An outbreak of two strains of OXA-48 producing Klebsiella pneumoniae in a teaching hospital.

OXA-48 producers can be difficult to detect in clinical specimens due to phenotypic low-level resistance to carbapenems. Additionally, low infection rates make clinical specimens poor sentinels for the presence of OXA-48 producers within a healthcare institution. We report an outbreak of OXA-48-producing Klebsiella pneumoniae (OXAKp) that was discovered following culture of OXAKp in a urine specimen from a patient with no known risk factors for acquisition. Widespread screening across medical wards in the trust revealed evidence of transmission across several wards. Samples from 60 patients were positive for OXAKp. Five patients had OXAKp clinical infection, four of whom were treated with ceftazidime/avibactam. Variable number tandem repeat analysis of the OXAKp isolates revealed two predominant strain types clustered around two groups of wards. Infection prevention measures included isolation and cohort nursing of infected and colonized patients, restriction of affected ward areas to new admissions, stringent hand hygiene and use of personal protective equipment. Environmental cleaning of patient areas was carried out using chlorine-releasing disinfectants and hydrogen peroxide vapour. Entire wards were decanted to enable effective cleaning of empty ward areas. The outbreak lasted almost five months and is estimated to have cost around £400 000. During the course of the outbreak, there were five reported prescription and administration incidents related to confusion between ceftazidime and ceftazidime/avibactam. No patient harm resulted from these incidents and the implementation of brand name prescribing for ceftazidime/avibactam prevented further incidents.

Super-resolution microscopy as a potential approach to diagnosis of platelet granule disorders.

BACKGROUND: Many platelet functions are dependent on bioactive molecules released from their granules. Deficiencies of these granules in number, shape or content are associated with bleeding. The small size of these granules is such that imaging them for diagnosis has traditionally required electron microscopy. However, recently developed super-resolution microscopes provide sufficient spatial resolution to effectively image platelet granules. When combined with automated image analysis, these methods provide a quantitative, unbiased, rapidly acquired dataset that can readily and reliably reveal differences in platelet granules between individuals. OBJECTIVE: To demonstrate the ability of structured illumination microscopy (SIM) to efficiently differentiate between healthy volunteers and three patients with Hermansky-Pudlak syndrome. METHODS: Blood samples were taken from three patients with Hermansky-Pudlak syndrome and seven controls. Patients 1-3 have gene defects in HPS1, HPS6 and HPS5, respectively; all controls were healthy volunteers. Platelet-rich plasma was isolated from blood and the platelets fixed, stained for CD63 and processed for analysis by immunofluorescence microscopy, using a custom-built SIM microscope. RESULTS: SIM can successfully resolve CD63-positive structures in fixed platelets. A determination of the number of CD63-positive structures per platelet allowed us to conclude that each patient was significantly different from all of the controls with 99% confidence. CONCLUSIONS: A super-resolution imaging approach is effective and rapid in objectively differentiating between patients with a platelet bleeding disorder and healthy volunteers. CD63 is a useful marker for predicting Hermansky-Pudlak syndrome and could be used in the diagnosis of patients suspected of other platelet granule disorders.

Rapid and highly sensitive qualitative real-time assay for detection of respiratory syncytial virus A and B using NASBA and molecular beacon technology.

The performance of a sensitive and specific qualitative respiratory syncytial virus (RSV) assay based on NASBA technology and real-time molecular beacon detection is presented. Very low detection limits for both RSV A and RSV B were determined: 95% detection hit-rate of 95 and 47 copies/input in isolation for RSV A and RSV B, respectively. RSV was detected in a wide variety of clinical samples including respiratory swabs, nasopharyngeal aspirates (NPA), bronchoalveolar lavages (BAL), endotracheal secretions, and sputum samples. In total 779 clinical samples were tested and a valid result was obtained for 765 (RSV NASBA assay), 765 (cell culture), and 529 (rapid direct immunofluorescence testing (IF)) samples. Of these samples, 229 (RSV NASBA assay), 61 (cell culture), and 122 (IF) samples were positive for RSV. In addition, 106 samples were reported as RSV negative using the NOW RSV assay (Binax). Subsequent testing using the RSV NASBA assay demonstrated that 32 (30%) of these samples were RSV positive. The RSV NASBA assay includes a homologous internal control, which offers a high degree of standardization and quality control. When the RSV NASBA assay was performed on the NucliSens EasyQ platform (bioMérieux), test results of 48 sample extracts were obtained in less than 2h.

