title: Comparison of concentration methods for rapid detection of hookworm ova in wastewater matrices using quantitative PCR.
abstract: Hookworm infection contributes around 700 million infections worldwide especially in developing nations due to increased use of wastewater for crop production. The effective recovery of hookworm ova from wastewater matrices is difficult due to their low concentrations and heterogeneous distribution. In this study, we compared the recovery rates of (i) four rapid hookworm ova concentration methods from municipal wastewater, and (ii) two concentration methods from sludge samples. Ancylostoma caninum ova were used as surrogate for human hookworm (Ancylostoma duodenale and Necator americanus). Known concentration of A. caninum hookworm ova were seeded into wastewater (treated and raw) and sludge samples collected from two wastewater treatment plants (WWTPs) in Brisbane and Perth, Australia. The A. caninum ova were concentrated from treated and raw wastewater samples using centrifugation (Method A), hollow fiber ultrafiltration (HFUF) (Method B), filtration (Method C) and flotation (Method D) methods. For sludge samples, flotation (Method E) and direct DNA extraction (Method F) methods were used. Among the four methods tested, filtration (Method C) method was able to recover higher concentrations of A. caninum ova consistently from treated wastewater (39-50%) and raw wastewater (7.1-12%) samples collected from both WWTPs. The remaining methods (Methods A, B and D) yielded variable recovery rate ranging from 0.2 to 40% for treated and raw wastewater samples. The recovery rates for sludge samples were poor (0.02-4.7), although, Method F (direct DNA extraction) provided 1-2 orders of magnitude higher recovery rate than Method E (flotation). Based on our results it can be concluded that the recovery rates of hookworm ova from wastewater matrices, especially sludge samples, can be poor and highly variable. Therefore, choice of concentration method is vital for the sensitive detection of hookworm ova in wastewater matrices.
authors.affiliation: CSIRO Land and Water, Ecosciences Precinct, 41 Boggo Road, QLD 4102, Australia; School of Public Health, University of Queensland, Herston Road, QLD 4006, Australia. Electronic address: pradip.gyawali@csiro.au.
authors.forename: P
authors.initials: P
authors.lastname: Gyawali
authors.affiliation: CSIRO Land and Water, Ecosciences Precinct, 41 Boggo Road, QLD 4102, Australia; Faculty of Science, Health and Education, University of the Sunshine Coast, Maroochydore, DC, QLD 4558, Australia.
authors.forename: W
authors.initials: W
authors.lastname: Ahmed
authors.affiliation: School of Public Health, University of Queensland, Herston Road, QLD 4006, Australia.
authors.forename: P
authors.initials: P
authors.lastname: Jagals
authors.affiliation: CSIRO Land and Water, Ecosciences Precinct, 41 Boggo Road, QLD 4102, Australia; School of Public Health, University of Queensland, Herston Road, QLD 4006, Australia.
authors.forename: J P S
authors.initials: JP
authors.lastname: Sidhu
authors.affiliation: CSIRO Land and Water, Ecosciences Precinct, 41 Boggo Road, QLD 4102, Australia; School of Public Health, University of Queensland, Herston Road, QLD 4006, Australia.
authors.forename: S
authors.initials: S
authors.lastname: Toze
mesh_terms: D000722:Ancylostoma / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D002498:Centrifugation / Q000592:standards; D018509:DNA, Helminth / Q000302:isolation & purification; D021903:DNA, Ribosomal Spacer / Q000032:analysis; D004285:Dogs; D005243:Feces / Q000469:parasitology; D005374:Filtration / Q000592:standards; D006801:Humans; D010063:Ovum; D011793:Queensland; D012340:RNA, Ribosomal, 5.8S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012722:Sewage / Q000469:parasitology; D014462:Ultrafiltration / Q000379:methods / Q000592:standards; D062065:Wastewater / Q000469:parasitology*; D018508:Water Purification / Q000379:methods / Q000592:standards*; D014904:Western Australia
chemical_list: D018509:DNA, Helminth; D021903:DNA, Ribosomal Spacer; D012340:RNA, Ribosomal, 5.8S; D012722:Sewage; D062065:Wastewater
keywords: Concentration methods; Hookworm ova; Public health; Recovery rate; Wastewater and sludge