Wilderness Medical Society snowmass 2005 Page 372
Abstract Presentations at the 2005 Wilderness Medical Society Meeting, Snowmass,
Colorado, July 23 to 27, 2005
Oral Presentation: Ashley Laird, WMS Houston Award Winner
Rapid detection techniques for Cryptosporidium and Giardia: Feasibility of use to
assess risk from drinking wilderness waters
Ashley R. Laird¹, Alexandria B. Boehm, PhD²
¹ Medical Student, Stanford University School of Medicine
² Assistant Professor, Department of Civil and Environmental Engineering, Stanford
University
Introduction: A quantitative PCR (QPCR) protocol for detection of Cryptosporidium
and Giardia was developed and applied to natural waters collected from Yosemite
National Park. The protocol involved concentrating (oo)cysts seeded into a target water
onto nitrocellulose filters, extracting DNA from the filters, and subsequently amplifying
(oo)cyst-specific genes for organism quantification.
Methods: Numerous methods for improving DNA extraction efficiency were evaluated,
including washing filters to remove organisms and then concentrating wash water prior to
DNA extraction, directly extracting DNA from filters using commercial extraction kits,
and adding carrier RNA and Geotrichium candidum cells to samples. Methods for coping
with inhibition were also assessed, including addition of BSA and Chelex, utilization of
extraction kits designed to remove PCR inhibitors, and dilution of extraction eluates prior
to QPCR amplification.
Results: Using MILLIQ (ultra-pure water) as the target water, we could detect
concentrations as low as 100 Giardia cysts per liter, but Cryptosporidium oocysts could
not be detected even at concentrations of 1000 oocysts per liter. When the target water
was a natural surface water, the assay sensitivity decreased, as our detection limit rose to
1000 Giardia cysts. Giardia and Cryptosporidium organisms were not detected in any
of the 34 samples collected from Yosemite. Since the inoculum required to establish
infection in humans is only 10-100 organisms, the detection limit of our protocol is too
high to calculate the risk of infection associated with drinking wilderness surface waters.
Conclusion: We conclude that at present, QPCR is not sensitive enough to detect
(oo)cysts in environmental waters with the goal of protecting human health. Although
QPCR is a promising approach for quantification of pathogens in natural waters, more
work is needed to improve sensitivity. Future research should focus on improving
methods of (oo)cyst collection, DNA extraction and purification, and QPCR
amplification to allow detection of low levels of pathogens in environmental waters.