Evaluation of molecular techniques in prediction and diagnosis of cytomegalovirus disease in immunocompromised patients.

OBJECTIVES: To evaluate selected molecular tests in diagnosis and screening of cytomegalovirus (CMV) infection in immunosuppressed patients. DESIGN: Clinical and cost-effectiveness were assessed through a prospective two-stage trial of CMV screening regimes in a routine service setting. Different molecular test results were fed back to clinicians in each stage, plus antigenaemia results. The technical performance of the molecular methods was assessed through an independent masked comparison of each molecular test against the established (antigenaemia) test. Scientists performing a particular test were blind to the other test results for that sample. Diagnostic and therapeutic impact were recorded prospectively for all tests, to include any effect on diagnostic certainty, changes to CMV therapy and any other reported impact on patient management. The cost of each test was estimated under different laboratory conditions. Prospective patients undergoing CMV screening were compared with consecutive historical controls in the same unit. Towards the end of the study, a survey of all UK virology laboratories was undertaken to identify current CMV screening practice and test preferences. In addition, all UK renal transplant surgeons and haematology transplant centres were surveyed in order to identify current clinical practice and perceptions of the benefits of CMV screening. SETTING: Study patients were recruited from University Hospital Wales (UHW), Cardiff. Staff in the Cardiff Public Health Laboratory Service virology laboratory performed the tests. PARTICIPANTS: A consecutive series of transplant patients was recruited to the prospective study over a 42-month period, totalling 98 renal and 140 haematology patients. A consecutive series of historical controls was identified, with 199 renal and 136 haematology patients who underwent transplants in the UHW during the 29 months prior to the prospective CMV screening trial. INTERVENTIONS: A predefined CMV screening protocol was applied to all patients in the prospective trial. Renal patients were tested every 4 weeks until 16 weeks post-transplant (five tests in total). Haematology patients were tested every 2 weeks until 12 weeks post-transplant, and then every 4 weeks until 24 weeks (10 tests in total). The assays used for CMV screening were as follows: non-molecular test, (1) pp65 antigenaemia assay; molecular tests, semi-quantitative in-house polymerase chain reaction (PCR), (2) single-round (PCR1) and (3) two-round, nested (PCR2); and qualitative commercial tests, (4) Roche Amplicor Assay (Amplicor) and (5) pp67 NASBA assay (NASBA). MAIN OUTCOME MEASURES: Test failure rates, sensitivity/specificity values and positive predictive value (PPV) and negative predictive value (NPV) were measured for each assay. The laboratory cost of undertaking various CMV tests was measured and other NHS costs associated with false-positive or false-negative test results were estimated. The likelihood of CMV disease and the likely impact of positive or negative test result on therapy and further investigations were recorded. On receipt of the test result, interim outcome measures were recorded to include the impact of test result on diagnostic certainty, changes to planned patient management (e.g. therapy, investigations) and perceived benefit. All definitive diagnoses of CMV disease, prescribing of CMV therapy and interim patient outcome at the end of the screening period were recorded. RESULTS: In haematology and renal transplant patients, all tests had a similar NPV (0.976--0.997 and 0.935--0.995, respectively) when used in CMV screening. PCR1 is the least expensive molecular test (7.80-13.70 UK pounds). Commercial tests, NASBA and Amplicor, are both more expensive (22.50-34.70 UK pounds NASBA; 23.20-29.20 UK pounds Amplicor). Antigenaemia costs 12.50-27.40 pounds depending on staff grade and batch size. Quantitative PCR (COBAS) is the most expensive at around 50 UK pounds per sample. No clear link between screening test results and CMV prescribing was detected; clinicians appear to consider screening results in the context of other factors. There was no evidence that the introduction of CMV screening led to reductions in CMV deaths or improved transplant success rates. For cost per positive test result, PCR1 was the most cost-effective screening test on this indicator (renal patients 116 UK pounds per true positive, haematology patients 518 pounds). Antigenaemia was the least cost-effective screening test (renal patients 643 UK pounds per true positive, haematology patients 2475 pounds). Cost-effectiveness analysis and cost per "beneficial result" (as judged by clinicians) confirmed that PCR1 remained the most cost-effective test. Modelling outputs for targeted screening protocols also supported this. CONCLUSIONS: The study findings offer some evidence that a CMV screening regime is more cost-effective than diagnostic testing alone, based on the cost per true positive detected and interim outcome such as changes in patient management. However, the study was unable to demonstrate any benefits in terms of longer term patient outcomes. If CMV screening is introduced, the use of antigenaemia pp65 is clearly less cost-effective than the use of molecular tests. The study identified the optimum test for CMV screening as an in-house molecular test (single-round PCR test). This test was less costly to perform and also resulted in lower costs linked to false positives and negatives than other tests. The in-house, semi-quantitative test was two to three times more cost-effective than the commercial molecular tests assessed; however changes to European Union legislation may mean that it may not be feasible to use in-house tests. The use of targeted screening (limiting CMV screening to high-risk transplants) as opposed to universal screening offers a significant improvement in the cost-effectiveness ratio for haematology transplant patients, but has limited impact in the case of renal transplants. Economic analyses could be expanded to model the cost-effectiveness of more frequent screening tests (as reported nationally), and screening in other "at risk" groups. Subgroup specific disease groups should be investigated across a larger population to allow more accurate modelling of the impact of CMV screening on disease progression. Further studies of CMV screening programmes should address a range of outcome measures, including patient outcomes.

