Eight pairs of published PCR primers were evaluated for the specific detection of Cryptosporidium parvum and Giardia lamblia in water. Detection sensitivities ranged from 1 to 10 oocysts or cysts for purified preparations and 5 to 50 oocysts or cysts for seeded environmental water samples. Maximum sensitivity was achieved with two successive rounds of amplification and hybridization, with oligonucleotide probes detected by chemiluminescence. Primer annealing temperatures and MgCl 2 concentrations were optimized, and the specificities of the primer pairs were determined with closely related species. Some of the primers were species specific, while others were only genus specific. Multiplex PCR for the simultaneous detection of Cryptosporidium and Giardia was demonstrated with primers amplifying 256-and 163-bp products from the 18S rRNA gene of Cryptosporidium and the heat shock protein gene of Giardia, respectively. The results demonstrate the potential utility of PCR for the detection of pathogenic protozoa in water but emphasize the necessity of continued development.

The presence of Cryptosporidium in drinking water supplies is a significant problem faced by the water industry. Although a variety of methods exist for the detection of waterborne oocysts, water utilities currently have no way of assessing the infectivity of detected oocysts and consequently are unable to accurately determine the risks posed to public health by waterborne Cryptosporidium. In this paper, the development of an infectivity assay for waterborne Cryptosporidium parvum is described. Oocysts were inoculated onto monolayers of Caco-2 cells and grown on microscope slides, and infections were detected by C. parvum specific reverse transcriptase PCR of extracted mRNA, targeting the heat shock protein 70 (hsp70) gene. A single infectious oocyst was detected by this experimental procedure. The use of concentrated samples obtained from 250 liters of finished water had no observable effect on the integrity of cell monolayers or on the infectivity of oocysts seeded into the concentrate. Intracellular developmental stages of the parasite were also detected by using fluorescently labeled antibodies. One pair of PCR primers targeting the hsp70 gene was specific for C. parvum, while a second pair recognized all species of Cryptosporidium tested. The C. parvum-specific primers amplified DNA from 1 to 10 oocysts used to seed 65 to 100 liters of concentrated environmental water samples and were compatible with multiplex PCR for the simultaneous detection of C. parvum and Giardia lamblia. This paper confirms the utility of PCR for the detection of waterborne C. parvum and, most importantly, demonstrates the potential of an in vitro infectivity assay. Cryptosporidium was confirmed as a significant waterborne public health threat by the 1993 outbreak in Milwaukee, Wis., in which an estimated 400,000 people became ill (23). Since 1983, there have been at least 31 reported outbreaks of cryptosporidiosis associated with either drinking or recreational water in the United States, the United Kingdom, Canada, Australia, and Japan, and over 25,000 cases in Canada and Japan were linked to drinking water in the first 6 months of 1996. Up to 87% of untreated raw water and 24% of finished drinking water contained Cryptosporidium oocysts, according to one survey (19), and analysis of 347 surface water samples between 1988 and 1993 demonstrated that 60% of the samples contained oocysts (20). The oocysts are resistant to chlorine at concentrations commonly used for drinking water treatment (typical residual concentrations range from 0.5 to 2 mg/liter), there is currently no effective anticryptosporidial agent for afflicted individuals (although cryptosporidiosis is usually selflimiting in otherwise healthy individuals), and the disease has potentially fatal consequences for immunocompromised individuals who become infected. As few as 30 oocysts caused infection in 20% of healthy volunteers tested, and the 50% infectious dose was determined to be 132 oocysts (8). Consequently, reliable detection methods would better enable water utilit...

Two commercial immunomagnetic separation (IMS) kits forCryptosporidium were compared for recovery of oocysts from environmental samples. Oocyst recovery efficiencies with the Dynal and Crypto-Scan kits ranged from 62 to 100% and 34 to 74%, respectively, for seeded environmental water concentrates (turbidity of 210 to 11,480 nephelometric turbidity units). Recovery efficiencies were dependent on the mechanism of agitation during the magnetic capture procedure. An assay combining in vitro cell culture and reverse transcriptase PCR demonstrated that oocysts recovered by IMS retained their infectivity.

