Quantification of hookworm ova from wastewater matrices using quantitative PCR

A quantitative PCR (qPCR) assay was used to quantify Ancylostoma caninum ova seeded into treated wastewater and unseeded wastewater and sludge samples. We have estimated the average gene copy numbers for a single ovum using a mixed population of ova. The average gene copy numbers derived from the mixed population were used to estimate numbers of hookworm ova in A. caninum seeded and unseeded wastewater and sludge samples. The newly developed qPCR assay estimated an average of 3.7 × 10³ gene copies per ovum, which was then validated by seeding known numbers of hookworm ova into treated wastewater. The qPCR estimated an average of (1.1 ± 0.1), (8.6 ± 2.9) and (67.3 ± 10.4) ova for treated wastewater that was seeded with (1 ± 0), (10 ± 2) and (100 ± 21) ova, respectively. The further application of the qPCR assay for the quantification of A. caninum ova in unseeded wastewater matrices indicated that 50%, 90% and 67% of treated wastewater (1 L), raw wastewater (1 L) and sludge (~ 4 g) samples had variable numbers of A. caninumgene copies present. After conversion of the qPCR estimated gene copy numbers to ova numbers for unseeded wastewater samples treated wastewater, raw wastewater, and sludge samples had an average of 0.02, 1.24 and 67 ova, respectively. The result of this study indicated that qPCR can be used for the quantification of hookworm ova from wastewater and sludge samples; however, caution is advised in interpreting qPCR generated data for health risk assessment because of variable numbers of gene copies in an ovum depending on the cell development stage of an ovum.