Simon D. Goldenberg

Lack of association of tcdC type and binary toxin status with disease severity and outcome in toxigenic Clostridium difficile

The production of binary toxin and presence of truncating mutations in the putative toxin repressor gene, tcdC, have been associated with the increased virulence and spread of Clostridium difficile, especially ribotype 027. We analysed the prevalence of binary toxin genes and tcdC mutations in 207 clinical C. difficile isolates collected between 2008-2010. The majority (83%) belonged to one of five tcdC types and 8% were ribotype 027. There was little evidence of epidemic spread but there was a high prevalence of both predicted tcdC truncating mutations (15%) and binary toxin genes (28%), which occurred in both 027 and other ribotypes. We measured risk factors (age and laboratory markers) and patient outcomes (severity of disease, ICU admission, mortality, recurrence and length of stay) for patients infected with C. difficile strains with and without these mutations and genes. There was a significantly higher serum C-reactive protein and total peripheral white cell count in the group with predicted tcdC truncating mutations, but no difference in patient outcome. The group with binary toxin genes had a significantly higher total peripheral white cell count and 30 day all cause mortality. We have demonstrated a high prevalence of both predicted tcdC truncating mutations and binary toxin genes in a variety of C. difficile ribotypes, however neither of these factors by themselves predicted clinical virulence. This and other work show that commonly described deletions and truncating mutations do not by themselves explain the virulence of ribotype 027 and other C. difficile strains and further work is required to explain why some isolates appear to produce more severe disease than others.

Surface-attached cells, biofilms and biocide susceptibility: implications for hospital cleaning and disinfection

Microbes tend to attach to available surfaces and readily form biofilms, which is problematic in healthcare settings. Biofilms are traditionally associated with wet or damp surfaces such as indwelling medical devices and tubing on medical equipment. However, microbes can survive for extended periods in a desiccated state on dry hospital surfaces, and biofilms have recently been discovered on dry hospital surfaces. Microbes attached to surfaces and in biofilms are less susceptible to biocides, antibiotics and physical stress. Thus, surface attachment and/or biofilm formation may explain how vegetative bacteria can survive on surfaces for weeks to months (or more), interfere with attempts to recover microbes through environmental sampling, and provide a mixed bacterial population for the horizontal transfer of resistance genes. The capacity of existing detergent formulations and disinfectants to disrupt biofilms may have an important and previously unrecognized role in determining their effectiveness in the field, which should be reflected in testing standards. There is a need for further research to elucidate the nature and physiology of microbes on dry hospital surfaces, specifically the prevalence and composition of biofilms. This will inform new approaches to hospital cleaning and disinfection, including novel surfaces that reduce microbial attachment and improve microbial detachment, and methods to augment the activity of biocides against surface-attached microbes such as bacteriophages and antimicrobial peptides. Future strategies to address environmental contamination on hospital surfaces should consider the presence of microbes attached to surfaces, including biofilms.

Two-step glutamate dehydrogenase antigen real-time polymerase chain reaction assay for detection of toxigenic Clostridium difficile

Current diagnosis of Clostridium difficile infection (CDI) relies upon detection of toxins A/B in stool by enzyme immunoassay [EIA(A/B)]. This strategy is unsatisfactory because it has a low sensitivity resulting in significant false negatives. We investigated the performance of a two-step algorithm for diagnosis of CDI using detection of glutamate dehydrogenase (GDH). GDH-positive samples were tested for C. difficile toxin B gene (tcdB) by polymerase chain reaction (PCR). The performance of the two-step protocol was compared with toxin detection by the Meridian Premier EIA kit in 500 consecutive stool samples from patients with suspected CDI. The reference standard among samples that were positive by either EIA(A/B) or GDH testing was culture cytotoxin neutralisation (culture/CTN). Thirty-six (7%) of 500 samples were identified as true positives by culture/CTN. EIA(A/B) identified 14 of the positive specimens with 22 false negatives and two false positives. The two-step protocol identified 34 of the positive samples with two false positives and two false negatives. EIA(A/B) had a sensitivity of 39%, specificity of 99%, positive predictive value of 88% and negative predictive value of 95%. The two-step algorithm performed better, with corresponding values of 94%, 99%, 94% and 99% respectively. Screening for GDH before confirmation of positives by PCR is cheaper than screening all specimens by PCR and is an effective method for routine use. Current EIA(A/B) tests for CDI are of inadequate sensitivity and should be replaced; however, this may result in apparent changes in CDI rates that would need to be explained in national surveillance statistics.

