Quantification of Giardia transcripts during in vitro excystation: interest for the estimation of cyst viability.

The aim of the present study was to evaluate the potential of transcript quantification as an indicator of Giardia cyst viability. The variations of beta-giardin, EF1A and ADHE mRNAs were quantified during excystation by real-time RT-PCR assays and compared with the percentages of viability estimated using propidium iodide staining and in vitro excystation. The first experiments were performed with purified G. duodenalis assemblage B cysts. When 55% of excysting protozoa were observed, the increase of the selected transcripts ranged from 0.40+/-0.13 to 0.97+/-0.11 log10 after 1h of incubation in excystation medium. Purified cysts were also stored at 4 degrees C for up to 56 days and analysed at several sampling times. Significant correlations were observed between the variations of the selected mRNAs and the percentages of viability estimated with staining and excystation methods. Among the three transcripts, beta-giardin appeared to be the most appropriate to study the viability of Giardia cysts concentrated from wastewater samples.

Detection and genotyping of Giardia duodenalis in wastewater: relation between assemblages and faecal contamination origin.

Among the seven assemblages identified in Giardia duodenalis species, only assemblages A and B infect humans and numerous other mammals as well. On the other hand, assemblage E is considered to be host restricted to livestock. The aim of the present study was to compare the presence of G. duodenalis assemblages A, B and E in wastewater samples from two municipal treatment plants (n=24) and one slaughterhouse (n=12). Thus, PCR assays targeting the tpi gene were developed to detect specifically these three G. duodenalis assemblages. Assemblages A and B were detected in urban wastewater with a predominance of assemblage A, especially for one treatment plant. Concerning slaughterhouse wastewater, assemblage A was found in 58% of the samples, whereas assemblage B was not detected. Assemblage E was not detected in urban wastewater, but was found in 92% of the samples from slaughterhouse. Thus, combination of assemblages A and B seemed to indicate a human contamination origin, while combination of assemblages A and E appeared to correspond to a livestock contamination origin.

Integrated analysis of established and novel microbial and chemical methods for microbial source tracking.

Several microbes and chemicals have been considered as potential tracers to identify fecal sources in the environment. However, to date, no one approach has been shown to accurately identify the origins of fecal pollution in aquatic environments. In this multilaboratory study, different microbial and chemical indicators were analyzed in order to distinguish human fecal sources from nonhuman fecal sources using wastewaters and slurries from diverse geographical areas within Europe. Twenty-six parameters, which were later combined to form derived variables for statistical analyses, were obtained by performing methods that were achievable in all the participant laboratories: enumeration of fecal coliform bacteria, enterococci, clostridia, somatic coliphages, F-specific RNA phages, bacteriophages infecting Bacteroides fragilis RYC2056 and Bacteroides thetaiotaomicron GA17, and total and sorbitol-fermenting bifidobacteria; genotyping of F-specific RNA phages; biochemical phenotyping of fecal coliform bacteria and enterococci using miniaturized tests; specific detection of Bifidobacterium adolescentis and Bifidobacterium dentium; and measurement of four fecal sterols. A number of potentially useful source indicators were detected (bacteriophages infecting B. thetaiotaomicron, certain genotypes of F-specific bacteriophages, sorbitol-fermenting bifidobacteria, 24-ethylcoprostanol, and epycoprostanol), although no one source identifier alone provided 100% correct classification of the fecal source. Subsequently, 38 variables (both single and derived) were defined from the measured microbial and chemical parameters in order to find the best subset of variables to develop predictive models using the lowest possible number of measured parameters. To this end, several statistical or machine learning methods were evaluated and provided two successful predictive models based on just two variables, giving 100% correct classification: the ratio of the densities of somatic coliphages and phages infecting Bacteroides thetaiotaomicron to the density of somatic coliphages and the ratio of the densities of fecal coliform bacteria and phages infecting Bacteroides thetaiotaomicron to the density of fecal coliform bacteria. Other models with high rates of correct classification were developed, but in these cases, higher numbers of variables were required.

