The major waterborne protozoan diseases are those caused by Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica. We studied the performance of a commercial multiplex real-time polymerase chain reaction (MRT-PCR) kit – applied in fecal samples – for the detection of intestinal protozoa in sewage. The MRT-PCR was assessed against direct immunofluorescence assay (DFA); and separate, nested PCRs (nPCRs) for the detection of G. lamblia, Cryptosporidium spp., and E. histolytica. MRT-PCR proved to be highly specific, enabling the detection of E. histolytica and a subset of Cryptosporidium spp. including those mainly responsible for human infections. MRT-PCR was also highly sensitive, finding 10 times more samples contaminated with G. lamblia than DFA. Compared with nPCR for G. lamblia, MRT-PCR was highly accurate. At a cutoff cycle threshold value of 37.6, it showed high sensitivity and specificity in detecting G. lamblia, while reaching substantial agreement with nPCR. Despite variable sensitivity by target DNA, its high specificity made the test a suitable alternative for fast, simultaneous screening for intestinal protozoa of public health importance, revealing co-contamination in five sewage samples. Its high throughput capacity may facilitate informed decision-making for drawing up a sewage monitoring plan and taking appropriate public health measures to minimize the public health risk posed by sewage reuse.
Bibliographical noteFunding Information:
The authors would like to acknowledge Aftab Jasir, European Centre for Disease Prevention and Control, Stockholm, and Silvia Herrera-León, Instituto de Salud Carlos III, Madrid, for their input to the project proposal and reviewing the manuscript. We would also like to thank Fotis Tsagkarakis, Greek Educational System, Athens, for reviewing mathematical equations and formulas. We also take the opportunity to thank Ioannis G. Koutelekos, Technological Educational Institute, Athens, for his support in preparing the manuscript.
- Cryptosporidium spp
- Entamoeba histolytica
- Giardia lamblia
- Multiplex real-time PCR
- Sewage monitoring