Water samples collected throughout several reclamation facilities were analyzed for the presence of infectious Cryptosporidium parvum by the focus detection method-most-probable-number cell culture technique. Results revealed the presence of infectious C. parvum oocysts in 40% of the final disinfected effluent samples. Sampled effluent contained on average seven infectious oocysts per 100 liters. Thus, reclaimed water is not pathogen free but contains infectious C. parvum.Reclaimed water (treated wastewater) is being utilized in the United States and throughout the world as an alternative nonpotable water source. In the United States, 18 states currently have standards and another 18 have guidelines for reclaimed water (8). These standards, for the most part, are based on total suspended solids and fecal coliforms. A variety of microbial pathogens are present in wastewater and can be detected in reclaimed water. Therefore, advanced treatment, including filtration and disinfection, is required to produce reclaimed water that does not have a negative impact on public health. In regulatory language, this means that pathogens are to be less than the limit of detection of the assay (8).Cryptosporidium parvum, a coccidian protozoan parasite, is a potential contaminant of reclaimed water. C. parvum oocysts have been found to be persistent in the environment and resistant to chlorination. Because of this, physical removal by chemical pretreatment and filtration is the primary means of reducing the level of oocysts in environmental water (6). A possible risk to human health exists if filtration fails to function efficiently. This risk is greater still with reclaimed water, as to date no monitoring for C. parvum oocysts has been required and little information is available on the filtration efficiency in these facilities. Recently, in the state of Florida, monitoring for protozoan parasites, including Cryptosporidium, once every 2 years for larger facilities and once every 5 years for smaller facilities has been mandated. Sampling is recommended at a single point following disinfection (2).In one study, C. parvum oocysts were detected in untreated wastewater (67% of the samples were positive) and in reclaimed water (25% of final effluent samples were positive) (5). However, only the presence of the oocysts was evaluated using fluorescence microscopy. Robertson et al. evaluated wastewater samples for viable C. parvum by using vital stains; 35% of the influent samples and 46% of the effluent samples contained viable oocysts (4). In the last few years, the focus detection method-most-probable-number (FDM-MPN) cell culture technique has been developed to test the oocyst infectivity because the previously employed methods did not accurately reflect the infectious nature of the oocysts (7). The objective of the present study was to demonstrate the presence of infectious C. parvum oocysts in final reclaimed effluent from six reclamation facilities in the United States by using the FDM-MPN cell culture technique.Samples were coll...

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Wastewater disinfection is practiced with the goal of reducing risks of human exposure to pathogenic microorganisms. In most circumstances, the efficacy of a wastewater disinfection process is regulated and monitored based on measurements of the responses of indicator bacteria. However, inactivation of indicator bacteria does not guarantee an acceptable degree of inactivation among other waterborne microorganisms (e.g., microbial pathogens).Undisinfected effluent samples from several municipal wastewater treatment facilities were collected for analysis. Facilities were selected to provide a broad spectrum of effluent quality, particularly as related to nitrogenous compounds. Samples were subjected to bench-scale chlorination and dechlorination and UV irradiation under conditions that allowed compliance with relevant discharge regulations and such that disinfectant exposures could be accurately quantified. Disinfected samples were subjected to a battery of assays to assess the immediate and long-term effects of wastewater disinfection on waterborne bacteria and viruses.In general, (viable) bacterial populations showed an immediate decline as a result of disinfectant exposure; however, incubation of disinfected samples under conditions that were designed to mimic the conditions in a receiving stream resulted in substantial recovery of the total bacterial community. The bacterial groups that are commonly used as indicators do not provide an accurate representation of the response of the bacterial community to disinfectant exposure and subsequent recovery in the environment. UV irradiation and chlorination/dechlorination both accomplished measurable inactivation of indigenous phage; however, the extent of inactivation was fairly modest under the conditions of disinfection used in this study. UV irradiation was consistently more effective as a virucide than chlorination/dechlorination under the conditions of application, based on measurements of virus (phage) diversity and concentration.Taken together, and when considered in conjunction with previously published research, the results of these experiments illustrate several important limitations of common disinfection processes as applied in the treatment of municipal wastewaters. In general, it is not clear that conventional disinfection processes, as commonly implemented, are effective for control of the risks of disease transmission, particularly those associated with viral pathogens. Microbial quality in receiving streams may not be substantially improved by the application of these disinfection processes; under some circumstances, an argument can be made that disinfection may actually yield a decrease in effluent and receiving water quality. Decisions regarding the need for effluent disinfection must account for site-specific characteristics, but it is not clear that disinfection of municipal wastewater effluents is necessary or beneficial for all facilities. When direct human contact or ingestion of municipal wastewater effluents is likely, disinfection may be ne...

Cryptosporidium remains at the forefront of studies on waterborne disease transmission and abatement. The impact of environmental land use patterns which contribute animal and human waste, climatic precipitation leading to a strong association with outbreaks, and community infrastructure and water treatment are now recognized as contributing factors in the potential for waterborne spread of the protozoan. Advances in detection methodologies, including the ability to genotype various strains of this organism, have shown that human wastes are often the source of the contamination and cell culture techniques have allowed insight into the viability of the oocyst populations. Currently water treatment has focused on UV and ozone disinfection as most promising for the inactivation of this protozoan pathogen.

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Cryptosporidium is globally established as a contaminant of drinking and recreational waters. A previously described cell culture infectivity assay capable of detecting infectious oocysts was adapted to quantify viable oocysts through sporozoite invasion and clustering of foci. Eight experiments were performed by using oocysts less than 4 months of age to inoculate host HCT-8 cell monolayers. Oocysts were diluted in a standard 5- or 10-fold multiple dilution format, levels of infection and clustering were determined, and the most probable number (MPN) of infectious oocysts in the stock suspension was calculated. The MPN was compared to the initial oocyst inoculum to determine the level of correlation. For oocysts less than 30 days of age, the correlation coefficient (r) was 0.9726 (0.9306 to 0.9893; n = 20). A two-tailed Pvalue (alpha = 0.05) indicated that P was less than 0.0001. This strong correlation suggests that the MPN can be used to effectively enumerate infectious oocysts in a cell culture system. Age affected the degree of oocyst infectivity. Oocyst infectivity was tested by the focus detection method (FDM)-MPN assay and in BALB/c mice before and after treatment with pulsed white light (PureBrite). The FDM-MPN assay and animal infectivity assays both demonstrated more than a 4 log10 inactivation. Municipal water systems and a host of other water testing organizations could utilize the FDM-MPN assay for routine survival and disinfection studies.

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This paper illustrates the implementation of a new theoretical model for rapid quantitative analysis of the Vis-NIR diffuse reflectance spectra of blood cultures. This new model is based on the photon diffusion theory and Mie scattering theory that have been formulated to account for multiple scattering populations and absorptive components. This study stresses the significance of the thorough solution of the scattering and absorption problem in order to accurately resolve for optically relevant parameters of blood culture components. With advantages of being calibration-free and computationally fast, the new model has two basic requirements. First, wavelength-dependent refractive indices of the basic chemical constituents of blood culture components are needed. Second, multi-wavelength measurements or at least the measurements of characteristic wavelengths equal to the degrees of freedom, i.e. number of optically relevant parameters, of blood culture system are required. The blood culture analysis model was tested with a large number of diffuse reflectance spectra of blood culture samples characterized by an extensive range of the relevant parameters.