Biomedical subjects

S Monpoeho

Publications and source records attributed to S Monpoeho.

7 recordsLinked to original sources

PubMed2005

Decrease of enteric micro-organisms from rural sewage sludge during their composting in straw mixture.

AIMS: To study the decrease of enteric micro-organisms including viable nematode eggs, enteroviruses, faecal indicators (Escherichia coli and enterococci) and pathogenic bacteria (Listeria monocytogenes, Salmonella sp. and Clostridium perfringens) of a rural sewage sludge when it is composted for 7 months in mixture with straw. METHODS AND RESULTS: Numbers of the test organisms and the physico-chemical parameters were measured on a monthly basis on the mixture, on the compost after being turned, and on the pile in three positions representing the part by which air is incoming, the bottom of the pile and the part through which air is outgoing. The lowest temperature in the pile was observed at the bottom, where it did not exceed 50 degrees C against 66 degrees C in the two other areas. There were no significant differences between the three areas in terms of micro-organism survival. Infectious enteroviruses were inactivated rapidly and were not found after the first turning whereas some genomes were detected until after the third turning. Escherichia coli and enterococci presented a similar survival rate and their number decreased by 4 log(10) whereas Salmonella decayed at a greater rate than L. monocytogenes. The numbers of C. perfringens decreased gradually to reach a final concentration in the mature compost of about 10(2) CFU g(-1) dry matter (d.m.), which was similar to that of the faecal indicators. CONCLUSIONS: The hygienic effect of sludge composting in mixture with straw results in a significant reduction of enteric micro-organisms, the concentration of the faecal indicators in the final product being < 64 most probable number g(-1) d.m. The concentrations of Salmonella, enteroviruses and viable nematode eggs in the final product were not detectable which is in accordance with the French legislation. SIGNIFICANCE AND IMPACT OF THE STUDY: The results which pointed out the different behaviour of the test micro-organisms reflect the difficulty to propose a relevant indicator of hygienization. Otherwise, they show that composting is an efficient means for hygienization of sludge of rural wastewater treatment, where the straw is available close to their place of production.

Animals↗

PubMed2004

Clearance of human-pathogenic viruses from sludge: study of four stabilization processes by real-time reverse transcription-PCR and cell culture.

Sludges derived from wastewater treatment are foul-smelling, biologically unstable substances. As well as containing numerous pathogenic microorganisms, they also consist of organic matter that can be used as agricultural fertilizer. Legislation nevertheless requires sludges to be virologically tested prior to spreading by the counting of infectious enterovirus particles. This method, based on culture of enterovirus on BGM cells, is lengthy and not very sensitive. The aim of this study was to propose an alternative method of genome quantification for all enteroviruses that is applicable to verifying the elimination of viruses in complex samples such as sludges. Our complete protocol was compared to the official method, consisting of enterovirus enumeration with the most probable number of cythopathic unit (MPNCU) assay through the study of four stabilization procedures: liming, composting, heat treatment, and mesophile anaerobic digestion. Enterovirus quantities at the start of the stabilization procedures were between 37 and 288 MPNCU/g on the one scale and between 4 and 5 log genome copies/g on the other. It was shown that all procedures except mesophile anaerobic digestion were highly effective in the elimination of enterovirus particles and genomes in wastewater sludges. Reduction of viruses by mesophile anaerobic digestion was by only 1 log (infectious particles and genomes). In conclusion, stabilization processes can indeed be checked by virological quality control of sludges with gene amplification. However, the infectivity of genomes needs to be confirmed with cell culture or a correlation model if the virological risk inherent in the agricultural use of such sludges is to be fully addressed.

Anaerobiosis↗

PubMed2002

Application of a real-time polymerase chain reaction with internal positive control for detection and quantification of enterovirus in cerebrospinal fluid.

A quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method based on TaqMan technology was developed to determine the presence and amount of enterovirus RNA. In order to prevent false-negative results, a one-step multiplex RT-PCR was optimized. It contains two dual-labelled fluorogenic probes to quantify the 5' noncoding region of enterovirus and detect an internal positive control. In the present study, 104 cerebrospinal fluid samples collected during an outbreak of enteroviral meningitis were analyzed using this method. Amplification of the internal positive control was effective in all but two specimens, confirming the absence of PCR inhibitors and allowing the results of amplification to be validated. The sensitivity of the RT-PCR was 96.8%, while that of cell culture was 34.9%. Genomic viral loads found ranged between 3.3 and 5.9 log(10) copies per milliliter of cerebrospinal fluid (mean, 4.8 log(10) copies/ml). This fluorogenic enterovirus RT-PCR allows large numbers of samples to be screened rapidly. Moreover, its sensitivity and reproducibility make it highly reliable. With these characteristics, the enterovirus RT-PCR can be a useful tool that may offer considerable benefit in the clinical management of patients with enteroviral infections.

