New tools for cryptosporidium monitoring and inactivation

US researchers Susan B Rivera and George Bajszar of the MIOX Corporation have found that chlorine triggers a defence reaction in cryptosporidia oocysts, here we reveal their findings.

Chlorine-based oxidants have been widely used in water disinfection for over a century. Over time, microorganisms have developed some resistance to common disinfection methods. As policy and technology improvements evolve to overcome increased resistance and improve water quality, on-site generation of disinfectants is advancing to meet the safety, cost reduction, and environmental footprint needs of customers.

In this case, efforts to understand the enhanced disinfection efficacy of on-site generated chlorine-based oxidants compared to chlorine gas or delivered bulk bleach have provided insight into a fundamental survival mechanism of one of the hardest-to-kill classes of water-borne parasites, the oocysts of Cryptosporidia.

A recent publication by MIOX, which compared a molecular viability assay with a live cell-based assay, also provided a potential path forward for bringing molecular diagnostics into the mainstream for Cryptosporidium monitoring and compliance testing. In general, molecular methods are faster and less expensive than conventional methods based on cell culture infectivity. Results were published in Applied and Environmental Microbiology.

Dual-phase inactivation

The paper, Stress-induced Hsp70 gene expression and inactivation of Cryptosporidium parvum oocysts by chlorine-based oxidants, uncovers for the first time that chlorine triggers a defensive molecular response to oxidative stress in C. parvum oocysts. This response likely contributes to the high resistance of these waterborne pathogens to chlorination.

Thus, when an oocyst is exposed to chlorine, a fundamental survival mechanism kicks in. Similar to an outnumbered army in battle, the oocysts eventually become overwhelmed and inactivated by the oxidant at moderate chlorine doses. Their physical structure, however, can remain intact for several days following inactivation, creating false positive results using the current field monitoring methods.

The relative biocidal effect of bleach and electrolytically generated MIOX mixed-oxidant solution (MOS) on C. parvum oocysts was compared at identical free chlorine concentrations. The results showed that MOS exhibits a higher efficacy in oocyst inactivation than hypochlorite.

In the shorter term, the information may allow engineers, regulators, and utility personnel to further develop disinfection protocols and plant processes to get more disinfection power from chemicals currently used in water treatment plants. Longer term research could involve developing new approaches, such as tricking the oocysts into entering the excystation
process, a state when they are virtually defenceless against chlorine at reasonable drinking water doses (1-5 mg/l).

Rapid viability assay

To quantify the biocidal action of chlorine-based oxidants against C. parvum oocysts, the research team developed a fast molecular diagnostic assay, based on the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) technique. The qRT-PCR assay can be applied in a broad variety of diagnostic tasks when viability of non-culturable waterborne pathogens needs to be determined.

Oocyst viability was also compared to a commonly used infectivity assay which uses live mammalian cells to estimate the infectivity of oocysts. The comparative assays gave consistent results. The latter method is faster and simpler to perform and could allow laboratories without cell culture capability to perform comparative disinfection assays.

While the qRT-PCR assay may not yet be used under current regulatory frameworks to monitor for C. parvum oocysts, it can be developed for rapid assessment of disinfection efficacy. This capability in the field could combat the complication that current field methods only detect presence and absence, not infectivity, or a measure of whether the oocysts can make someone sick upon ingestion.

With a faster assay, laboratories could achieve test results faster, allowing water treatment plant operators to adjust and optimise disinfection dose rates more rapidly in response to changing conditions. This capability would enable them to reduce the use of disinfection chemicals, thereby reducing disinfection by-product formation in the finished water.

Conversely, dose increases could be performed earlier if a health or compliance risk is suspected. Another benefit is that the molecular assay could significantly speed up the evaluation of new devices that must be tested by third party laboratories for efficacy before labelling claims may be made.

Potential outcomes of the research include short term disinfection process changes that could positively affect water quality with current methods and a longer term strategy to develop methods to outsmart the C. parvum oocysts and inactivate them.

Four important findings were communicated in the AEM paper. First, chlorine-based oxidants trigger a strong stress response in the oocysts of Cryptosporidium parvum, which likely contributes to their high chlorine tolerance. Secondly, MIOX MOS is a stronger biocide against C. parvum oocysts than bleach.

Third, C. parvum oocysts may remain physically intact for several days following disinfection treatment - even when they are already non-infective (dead). Finally, qRT-PCR assays for oocyst viability are comparable to the traditional cell culture-based infectivity assays. A shift in allowing this type of assay to be used by third party evaluation laboratories would save time, energy and money.

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