Environmental Science and Pollution Research

, Volume 23, Issue 18, pp 18639–18648 | Cite as

Quantitative detection of viable helminth ova from raw wastewater, human feces, and environmental soil samples using novel PMA-qPCR methods

  • P. GyawaliEmail author
  • W. Ahmed
  • J. P. S. Sidhu
  • S. V. Nery
  • A. C. Clements
  • R. Traub
  • J. S. McCarthy
  • S. Llewellyn
  • P. Jagals
  • S. Toze
Research Article


In this study, we have evaluated the efficacy of propidium monoazide quantitative polymerase chain reaction (PMA-qPCR) to differentiate between viable and non-viable Ancylostoma caninum ova. The newly developed method was validated using raw wastewater seeded with known numbers of A. caninum ova. Results of this study confirmed that PMA-qPCR has resulted in average of 88 % reduction (P < 0.05) in gene copy numbers for 50 % viable +50 % non-viable when compared with 100 % viable ova. A reduction of 100 % in gene copies was observed for 100 % non-viable ova when compared with 100 % viable ova. Similar reductions (79–80 %) in gene copies were observed for A. caninum ova-seeded raw wastewater samples (n = 18) collected from wastewater treatment plants (WWTPs) A and B. The newly developed PMA-qPCR method was applied to determine the viable ova of different helminths (A. caninum, A. duodenale, Necator americanus and Ascaris lumbricoides) in raw wastewater, human fecal and soil samples. None of the unseeded wastewater samples were positive for the above-mentioned helminths. N. americanus and A. lumbricoides ova were found in unseeded human fecal and soil samples. For the unseeded human fecal samples (1 g), an average gene copy concentration obtained from qPCR and PMA-qPCR was found to be similar (6.8 × 105 ± 6.4 × 105 and 6.3 × 105 ± 4.7 × 105) indicating the presence of viable N. americanus ova. Among the 24 unseeded soil samples tested, only one was positive for A. lumbricoides. The mean gene copy concentration in the positively identified soil sample was 1.0 × 105 ± 1.5 × 104 (determined by qPCR) compared to 4.9 × 104 ± 3.7 × 103 (determined by PMA-qPCR). The newly developed PMA-qPCR methods were able to detect viable helminth ova from wastewater and soil samples and could be adapted for health risk assessment.


Hookworm ova Viability Wastewater PMA-qPCR Public health 



The authors would like to appreciate financial support from the Water Corporation, WA, and the Commonwealth Scientific and Industry Research Organization (CSIRO). The authors are thankful to Lyn Knott, Laboratory Manager Faculty of Veterinary Science, University of Queensland, Australia, for providing A. caninum-positive dog fecal samples. We also thank Queensland Urban Utilities for providing access to the wastewater treatment plants for collecting samples and people in study communities in East Timor for providing their fecal samples.


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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • P. Gyawali
    • 1
    • 2
    Email author
  • W. Ahmed
    • 1
  • J. P. S. Sidhu
    • 1
    • 2
  • S. V. Nery
    • 3
  • A. C. Clements
    • 3
  • R. Traub
    • 4
  • J. S. McCarthy
    • 5
    • 6
  • S. Llewellyn
    • 5
  • P. Jagals
    • 2
  • S. Toze
    • 1
    • 2
  1. 1.CSIRO Land and WaterBrisbaneAustralia
  2. 2.School of Public HealthThe University of QueenslandHerstonAustralia
  3. 3.Research School of Population Health, College of Medicine, Biology and EnvironmentThe Australian National UniversityLinnaeus WayAustralia
  4. 4.Faculty of Veterinary and Agricultural SciencesThe University of MelbourneParkvilleAustralia
  5. 5.Clinical Tropical Medicine LaboratoryQIMR Berghofer Medical Research InstituteHerstonAustralia
  6. 6.School of MedicineThe University of QueenslandHerstonAustralia

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