Laboratory plasticware - Use at your own risk: Suitability of microcentrifuge tubes for spores' analysis of Clostridium difficile.

Clostridium difficile is a Gram-positive spore forming rod-shaped bacterium which causes mild to severe diarrhea. Spores play a key role in transmission of C. difficile in hospital environment. To investigate ability of spores to stay on fomites and to assess levels of contamination it is essential to prevent loss of spores collected for the analysis. Working with C. difficile spores we noticed a significant loss after vortexing of spore suspensions and investigated if it can be prevented by using a specific brand or type of microcentrifuge tubes. 7 types of microcentrifuge tubes from 3 manufacturers were tested. Spores of three types of C. difficile, NAP1/027, NAP4/014 and NAP7/078 (clinical isolates) were used. C. difficile was grown on Brucella Supplemented Agar for 9 days, spores were collected, washed and density of 3 suspensions was normalized to optical density (OD) 550 0.1 or 0.05. These suspensions (OD 0.1) were used in serial dilutions with 3 experimental conditions - pipetting, vortexing or vortexing in 3% albumin solution and in vortexing experiment when 150 µl were vortexed for 1 min at 1500 rpm per tube and loss of spores was measured by a decrease in dipicolinic acid (DPA) concentration measured by time-delayed terbium fluorescence. Inner surface of the tubes was visualized with microscopy to observe adhered spores. In serial dilution experiment, initial concentration of spores would be underestimated by up to 18X in case of vortexing for NAP1 strain and 9X for NAP4 strain. Presence of 3% albumin significantly decreases this effect but does not eliminate it completely. Comparison of 7 types of tubes shows that a single vortexing for 1 min of diluted spore suspension at concentrations of 1.8 × 107 spores per ml leads to a loss of up to 90% of spores in some tubes. Degree of spores' adhesion varied between brands and types of tubes and between tubes of the same type. In some brands there was a significant variability in adhesion between tubes from the same batch. Microscopy after vortexing shows a film of spores attached to the tube's wall. Adherence could be affected by the type of plastic, additives (plasticizers) used in manufacturing and quality of moulds (e.g."diamond polished"). To identify the most appropriate type of tubes for the experiment it is essential to test it beforehand as not every brand is suitable for this purpose. Using tubes with a high degree of adherence could significantly affect measurement of spores' concentration in serial dilutions, e.g. when quantifying spores production by a specific strain or when limits of detection are measured. Also, sensitivity of commercial tests for detection of C. difficile in clinical specimens can be decreased if an unsuitable type of plastic containers and tubes is used.

The outcome and long-term follow-up of 94 patients with recurrent and refractory Clostridium difficile infection using single to multiple fecal microbiota transplantation via retention enema.

Clostridium difficile infection (CDI) is one of the most frequent causes of healthcare-associated infections, and its rates are also increasing in the community. The management of CDI has become a major challenge, given growing rates of recurrences and failures with standard antibiotic therapy. Mounting evidence suggests that fecal microbiota transplantation (FMT) may be effective; however, as there is a paucity of data with regard to repeat FMT for primary non-response to this treatment, this study examined the outcome of multiple FMTs for recurrent CDI. Case records were reviewed for 94 patients who underwent FMT via retention enema for recurrent or refractory CDI during the period 2008-2012. Demographic information, treatment data, and clinical resolution rates were examined for single FMT and cumulative resolution was assessed for multiple FMTs in the context of ongoing symptoms. The cumulative clinical resolution following four or more FMTs was 86%. When antibiotic therapy was used between FMTs, the clinical resolution rate increased to 92%. There were no reported adverse events and no patients who were cured with FMT had further episodes of CDI at 6-24 months follow-up. Multiple FMTs administered through enemas is an effective, safe, and simple therapy for the management of recurrent or refractory CDI.

Detection of severe acute respiratory syndrome coronavirus in stool specimens by commercially available real-time reverse transcriptase PCR assays.

Three commercially available real-time reverse transcriptase PCR assays (the Artus RealArt HPA coronavirus LightCycler, the Artus RealArt HPA coronavirus Rotor-Gene, and the EraGen severe acute respiratory syndrome coronavirus POL assay) and three RNA extraction methodologies were evaluated for the detection of severe acute respiratory syndrome coronavirus RNA from 91 stool specimens. The assays' sensitivities were highest (58% to 75%) for specimens obtained 8 to 21 days after symptom onset. The assays were less sensitive when specimens were obtained less than 8 days or more than 21 days after the onset of symptoms. All assays were 100% specific.

Multicenter comparison of nucleic acid extraction methods for detection of severe acute respiratory syndrome coronavirus RNA in stool specimens.

The emergence of a novel coronavirus (CoV) as the cause of severe acute respiratory syndrome (SARS) catalyzed the development of rapid diagnostic tests. Stool samples have been shown to be appropriate for diagnostic testing for SARS CoV, although it has been recognized to be a heterogeneous and difficult sample that contains amplification inhibitors. Limited information on the efficiency of extraction methods for the purification and concentration of SARS CoV RNA from stool samples is available. Our study objectives were to determine the optimal extraction method for SARS CoV RNA detection and to examine the effect of increased specimen volume for the detection of SARS CoV RNA in stool specimens. We conducted a multicenter evaluation of four automated and four manual extraction methods using dilutions of viral lysate in replicate mock stool samples, followed by quantitation of SARS CoV RNA using real-time reverse transcriptase PCR. The sensitivities of the manual methods ranged from 50% to 100%, with the Cortex Biochem Magazorb method, a magnetic bead isolation method, allowing detection of all 12 positive samples. The sensitivities of the automated methods ranged from 75% to 100%. The bioMérieux NucliSens automated extractor and miniMag extraction methods each had a sensitivity of 100%. Examination of the copy numbers detected and the generation of 10-fold dilutions of the extracted material indicated that a number of extraction methods retained inhibitory substances that prevented optimal amplification. Increasing the volume of sample input did improve detection. This information could be useful for the extraction of other RNA viruses from stool samples and demonstrates the need to evaluate extraction methods for different specimen types.