Pierre Payment

Real-Time PCR for Quantification of Giardia and Cryptosporidium in Environmental Water Samples and Sewage

ABSTRACT The protozoan pathogens Giardia lamblia and Cryptosporidium parvum are major causes of waterborne enteric disease throughout the world. Improved detection methods that are very sensitive and rapid are urgently needed. This is especially the case for analysis of environmental water samples in which the densities of Giardia and Cryptosporidium are very low. Primers and TaqMan probes based on the β-giardin gene of G. lamblia and the COWP gene of C. parvum were developed and used to detect DNA concentrations over a range of 7 orders of magnitude. It was possible to detect DNA to the equivalent of a single cyst of G. lamblia and one oocyst of C. parvum. A multiplex real-time PCR (qPCR) assay for simultaneous detection of G. lamblia and C. parvum resulted in comparable levels of detection. Comparison of DNA extraction methodologies to maximize DNA yield from cysts and oocysts determined that a combination of freeze-thaw, sonication, and purification using the DNeasy kit (Qiagen) provided a highly efficient method. Sampling of four environmental water bodies revealed variation in qPCR inhibitors in 2-liter concentrates. A methodology for dealing with qPCR inhibitors that involved the use of Chelex 100 and PVP 360 was developed. It was possible to detect and quantify G. lamblia in sewage using qPCR when applying the procedure for extraction of DNA from 1-liter sewage samples. Numbers obtained from the qPCR assay were comparable to those obtained with immunofluorescence microscopy. The qPCR analysis revealed both assemblage A and assemblage B genotypes of G. lamblia in the sewage. No Cryptosporidium was detected in these samples by either method.

A randomized trial to evaluate the risk of gastrointestinal disease due to consumption of drinking water meeting current microbiological standards.

BACKGROUND This project directly and empirically measured the level of gastrointestinal (GI) illness related to the consumption of tapwater prepared from sewage-contaminated surface waters and meeting current water quality criteria. METHODS A randomized intervention trial was carried out; 299 eligible households were supplied with domestic water filters (reverse-osmosis) that eliminate microbial and chemical contaminants from their water, and 307 households were left with their usual tapwater without a filter. The GI symptomatology was evaluated by means of a family health diary maintained prospectively by all study families over a 15-month period. RESULTS The estimated annual incidence of GI illness was 0.76 among tapwater drinkers compared with 0.50 among filtered water drinkers (p less than 0.01). These findings were consistently observed in all population subgroups. CONCLUSION It is estimated that 35% of the reported GI illnesses among the tapwater drinkers were water-related and preventable. Our results raise questions about the adequacy of current standards of drinking water quality to prevent water-borne endemic gastrointestinal illness.

A prospective epidemiological study of gastrointestinal health effects due to the consumption of drinking water

The objective of this study was to assess if drinking water meeting currently accepted microbiological standards is the source of gastrointestinal illnesses and to attempt to identify the source(s) of these illnesses. A randomized prospective study was conducted over a period of 16 months (September 1993-December 1994) in a middle class suburban community served by a single water filtration plant. A representative sample of 1400 families were selected and randomly allocated in four groups of 350, to the following regimens: (1) tap water; (2) tap water from a continuously purged tap; (3) bottled plant water; (4) purified bottled water (tap water treated by reverse osmosis or spring water). The water treatment plant produced wather that met or exceeded current North American regulations for drinking water quality. The distribution system was found to be in compliance for both coliforms and chlorine. Using the purified water group as the baseline, the excess of gastrointestinal illness associated with tap wa...

Disinfection efficiency of peracetic acid, UV and ozone after enhanced primary treatment of municipal wastewater