Comparison of two target genes for detection and genotyping of Giardia lamblia in human feces by PCR and PCR-restriction fragment length polymorphism.

A PCR assay targeting the tpi gene was developed to detect and to genotype Giardia lamblia in human feces. Our assay was specific and discriminated between G. lamblia assemblages A and B. G. lamblia cysts isolated from human feces were also analyzed with two previously described PCR-restriction fragment length polymorphism (RFLP) assays, which are based on the detection of tpi or gdh genes. These RFLP analyses distinguished groups I and II within assemblage A or groups III and IV within assemblage B. Among 26 fecal samples from patients with sporadic giardiasis diagnosed by hospital laboratories, the tpi gene was amplified from 25 (96%) with our PCR assay, whereas only 21 (81%) samples were positive when the gdh gene was targeted. Of the 25 positive samples, nine (36%) contained assemblage A and 16 (64%) contained assemblage B. Thus, RFLP analysis classified eight samples (32%) in assemblage A group II, eight (32%) in assemblage B group III, and five (20%) in assemblage B group IV. The group could not be specified for four samples. The tpi and gdh genes of G. lamblia assemblage B were amplified from 14 (93%) of 15 samples collected only from French soldiers coming back from the Ivory Coast. All of these contained assemblage B group III. The PCR method developed is sensitive, simple, and specific and shows that the tpi gene is well adapted for G. lamblia genotyping.

Quantitative and qualitative comparison of density-based purification methods for detection of Cryptosporidium oocysts in turbid environmental matrices.

Purification methods for Cryptosporidium oocysts are usually selected on the basis of recovery yield, but the amount of particulate debris in environmental matrices could limit efficiency of oocyst detection by microscopic examination or PCR detection. Previous studies have shown that the standard immunomagnetic separation (IMS) procedure would not be the most suitable method for oocyst purification from turbid matrices. We compared the capacity of Percoll-sucrose flotation and six other density-based purification methods to achieve selective separation of Cryptosporidium oocysts from particulate debris. Rate of oocyst recovery and particulate loading in the purified suspensions were chosen as comparison criteria for the different purification methods. In most earlier studies, the chemical treatments employed to obtain a purified oocyst suspension modify the surface properties of oocysts in spiked samples. Assuming this produces unrealistic conditions affecting the evaluation of purification methods, we performed the present study with native oocysts. Flotation and gradient procedures were tested with and without formaldehyde ethyl acetate (FEA) separation. FEA separation was found to be unsuitable. Filtration and Percoll gradient did not allow selective oocyst separation from debris. Among the purification methods suitable for routine microscopic examination, Percoll-sucrose flotation provided the best recovery rates. For automated enumeration systems or PCR detection, potassium bromide and especially Nycodenz gradients appeared to be the most suitable purification methods. Potassium bromide and Nycodenz gradients provided the best balance between oocyst recovery and particulate load.

Improved specificity for Giardia lamblia cyst quantification in wastewater by development of a real-time PCR method.

The protozoan parasite Giardia lamblia is the most common cause of waterborne disease outbreaks associated with drinking water in the United States. The conventional method used for the enumeration of Giardia cysts in water is based on immunofluorescence with monoclonal antibodies. It is tedious and time-consuming and has the major drawback to be non-specific for the only species infecting humans, G. lamblia. We have developed a real-time polymerase chain reaction (PCR) method using fluorescent TaqMan technology, which improved the specificity of G. lamblia cyst quantification compared to the immunofluorescence assay (IFA). However, this PCR was not totally specific for G. lamblia species and amplified Giardia ardeae target as well. This method showed a sensitivity of 0.45 cysts per reaction and an efficiency of 95% in purified suspensions. We have then applied this quantification method to raw wastewater, a medium containing numerous debris, particles and PCR inhibitors. The adaptation to these environmental samples was realized by a screening of three cyst purification methods and six DNA extraction protocols. Real-time quantification was accomplished by the simultaneous amplification of unknown samples and a tenfold serial dilution of purified G. lamblia cysts. For all samples, the concentrations observed with TaqMan PCR method were compared to the IFA values. Giardia spp. cysts were detected in all non-spiked raw wastewater samples with IFA procedure and the concentrations of Giardia spp. cysts used for the comparison between the two methods ranged between 3.3x10(2)/l and 4.3x10(3)/l. The highest TaqMan PCR/IFA ratios were observed when Percoll/sucrose flotation was combined with DNA extraction protocol optimized for cyst wall lysis, impurities adsorption on a resin, and double step protein digestion and column purification. The concentrations observed with this TaqMan PCR method ranged from 2.5x10(2) to 2.4x10(3) G. lamblia cysts/l and only one sample resulted in a no amplification curve. Thus, we developed a TaqMan PCR method increasing the rapidity and specificity of G. lamblia cyst quantification. The combination of Percoll/sucrose flotation and DNA extraction optimized protocol before TaqMan assay has provided a good indication of the G. lamblia contamination level in raw sewage samples.