Diagnostic testing for Clostridium difficile: a comprehensive survey of laboratories in England

Recent studies have shown poor performance of commonly used toxin enzyme immunoassays (EIAs) for laboratory testing for Clostridium difficile infection (CDI). In 2009-2010, the UK Health Protection Agency and the European Society of Clinical Microbiology and Infectious Diseases stated that toxin EIA testing alone is suboptimal, and recommended a two-step testing protocol (i.e. screening with one method and confirming the results with another method). All acute English National Health Service trusts were surveyed to determine their testing methods and positivity rates using freedom of information requests. Replies were received from 168 of 170 trusts (99% response rate). Seventy percent of trusts were using a toxin EIA as a standalone testing method, with positive predictive values (PPVs) as low as 20% in some cases. The mean positivity rate decreased from 6.45% in 2008 to 4.47% in 2009, which will have a negative effect on the PPVs of these tests. The UK Department of Health publishes CDI rates as a measure of quality of care and good infection control practice. However, this may not provide valid comparisons because of the wide disparity between testing methods. The present study demonstrates wide variation in testing practices for CDI in England, and laboratories should reconsider their current testing strategies.

Multiplex molecular testing for management of infectious gastroenteritis in a hospital setting: a comparative diagnostic and clinical utility study

Laboratory diagnosis and clinical management of inpatients with diarrhoea is complex and time consuming. Tests are often requested sequentially and undertaken in different laboratories. This causes prolonged unnecessary presumptive isolation of patients, because most cases are non-infectious. A molecular multiplex test (Luminex(®) Gastrointestinal Pathogen Panel (GPP)) was compared with conventional testing over 8xa0months to determine diagnostic accuracy, turnaround times, laboratory costs, use of isolation facilities and user acceptability. A total of 262 (12%) patients had a pathogen detected by conventional methods compared with 483 (22.1%) by GPP. Most additional cases were detected in patients developing symptoms in the first 4xa0days of admission. Additional cases were detected because of presumed improved diagnostic sensitivity but also because clinicians had not requested the correct pathogen. Turnaround time (41.8xa0h) was faster than bacterial culture (66.5xa0h) and parasite investigation (66.5xa0h) but slower than conventional testing for Clostridium difficile (17.3xa0h) and viruses (27xa0h). The test could allow simplified requesting by clinicians and a consolidated laboratory workflow, reducing the overall number of specimens received by the laboratory. A total of 154 isolation days were saved at an estimated cost of £30xa0800. Consumables and labour were estimated at £150xa0641 compared with £63xa0431 for conventional testing. Multiplex molecular testing using a panel of targets allowed enhanced detection and a consolidated laboratory workflow. This is likely to be of greater benefit to cases that present within the first 4xa0days of hospital admission.

Performance evaluation of the Verigene® (Nanosphere) and FilmArray® (BioFire®) molecular assays for identification of causative organisms in bacterial bloodstream infections

Molecular assays designed to provide bacterial identification and detection of resistance genes directly from positive blood cultures can significantly reduce the time to definitive results. This has the potential to improve patient management and antimicrobial stewardship. However, the extent of such an impact is yet to be fully assessed. We tested two such assays, the Verigene® System Bloodstream Infection Tests (Nanosphere, Inc., Northbrook, IL, USA) (both Gram-positive and Gram-negative cartridges) and the FilmArray® Blood Culture Identification Panel (BioFire® Diagnostics, Inc., Salt Lake City, UT, USA). We compared their accuracy and speed of organism and resistance gene identification to conventional culture-based methods for 173 positive blood cultures. We also retrospectively determined, for organisms deemed not to be contaminants, the potential impact on antimicrobial prescribing. Both the Verigene® and FilmArray® assays accurately identified organisms, on average, 27.95 and 29.17xa0h earlier than conventional methods, respectively. There were a significant number of false-positives for Pseudomonas aeruginosa with the FilmArray® assay, which may have been related to contamination of the bioMérieux BacT standard anaerobic blood culture bottles, which the manufacturer has acknowledged. Both panels provided results significantly faster than conventional methods. In our setting, the extent of the potential positive impact on antimicrobial prescribing was modest (9 out of 173 samples). However, this may be an underestimation, since probable contaminants were not included in this analysis. In conclusion, both panels gave accurate results with significantly improved turnaround times.