The City of Montreal Wastewater Treatment Plant uses enhanced physicochemical processes (ferric and/or alum coagulation) for suspended solids and phosphorus removal. The objective of this study was to assess the ability of peracetic acid (PAA), UV, or ozone to inactivate the indicator organisms fecal coliforms, Enterococci, MS-2 coliphage, or Clostridium perfringens in the effluent from this plant. PAA doses to reach the target fecal coliform level of 9000 CFU/100mL exceeded 6 mg/L; similar results were obtained for enterococci, and no inactivation of Clostridium perfringens was observed. However a 1-log reduction of MS-2 occurred at PAA doses of 1.5 mg/L and higher. It was expected that this effluent would have a high ozone demand, and would require relatively high UV fluences, because of relatively high effluent COD, iron and suspended solids concentrations, and low UV transmittance. This was confirmed herein. For UV, the inactivation curve for fecal coliforms showed the typical two-stage shape, with the target of 1000 CFU/100 mL (to account for photoreactivation) occurring in the asymptote zone at fluences >20 mJ/cm(2). In contrast, inactivation curves for MS-2 and Clostridium perfringens were linear. Clostridium perfringens was the most resistant organism. For ozone, inactivation was already observed before any residuals could be measured. The transferred ozone doses to reach target fecal coliform levels ( approximately 2-log reduction) were 30-50 mg/L. MS-2 was less resistant, but Clostridium perfringens was more resistant than fecal coliforms. The different behaviour of the four indicator organisms studied, depending on the disinfectant, suggests that a single indicator organism might not be appropriate. The required dose of any of the disinfectants is unlikely to be economically viable, and upstream changes to the plant will be needed.

Pathogens in Water: Value and Limits of Correlation with Microbial Indicators

This article discusses the value and limitations of using microbial indicators to predict occurrence of enteric pathogens in water. Raw or treated sewage is a primary source of fecal contamination of the receiving surface water or groundwater; hence, understanding the relationship between pathogens and indicators in sewage is an important step in understanding the correlation in receiving waters. This article presents three different datasets representing different concentrations of pathogens and microbial indicators: sewage containing high concentrations of pathogens and indicators, surface water with variable concentrations, and groundwater with low concentrations. In sewage, even with very high levels of microorganisms, no mathematical correlation can predict the type or concentration of any pathogen. After discharge in the environment, direct correlation becomes biologically improbable as dilution, transport, and different inactivation rates occur in various environments. In surface waters, advanced statistical methods such as logistic regression have provided some level of predictability of the occurrence of pathogens but not specific counts. In groundwater, the continuous absence of indicators indicates an improbable occurrence of pathogen. In contrast, when these indicators are detected, pathogen occurrence probability increases significantly. In groundwater, given the nature and dissemination pattern of pathogenic microorganisms, a direct correlation with fecal microbial indicators is not observed and should not be expected. However, the indicators are still useful as a measure of risk. In summary, many pathogens of public health importance do not behave like fecal microbial indicators, and there is still no absolute indicator of their presence, only a probability of their co-occurrence.

Removal of indicator bacteria, human enteric viruses, Giardia cysts, and Cryptosporidium oocysts at a large wastewater primary treatment facility

Pathogens and fecal indicator bacteria occurrence and removal were studied for a period of 6 months at the Montreal Urban Community wastewater treatment facility. With a capacity of about 7.6 million cubic metres per day (two billion U.S. gallons per day), it is the largest primary physico-chemical treatment plant in America. The plant discharges a nondisinfected effluent containing about 20 mg/L of suspended matter and 0.5 mg/L of total phosphorus on the basis of average annual concentrations. BDO5 (annual mean) is 75 mg/L before treatment and 32 mg/L after treatment. Samples were collected for a period of 6 months, and they demonstrated that the plant was not efficient at removing indicator bacteria and the pathogens tested. Fecal coliforms were the most numerous of the indicator bacteria and their removal averaged 25%. Fecal streptococci removal was 29%, while Escherichia coli removal was 12%. In untreated sewage, fecal coliforms, E. coli, and human enteric viruses were more numerous in summer and early autumn. Fecal streptococci counts remained relatively similar throughout the period. Clostridium perfringens removal averaged 51%. Giardia cysts levels were not markedly different throughout the study period, and 76% of the cysts were removed by treatment. Cryptosporidium oocyst counts were erratic, probably due to the methods, and removal was 27%. Human enteric viruses were detected in all samples of raw and treated wastewater with no removal observed (0%). Overall, the plant did not perform well for the removal of fecal indicator bacteria, human enteric viruses, or parasite cysts. Supplementary treatment and disinfection were recommended to protect public health. Various alternatives are being evaluated.