Adolescent↗

PubMed2002

Quantification and duration of viraemia during hepatitis A infection as determined by real-time RT-PCR.

Human transmission studies and molecular techniques have provided evidence that transient viraemia occurs during infection with hepatitis A virus (HAV). However, the duration of its presence and levels during the phases of clinical disease and convalescence has not yet been well studied in human patients. Real-time RT-PCR techniques are increasingly used to quantify RNA viruses for diagnosis and/or research purposes. We have optimized a one-step RT-PCR that contains a dual-labelled fluorogenic probe to quantify the 5' noncoding region (5' NCR) of HAV. This method has a dynamic range (5-5 x 10(6) copies). The coefficient of regression of the standard curve was, on average 0.978. Intra-assay CVs% varied from 6.1% to 0.98%, and interassay CVs% from 6.46% to 2.1%. In the currently reported study 41 HAV IgM positive serum samples and 200 serum samples from healthy blood donors were tested by the quantitative RT-PCR method. The mean values on the first day of diagnosis found was 6.38 x 10(5) copies/mL. In a longitudinal study, viraemia persisted for an average of 60 days after clinical onset. These results show that viraemia in HAV infection lasts for many weeks.

Adolescent↗

PubMed2001

Genetic analysis of hepatitis A virus outbreak in France confirms the co-circulation of subgenotypes Ia, Ib and reveals a new genetic lineage.

Genetic analysis of selected genome regions of hepatitis A Virus (HAV) suggested that distinct genotype could be defined in different geographic locations. In order to study the degree of genetic variability among HAV isolated during a single epidemic outbreak, sequences from a 148 base pair segment within the VP1 amino terminal region were obtained for eight distinct HAV isolates from an outbreak that occurred in North Bretagne (France). These sequences were compared among themselves and with published sequences from 30 different strains that represented different HAV sub-genotypes that were isolated all over the world. Phylogenetic analysis revealed an extensive genetic heterogeneity among strains belonging to the same outbreak and revealed co-circulation of sub-genotype IA, IB, and the presence of IIIA sub-genotype for the first time in a Mediterranean country.

Adult↗

PubMed2001

Best viral elution method available for quantification of enteroviruses in sludge by both cell culture and reverse transcription-PCR.

The aim of this study was to select one or several virus extraction techniques that enable simultaneous detection of enterovirus genomes and infectious particles in different types of urban sludge. Eight techniques were compared by using 16 different liquid and solid sludge samples. The numbers of infectious enteroviruses in cell cultures were determined by using the most-probable-number method. The enterovirus genome was quantified by a single-tube reverse transcription-PCR using TaqMan technology. The results were statistically analyzed by Friedman's test, a nonparametric test for analysis of randomized block data using only ranks in terms of extraction technique efficiency. Two techniques seemed to yield higher viral titers as determined by simultaneous detection by cell culture and PCR. The first involved a 10% beef extract solution at pH 9 and sonication; the second involved a 0.3 M NaCl-7% beef extract solution at pH 7.5 followed by Freon treatment. In solid sludge, no significant differences were observed among the eight techniques tested. Both of the best techniques can be used for simultaneous detection of infectious enterovirus particles and genomes in any type of urban sludge.

Enterovirus↗

PubMed2000

Quantification of enterovirus RNA in sludge samples using single tube real-time RT-PCR.

We have developed a quantitative RT-PCR method that can be used to determine the amount of enterovirus RNA in urban sludge samples. This method combines Taq-Man technology with the ABI Prism 7700 real-time sequence detection system. We optimized a one-step RT-PCR that uses a dual-labeled fluorogenic probe to quantify the 5' noncoding region of enteroviruses. For accurate quantification of the number of copies, a Mahoney type 1 poliovirus RNA standard was designed and produced using genetic engineering. This fragment, quantified using the Ribogreen method, was used in serial dilutions as an external standard. The method had a 7-log dynamic range (5 to 2 x 10(7)). PCR inhibitors were removed by extracting viral RNA (after virus concentration) using the RNeasy mini kit with added polyvinylpyrrolidone (PVP) and running the amplification reaction with a mixture containing PVP and T4 gene 32 protein. This real-time quantification of enterovirus RNA allows large numbers of samples to be screened. Its sensitivity, simplicity and reproducibility render it suitable as a screening method with which to characterize enteroviruses, the presence of infectious particles being subsequently confirmed by cell culture.

Enterovirus↗