Use of microfluidic chips for concentrating Toxoplasma oocysts in water samples

5.1.1. Preliminary tests with Trilobite® chips using Giardia cysts as a model organism

Initial work used Giardia cysts rather than Toxoplasma oocysts, as a supply of this protozoan parasite was more readily available. Preliminary results were not promising, with only a few cysts recovered in the outlet fractions, indicating poor performance. As cysts were not detected at either outlet, it appeared that cyst losses had occurred within the chip system. Microscopic examination of the used chips revealed that salt crystals had been deposited inside the chip, apparently clogging the system; this may have caused a build-up of pressure, resulting in the chip cracking (Figure 11). The source of the salt precipitation could not be identified, as the cysts had been washed several times in laboratory grade water before being used in the spiking experiments, and could have been due to a manufacturing error.

Figure 11. Broken chip (white arrow) as a result of increased pressure

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Although our experiments indicated limited use of this system for concentration of Giardia cysts from water, previous studies have reported high recoveries of both Cryptosporidium parvum oocysts and Giardia cysts, and have suggested that this technology could be used as a pre-filter to improve recovery of Cryptosporidium oocysts from turbid water samples (Pires & Dong 2013;

Pires & Dong 2014). However, we were unable to replicate these experiments and obtain equivalent results, and it was clear that the issue of salt precipitation should be addressed. It is possibly of relevance that the experiments of (e.g.Pires & Dong 2013; 2014) were conducted using chips from a different manufacturer, but of the same design, in China, and therefore are not entirely equivalent.

55.1.2. Preliminary tests with Trilobite® chips for concentrating Toxoplasma oocysts

Despite the lack of success with Giardia cysts, some preliminary trials were also conducted with Toxoplasma oocysts. The original Toxoplasma oocyst suspension (50 mL) was reduced to 7.5 mL following two rounds of chip processing. Although more successful than the experiments with Giardia cysts, Toxoplasma oocyst recovery was also low, with higher or more reproducible results being achieved by either membrane filtration or direct centrifugation (the control) (Figure 12 and Figure 13). With experiments using 500 oocysts, the average number of oocysts and (%) recoveries were 120 (24 %) using the chip, 181 (36 %) using membrane filtration, and 360 (72 %) for the control. When samples were spiked with 1000 oocysts, the equivalent recoveries were 427 (43 %), 445 (45 %) and 560 (56 %) oocysts, respectively. Thorough examination of the eluate fraction and the chip after processing by microscopy were negative for oocysts.

Although no significant difference in oocyst recovery efficiencies could be observed between the concentration methods (chip treatment and membrane filtration) and the control when 1000 oocysts were used in the spike, when 500 oocysts were used, the chip treatment performed relatively poorly. As 2 logs fewer oocysts would be expected in environmental samples, and the results tended to be inconsistent, further research on this technology as a concentration step was halted.

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Figure 12. Comparison of recovery of Toxoplasma oocysts (spike size: 500 oocysts) by Trilobite®

chips and membrane filtration with recovery from controls (suspensions concentrated to 1 mL by centrifugation).

Figure 13. Comparison of recovery of Toxoplasma oocysts (spike size: 1000 oocysts) by Trilobite® chips and membrane filtration with recovery from controls (suspensions concentrated to 1 mL by centrifugation).

The efforts to explore the application of Trilobite® chips to concentrating Toxoplasma oocysts from water indicated that this technology is currently not suitable for this purpose, as demonstrated by the very low recovery efficiencies for the model organism (Giardia cysts) used initially, and the low recovery seen with Toxoplasma oocysts. Whereas for the Giardia cyst, cracking of the chips may have been partially to blame for the lack of success, this did not seem

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Chip treated Membrane filtered Control

recovered oocysts

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Chip treated Membrane filtered Control

recovered oocysts

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to be the case for Toxoplasma. For both parasites, it seems likely that the poor recovery is due to loss of the parasites in the tubing system in and out of the chip. Although efforts were made to reduce possible points of oocyst trappings, it seems possible that they might adhere to the tubes themselves and also accumulate at the junctions connecting the micro-tubing to the tubing system of the pump itself. Micro-tubing could also be blocked by particles, and these were regularly rinsed throughout the treatments. Another possibility for losses is from leakage at these connection points. Although attempts were made to reduce the possibility of leakage by using hydrophobic silicone paste to seal the junctions, the parasites could also be lost due to adherence to the silicone paste.

Bubble formation in the larger tubing occurred frequently during the experiment, and might have contributed to losses, by providing extended surfaces for adherence. It should also be noted that the post-concentration centrifugation step, in which the volume was further reduced prior to enumeration, could also result in losses, as the recovery efficiency in the control was also low.

Another limitation of the technique was the limited capacity of the peristaltic pump (approximately 3-6 mL/min) and the handling volume of a single chip. Thus, the volume of the samples concentrated using the Trilobite® chip was only 0.05 L, whereas with membrane filtration the volume can be at least twenty times higher at 10 L.

Although the Trilobite® chip seems to have application at removing liquid from a sample of greater volume, and has been used successfully (at lab scale) to dewater algae (Hønsvall et al.

2016), in its current format, it seems less successful for concentrating organisms in which retention and recovery of all organisms is the aim, rather than removing excess fluid.

According to Ganz et al. (2015), 10 mL suspensions of Giardia duodenalis cysts were reduced to less than 1 mL in about 30 min by running the sample 6 times through the unit using an inertial microfluidic separation system. Using the system, they concentrated and detected G.

duodenalis cysts with a recovery rate of 68.4 % for 1000 oocysts in 10 mL. Compared with, for example, immunomagnetic separation technology, this is not a very high recovery efficiency. In our experiment, we were able to use the Trilobite® chip to reduce the volumes from 50 mL to 7.5 mL by running the sample twice through the chip in 10 min. This reduction in volume is

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comparable or better to that achieved by Ganz et al. (2015). However, our recovery rate was lower, presumably due to difference in the equipment set up and protocol.

Should further work on water analysis using microfluidic chips be attempted, upscaling the process by making larger chips, or putting several chips together into a system would be recommended such that larger volumes of water could be processed.