood fluke Schistosoma japonicum Katsurada. Philipp J Sci. 1932; 295–304.

6. Blas BL (Ed). Handbook for the control of schistosomiasis japonica: Part II guide to control operations. DOH Schistosomiasis Control Service. 1988–89: 22–24.

7. Leonardo L, Chigusa Y, Kikuchi M, Kato-Hayashi N, Kawazu S-I, Angeles JM, et al. Schistosomiasis in the Philippines: Challenges and some successes in control. Southeast Asian J Trop Med Public Health. 2016;47.

8. Blas BL, Rosales MI, Lipayon IL, Yasuraoka K, Matsuda H, Hayashi M. The schistosomiasis problem in the Philippines: a review. Parasitol Int. 2004;53: 127–134. doi: 10.1016/j.parint.2004.01.003 15081944

9. Rabinowitz PM, Kock R, Kachani M, Kunkel R, Thomas J, Gilbert J, et al. Toward proof of concept of a one health approach to disease prediction and control. Emerg Infect Dis. 2013;19.

10. Gryseels B, Polman K, Clerinx J, Kestens L. Human schistosomiasis. Lancet. 2006;368: 1106–1118. doi: 10.1016/S0140-6736(06)69440-3 16997665

11. Colley DG, Bustinduy AL, Secor WE, King CH. Human schistosomiasis. Lancet. 2014;383: 2253–2264. doi: 10.1016/S0140-6736(13)61949-2 24698483

12. Leonardo L, Rivera P, Saniel O, Villacorte E, Lebanan MA, Crisostomo B, et al. A national baseline prevalence survey of schistosomiasis in the Philippines using stratified two-step systematic cluster sampling design. J Trop Med. 2012;2012.

13. Leonardo L, Rivera P, Saniel O, Solon JA, Chigusa Y, Villacorte E, et al. New endemic foci of schistosomiasis infections in the Philippines. Acta Trop. 2015;141: 354–360. doi: 10.1016/j.actatropica.2013.03.015 23583862

14. Driscoll AJ, Kyle JL, Remais J. Development of a novel PCR assay capable of detecting a single Schistosoma japonicum cercaria recovered from Oncomelania hupensis. Parasitology. 2005;131: 497–500. doi: 10.1017/S0031182005007961 16174414

15. Hung YW, Remais J. Quantitative detection of Schistosoma japonicum cercariae in water by real-time PCR. PLoS Negl Trop Dis. 2008;2: e337. doi: 10.1371/journal.pntd.0000337 19015722

16. Worrell C, Xiao N, Vidal JE, Chen L, Zhong B, Remais J. Field detection of Schistosoma japonicum cercariae in environmental water samples by quantitative PCR. Appl Environ Microbiol. 2011;77: 2192–2195. doi: 10.1128/AEM.01561-10 21278276

17. Ficetola GF, Miaud C, Pompanon F, Taberlet P. Species detection using environmental DNA from water samples. Biol Lett. 2008;4: 423–425. doi: 10.1098/rsbl.2008.0118 18400683

18. Minamoto T, Yamanaka H, Takahara T, Honjo MN, Kawabata Z. Surveillance of fish species composition using environmental DNA. Limnology. 2012;13: 193–197.

19. Bass D, Stentiford GD, Littlewood DTJ, Hartikainen H. Diverse applications of environmental DNA methods in parasitology. Trends Parasitol. 2015;31: 499–513. doi: 10.1016/ 26433253

20. Sato MO, Rafalimanantsoa A, Ramarokoto C, Rahetilahy AM, Ravoniarimbinina P, Kawai S, et al. Usefulness of environmental DNA for detecting Schistosoma mansoni occurrence sites in Madagascar. Int J Infect Dis. 2018;76: 130–136. doi: 10.1016/j.ijid.2018.08.018 30201503

21. Hashizume H, Sato M, Sato MO, Ikeda S, Yoonuan T, Sanguankiat S, et al. Application of environmental DNA analysis for the detection of Opisthorchis viverrini DNA in water samples. Acta Trop. 2017;169: 1–7. doi: 10.1016/j.actatropica.2017.01.008 28108370

22. Jones RA, Brophy PM, Davis CN, Davies TE, Emberson H, Stevens PR, et al. Detection of Galba truncatula, Fasciola hepatica and Calicophoron daubneyi environmental DNA within water sources on pasture land, a future tool for fluke control? Parasit Vectors. 2018;11: 342. doi: 10.1186/s13071-018-2928-z 29884202

23. McManus DP, Gordon C, Weerakoon KGAD. Testing of water samples for environmental DNA as a surveillance tool to assess the risk of schistosome infection in a locality. Int J Infect Dis. 2018;76: 128–129. doi: 10.1016/j.ijid.2018.09.022 30266632

24. Fornillos RJC, Fontanilla IKC, Chigusa Y, Kikuchi M, Kirinoki M, Kato-Hayashi N, et al. Infection rate of Schistosoma japonicum in the snail Oncomelania hupensis quadrasi in endemic villages in the Philippines: Need for snail surveillance technique. Trop Biomed. 2019;36: 402–411.

