SENSITIVE AND RAPID DETECTION OF VIABLE GIARDIA CYSTS AND CRYPTOSPORIDIUM-PARVUM OOCYSTS IN LARGE-VOLUME WATER SAMPLES WITH WOUND FIBERGLASS CARTRIDGE FILTERS AND REVERSE TRANSCRIPTION-PCR

We recently described a reverse transcription-PCR (RT-PCR) for detecti ng low numbers of viable Cryptosporidium parvum oocysts spiked into cl arified environmental water concentrates. We have now modified the ass ay for direct analysis of primary sample concentrates with simultaneou s detection of viable C. parvum oocysts, Giardia cysts, and a novel ty pe of internal positive control (IPC). The IPC was designed to assess both efficiency of mRNA isolation and potential RT-PCR inhibition. Sen sitivity testing showed that low numbers of organisms, in the range of a single viable cyst and oocyst, could be detected when spiked into 1 00-mu l packed pellet volumes of concentrates from creek and river wat er samples. The RT-PCR was compared with an immunofluorescence (IF) as say by analyzing 29 nonspiked environmental water samples. Sample volu mes of 20 to 1,500 liters were concentrated with a wound fiberglass ca rtridge filter. Frequency of detection for viable Giardia cysts increa sed from 24% by IF microscopy to 69% by RT-PCR. Viable C. parvum oocys ts were detected only once by RT-PCR (3%) in contrast to detection of viable Cryptosporidium spp. in four samples by IF microscopy (14%), su ggesting that Cryptosporidium species other than C. parvum were presen t in the water. This combination of the large-volume sampling method w ith RT-PCR represents a significant advance in terms of protozoan path ogen monitoring and in the wider application of PCR technology to this field of microbiology.