A comparison of qPCR and microscopy for the detection and enumeration of Cryptosporidium oocysts from drinking water

Abstract

Introduction. Cryptosporidium presents one of the main waterborne public health threats due to its resistance to chlorine disinfection and ability to cause large-scale outbreaks. The standard method used in the UK water industry for detection and enumeration of Cryptosporidium is based on fluorescence microscopy and is laborious and expensive. Molecular methods such as quantitative polymerase chain reaction (qPCR) can be more amenable to streamlining through automation, improving workflows and standardizing procedures. Hypothesis. The null hypothesis was that there was no difference in the detection or enumeration between the standard method and a qPCR. Aim. We aimed to develop and evaluate a qPCR for the detection and enumeration of Cryptosporidium in drinking water, and to compare the assay with the standard method used in the UK. Methodology. We first developed and evaluated a qPCR method by incorporating an internal amplification control and calibration curve into a real-time PCR currently used for Cryptosporidium genotyping. Then we compared the qPCR assay with the standard method of immunofluorescent microscopy for the detection and enumeration of 10 and 100 Cryptosporidium oocysts in 10 l of artificially contaminated drinking water. Results. The results demonstrated that detection of Cryptosporidium by this qPCR was reliable at low numbers of oocysts; however, enumeration was less reliable and more variable than immunofluorescence microscopy. Conclusions. Despite these results, qPCR offers practical advantages over microscopy. There is potential for the use of PCR-based methods for Cryptosporidium analysis if parts of the upstream sample preparation are revised, and alternative technologies for enumeration (such as digital PCR) are also explored to improve analytical sensitivity.