25. Uchii K, Doi H, Minamoto T. A novel environmental DNA approach to quantify the cryptic invasion of non-native genotypes. Mol Ecol Resour. 2016;16: 415–422. doi: 10.1111/1755-0998.12460 26307935

26. Thompson JD, Higgins DG, Gibson TJ. Using CLUSTAL for multiple sequence alignments. Methods Enzymol. 1996;266:383–402. doi: 10.1016/s0076-6879(96)66024-8 8743695

27. Hall TA, others. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic acids symposium series. 1999. pp. 95–98.

28. Staden R, Beal KF, Bonfield JK. The staden package, 1998. Bioinformatics methods and protocols. Springer; 2000. pp. 115–130.

29. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215: 403–410. doi: 10.1016/S0022-2836(05)80360-2 2231712

30. Taberlet P, Coissac E, Hajibabaei M, Rieseberg LH. Environmental DNA. Mol Ecol. 2012;21: 1789–1793. doi: 10.1111/j.1365-294X.2012.05542.x 22486819

31. Deiner K, Bik HM, Mächler E, Seymour M, Lacoursière-Roussel A, Altermatt F, et al. Environmental DNA metabarcoding: Transforming how we survey animal and plant communities. Mol Ecol. 2017;26: 5872–5895. doi: 10.1111/mec.14350 28921802

32. Barnes MA, Turner CR, Jerde CL, Renshaw MA, Chadderton WL, Lodge DM. Environmental conditions influence eDNA persistence in aquatic systems. Environ Sci Technol. 2014;48: 1819–1827. doi: 10.1021/es404734p 24422450

33. Goldberg CS, Turner CR, Deiner K, Klymus KE, Thomsen PF, Murphy MA, et al. Critical considerations for the application of environmental DNA methods to detect aquatic species. Methods Ecol Evol. 2016;7: 1299–1307.

34. Sassoubre LM, Yamahara KM, Gardner LD, Block BA, Boehm AB. Quantification of environmental DNA (eDNA) shedding and decay rates for three marine fish. Environ Sci Technol. 2016;50: 10456–10464. doi: 10.1021/acs.est.6b03114 27580258

35. McMullen DB. The Control of Schistosomiasis japonica: I. Observations on the habits, ecology and life cycle of Oncomelania quadrasi, the molluscan intermediate host of Schistosoma japonicum in the Philippine Islands. Am J Epidemiol. 1947;45: 259–273.

36. Ohmae H, Iwanaga Y, Nara T, Matsuda H, Yasuraoka K. Biological characteristics and control of intermediate snail host of Schistosoma japonicum. Parasitol Int. 2003;52: 409–417. 14665400

37. World Health Organization. Expert Consultation to accelerate elimination of schistosomiasis. Shanghai, China. 22–23 May 2017. Available from: Cited 07 September 2018.

38. Shi Y, Qiu J, Li R, Shen Q, Huang D. Identification of potential high-risk habitats within the transmission reach of Oncomelania hupensis after floods based on SAR techniques in a plane region in china. Int J Environ Res Public Health. 2017;14: 986.

39. McCreesh N, Booth M. Challenges in predicting the effects of climate change on Schistosoma mansoni and Schistosoma haematobium transmission potential. Trends Parasitol. 2013;29: 548–555. doi: 10.1016/ 24064438

40. Chua JC, Tabios IK, Tamayo PG, Leonardo LR, Fontanilla IKC, Fornillos RJC, et al. Genetic Comparison of Oncomelania hupensis quadrasi (Möllendorf, 1895) (Gastropoda: Pomatiopsidae), the Intermediate Host of Schistosoma japonicum in the Philippines, Based on 16S Ribosomal RNA Sequence. Sci. Diliman. 2017;292: 32–50.

41. Calata FIC, Caranguian CZ, Mendoza JEM, Fornillos RJC, Tabios IKB, Fontanilla IKC, et al. Analysis of Environmental DNA and Edaphic Factors for the Detection of the Snail Intermediate Host Oncomelania hupensis quadrasi. Pathogens. 2019;8: 160.

Článek vyšel v časopise


2019 Číslo 11