Bacteriological analyses were performed on the effluent from a conventional water treatment pilot plant in which granular activated carbon (GAC) had been used as the final process to assess the impact of GAC on the microbial quality of the water produced. Samples were collected twice weekly for 160 days from the effluents of six GAC columns, each of which used one of four different empty-bed contact times (7.5, 15, 30, and 60 min). The samples were analyzed for heterotrophic plate counts and total coliforms. Effluent samples were also exposed to chloramines and free chlorine for 60 min (pH 8.2, 23°C). Bacterial identifications were performed on the disinfected and nondisinfected effluents. Additional studies were conducted to assess the bacteriological * Corresponding author.

The Metropolitan Water District of Southern California recently completed construction of an 800,000 acre-ft (1 × 10 9 m 3 ) drinking water reservoir. Recreational activities, including swimming and other sports involving direct body contact (BC) with the water, have been the subject of considerable interest by local community members. Consequently, a modelingbased risk assessment study was conducted to assess the potential public health consequences to downstream potable water users consuming water from this reservoir if BC recreation was permitted. Results of the study indicated that the annual risk of waterborne illness would increase three times above background, despite conventional treatment.Moreover, the occurrence of high-loading pathogen events associated with BC recreation was observed to significantly increase the daily risk of waterborne illness to downstream consumers. The study also considered the cost of additional treatment that would be necessary to address the increased risk. The modeling approach used in this study provides guidance for policymakers and stakeholders who are examining issues associated with BC recreation and drinking water reservoirs.ater-based recreational sports-such as swimming, waterskiing, use of personal watercraft (PWC), sailing, and fishing-may be considered an asset to those communities with lakes or reservoirs available for such activities. In many cases, these same bodies of water are also used as the primary source for drinking water. Consequently, the desire for recreational opportunities can be in conflict when these activities compromise the quality of drinking water sources. Moreover, emerging regulatory requirements that place greater emphasis on source water protection, coupled with community interest in meeting growing recreational demands or enhancing eco-W watershed management Body-contact recreational activities such as swimming and waterskiing on lakes and reservoirs used as drinking water sources can compromise the quality of the water.

No abstract

The resistance of Klebsiella pneumoniae to inorganic monochloramine (1.5 mg/liter; 3:1 C12:N ratio, pH 8.0) was examined in relation to growth phase, temperature of growth, and growth under decreased nutrient conditions. Growth phase did not impact resistance to chloramines. Mid-exponential and stationary-phase cells, grown in a yeast extract-based medium, had CT99 values and standard deviations of 4.8 + 0.1 and 4.6-+-0.2 mg. min/liter, respectively. Growth temperature did not alter chloramine resistance at short contact times. CT99 values of cells grown at 15 and 23°C were 4.5 + 0.2 and 4.6 + 0.2 mg. min/liter, respectively. However, at longer contact times, CT99.99 values of cells grown at 15 and 23°C were 14 and 8 mg. min/liter, respectively, suggesting a small resistant subpopulation for cells grown at the lower temperature. Growth under decreased nutrient conditions resulted in a concomitant increase in resistance to chloramines. When K. pneumoniae was grown in undiluted Ristroph medium and Ristroph medium diluted by 1:100 and 1:1,000, the CT99 values were 4.6 0.2, 9.6 0.4, and 24 + 7.0 mg-min/liter, respectively. These results indicate that nutrient availability has a greater impact than growth phase or growth temperature in promoting the resistance of K. pneumoniae to inorganic monochloramine.

A number of approaches offer promise for addressing the limitations of current antibody‐based detection methods. This report was prepared by the Working Group on Waterborne Cryptosporidiosis (Technical Task Force E, Developmental Status of Environmental Sampling, Water Testing, and Surrogate Indicators). Methods for detecting Cryptosporidium oocysts in water have centered around microscopic examination of fluorescent antibody‐stained concentrates from large‐volume water samples. The limitations of these antibody‐based methods include the need for experienced analysts, lengthy analytical time, expense, lack of specificity, erratic efficiency, low precision, and difficulty in determining viability. A number of methods, assays, and procedures that have the potential for ameliorating some of these limitations are currently being evaluated. How successful such processes will be remains to be demonstrated by the scientific community.