A cost benefit analysis of the Luminex xTAG Gastrointestinal Pathogen Panel for detection of infectious gastroenteritis in hospitalised patients.

OBJECTIVESnRecent advances in the laboratory detection of infectious diarrhoea allow more rapid and sensitive identification of infected patients. Several commercial multiplex molecular panels are now available and may have significant advantages over culture based techniques. Faster and more sensitive testing of hospitalised patients with suspected infectious gastroenteritis could result in significant efficiencies in the utilisation of isolation facilities, however few studies have examined this potential benefit. We studied the potential clinical and cost benefits of a commercially available molecular panel.nnnMETHODSnAn eight-month parallel diagnostic study was conducted to measure potential economic benefits of testing hospitalised patients with the Luminex xTAG Gastrointestinal Pathogen Panel (GPP) compared with conventional laboratory testing (based on a combination of culture, microscopy and enzyme immunoassay). Laboratory testing costs and patient isolation costs were measured or estimated for 800 patients.nnnRESULTSnAlthough costing an additional £22,283, use of GPP could enable a reduction in isolation time from 2202 to 1447 days, a saving of £66,765, which more than offsets the additional laboratory testing costs.nnnCONCLUSIONnSyndromic testing of patients against a broad panel of organisms using a multiplex molecular panel can both improve detection rates and allow better laboratory workflow practices. Removing patients testing negative using this panel could result in significant patient isolation savings.

Performance of the GeneXpert CT/NG Assay Compared to That of the Aptima AC2 Assay for Detection of Rectal Chlamydia trachomatis and Neisseria gonorrhoeae by Use of Residual Aptima Samples

ABSTRACT There are currently no commercially available molecular assays for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in rectal swabs with regulatory approval. We compared the Cepheid GeneXpert CT/NG assay with the GenProbe Aptima Combo2 assay, using 409 rectal swabs. Using Aptima as the gold standard, the sensitivity, specificity, and positive and negative predictive values of GeneXpert for the detection of C. trachomatis and N. gonorrhoeae were 86%, 99.2%, 92.5%, and 98.4% and 91.1%, 100%, 100%, and 98.6%, respectively. Despite significant dilution of samples prior to GeneXpert testing, the assay performed well with excellent specificity.

Detection of toxigenic Clostridium difficile in diarrheal stools by rapid real-time polymerase chain reaction.

The Cepheid Xpert polymerase chain reaction assay (Sunnyvale, CA) had a sensitivity of 100%, specificity of 96.7%, and positive and negative predictive values of 90.5% and 100%, respectively, compared with toxigenic culture for the laboratory diagnosis of Clostridium difficile in diarrheal stool samples. This test appears to be a significant improvement to poorly performing enzyme immunoassays.

An early evaluation of clinical and economic costs and benefits of implementing point of care NAAT tests for Chlamydia trachomatis and Neisseria gonorrhoea in genitourinary medicine clinics in England

Objectives To estimate the costs and benefits of clinical pathways incorporating a point of care (POC) nucleic acid amplification test (NAAT) for chlamydia and gonorrhoea in genitourinary medicine (GUM) clinics compared with standard off-site laboratory testing. Method We simulated 1.2 million GUM clinic attendees in England. A simulation in Microsoft Excel was developed to compare existing standard pathways of management for chlamydia and gonorrhoea with a POC NAAT. We conducted scenario analyses to evaluate the robustness of the model findings. The primary outcome was the incremental cost-effectiveness ratio. Secondary outcomes included the number of inappropriate treatments, complications and transmissions averted. Results The baseline cost of using the point of POC NAAT was £103.9 million compared with £115.6 million for standard care. The POC NAAT was also associated with a small increase of 46 quality adjusted life years, making the new test both more effective and cheaper. Over 95u2005000 inappropriate treatments might be avoided by using a POC NAAT. Patients receive diagnosis and treatment on the same day as testing, which may also prevent 189 cases of pelvic inflammatory disease and 17u2005561 onward transmissions annually. Discussion Replacing standard laboratory tests for chlamydia and gonorrhoea with a POC test could be cost saving and patients would benefit from more accurate diagnosis and less unnecessary treatment. Overtreatment currently accounts for about a tenth of the reported treatments for chlamydia and gonorrhoea and POC NAATs would effectively eliminate the need for presumptive treatment.