Biofilms, infectious agents, and dental unit waterlines : a review

Aquatic biofilms, which are widespread not only in nature but also in medical and dental devices, can be the source of serious nosocomial infections. In these hardy microbial communities, pathogens like nontuberculous mycobacteria, Pseudomonas aeruginosa, Legionella pneumophila, and other bacteria not only survive but proliferate and lie in wait for susceptible hosts. Not only are these organisms intrinsically resistant to high temperatures and biocides, but the biofilms they inhabit enhance their resistance. This should be of concern to infection control practitioners. The bacterial colonization of dental unit waterlines can be used as a model to investigate the problem of waterborne biofilms in health care settings.

Waterborne Pathogen Detection by Use of Oligonucleotide-Based Microarrays

ABSTRACT A small-oligonucleotide microarray prototype was designed with probes specific for the universal 16S rRNA and cpn60 genes of several pathogens that are usually encountered in wastewaters. In addition to these two targets, wecE-specific oligonucleotide probes were included in the microarray to enhance its discriminating power within the Enterobacteriaceae family. Universal PCR primers were used to amplify variable regions of 16S rRNA, cpn60, and wecE genes directly in Escherichia coli and Salmonella enterica serovar Typhimurium genomic DNA mixtures (binary); E. coli, S. enterica serovar Typhimurium, and Yersinia enterocolitica genomic DNA mixtures (ternary); or wastewater total DNA. Amplified products were fluorescently labeled and hybridized on the prototype chip. The detection sensitivity for S. enterica serovar Typhimurium was estimated to be on the order of 0.1% (104S. enterica genomes) of the total DNA for the combination of PCR followed by microarray hybridization. The sensitivity of the prototype could be increased by hybridizing amplicons generated by PCR targeting genes specific for a bacterial subgroup, such as wecE genes, instead of universal taxonomic amplicons. However, there was evidence of PCR bias affecting the detection limits of a given pathogen as increasing amounts of a different pathogen were spiked into the test samples. These results demonstrate the feasibility of using DNA microarrays in the detection of waterborne pathogens within mixed populations but also raise the problem of PCR bias in such experiments.

Fecal coliforms, caffeine and carbamazepine in stormwater collection systems in a large urban area

Water samples from streams, brooks and storm sewer outfall pipes that collect storm waters across the Island of Montréal were analyzed for caffeine, carbamazepine and fecal coliforms. All samples contained various concentrations of these tracers, indicating a widespread sanitary contamination in urban environments. Fecal coliforms and caffeine levels ranged over several orders of magnitude with a modest correlation between caffeine and fecal coliforms (R(2) value of 0.558). An arbitrary threshold of 400 ng caffeine L(-1) allows us to identify samples with an elevated fecal contamination, as defined by more than 200 colony-forming units per 100 mL (cfu 100 mL(-1)) of fecal coliforms. Low caffeine levels were sporadically related to high fecal coliform counts. Lower levels of caffeine and fecal coliforms were observed in the brooks while the larger streams and storm water discharge points contained over ten times more. The carbamazepine data showed little or no apparent correlation to caffeine. These data suggest that this storm water collection system, located in a highly urbanized urban environment, is widely contaminated by domestic sewers as indicated by the ubiquitous presence of fecal contaminants as well as caffeine and carbamazepine. Caffeine concentrations were relatively well correlated to fecal coliforms, and could potentially be used as a chemical indicator of the level of contamination by sanitary sources. The carbamazepine data was not significantly correlated to fecal coliforms and of little use in this dataset.

A time series study of anti-diarrheal drug sales and tap-water quality

The City of Le Havre in France operates two water treatment plants and distributes water to 200,000 people. It exploits, as the source of its drinking water, karstic resources that are subjected to episodical microbiological quality degradations. The first plant only chlorinates the water, whereas the second plant normally uses direct sand filtration before chlorination but can also implement coagulation-settling when turbidity of the raw water exceeds 3 nephelometric turbidity units (NTU). During the study period there were several occurrences of failure to maintain residual chlorine as well as significant turbidity increases. The treated water still met all microbiological criteria for potable water in France and this study was undertaken to determine if public health was adequately protected. An ecological time series study was carried out on data collected between April 1993 and September 1996. The record of the sales of medications prescribed or self-selected for the treatment of gastroenteritis (GE)...