Tracking the origin of faecal pollution in surface water: an ongoing project within the European Union research programme.

The objectives of this study are to generate knowledge about methods to track the sources of faecal pollution in surface waters, with the aim of having one or a few easy procedures applicable to different geographic areas in Europe. For this, a first field study using already proposed methods (genotypes of F-specific RNA bacteriophages, bacteriophages infecting Bacteroides fragilis, phenotypes of faecal coliforms and enterococci, and sterols) has been done in five areas representing a wide array of conditions in Europe. The present faecal indicators (faecal coliforms, enterococci, sulfite reducing clostridia and somatic coliphages) have also been included in this first field study. At the same time some emerging methods have been settled or adapted to water samples and assayed in a limited number of samples. The results of this first field study indicate that no single parameter alone is able to discriminate the sources, human or non-human, of faecal pollution, but that a 'basket' of 4 or 5 parameters, which includes one of the present faecal indicators, will do so. In addition, numerical analysis of the data shows that this 'basket' will allow the successful building of predictive models. Both the statistical analyses and the studied predictive models indicate that genotype II of F-specific RNA bacteriophages, the coprostanol and the ratio coprostanol: coprostanol+epicoprostanol are, out of the studied parameters, those with a greater discriminating power. Either because unsuccessful adaptation of the methods to water samples or because the preliminary assays in water samples indicated low discriminating capability, only three (sorbitol-fermenting bifidobacteria, some species of bifidobacteria detected by PCR with specific primers and phages infecting Bacteroides tethaiotaomicron) of the newly assayed methods have been considered for a second field study, which is currently underway. Expectations are that these new tools will minimize the number of parameters in the 'basket', or at least minimize the difficulty in assaying them.

Parasite contamination (helminth eggs) in sludge treatment plants: definition of a sampling strategy.

The use of sludge in agriculture must be carried out according to many guidelines, especially regarding a precise knowledge of the pathogenic microorganisms it contains. The control of the produced sludge requires a sampling strategy that is representative of the contamination present in the sludge. Thus, we evaluated the distribution of helminth eggs in sludge to determine how to sample and at what frequency. Two plants were studied, firstly we studied sludge that was undergoing biological treatment (anaerobic digestion, prolonged aeration), secondly we evaluated the dehydration step (centrifugation and filter press). The helminth egg concentrations were measured over short periods (between 5 minutes and 7 hours) and for periods of over 24 hours (7 to 28 days). The results showed that there was much homogeneity in periods of less than 7 hours, thus it was advisable to take grab samples. An appropriate sample weight was 30 g dry matter, because this allowed an analysis in triplicate when testing treatment processes according to standards of France, (less than 3 viable eggs/10 g dry matter). Determination of the egg concentration in the plants during periods of over 24 hours showed that the parasite flow was stable. In some cases, large variations were due to the treatment processes (storage or thickening, mixing of different sludges). These results have been confirmed with the study of 6 other plants during a one year period. Thus, the recommended sampling frequency can be limited to every 3 to 6 months, by adapting the sampling methods to the characteristics of the plant.