Enhanced clinical utility of the NucliSens EasyQ RSV A+B Assay for rapid detection of respiratory syncytial virus in clinical samples.

The aim of the present study was to compare traditional methods for the detection of respiratory syncytial virus with a newly developed commercial assay based on real-time nucleic acid sequence based amplification. Respiratory syncytial virus is a major cause of severe respiratory infection in infants and in certain groups of older children and adults. Treatment options are limited, but a rapid diagnosis improves patient management and infection control. The rapid diagnosis of respiratory syncytial virus currently relies on antigen detection assays. These tests are limited to use in certain good-quality types of samples, which are rarely obtained from adult patients. Molecular-based assays for the detection of respiratory syncytial virus are shown to be highly sensitive, specific, and more rapid than cell culture techniques. This retrospective study compared traditional laboratory techniques for the detection of respiratory syncytial virus in 508 respiratory samples collected during the winter months of 2003-2004 against the recently developed, commercially available NucliSens EasyQ Respiratory Syncytial Virus A+B assay (bioMérieux, Marcy l'Etoile, France), which is based on real-time nucleic acid sequence based amplification using molecular beacons and an internal control. Using traditional techniques, the prevalence of respiratory syncytial virus in the samples tested was found to be 21%. Using the real-time nucleic acid sequence-based amplification assay, an additional 41 samples from patients with a clinically diagnosed respiratory illness were found to be positive for respiratory syncytial virus. The NucliSens EasyQ assay was shown to be sensitive and specific for the detection of respiratory syncytial virus A+B in different types of respiratory samples. Moreover, the time required to complete the assay was <4 h, so results could be obtained on the same day as sample receipt in the laboratory.

The value of follow-up after acute Q fever infection.

This is a case report of a 53-year-old woman involved in an outbreak of Q fever, in whom Q fever endocarditis was diagnosed 18 months after acute Q fever infection. At the time of diagnosis, she was completely asymptomatic and without screening for chronic Q fever, this severe potentially life-threatening infection would probably not have been recognised until significant valvular destruction had taken place. Early diagnosis enabled prompt, potentially curative medical treatment to start without the need for valvular heart surgery. The authors advocate that serological monitoring should be carried out every 4 months for a period of 2 years after acute Q fever and patients with high phase 1 IgG titres (>800) be investigated further and/or followed more closely depending on the clinical scenario. The case report also discusses the use of complement fixation testing in the diagnosis of Q fever endocarditis. The authors recommend that in cases of culture negative endocarditis, a single negative complement fixation test is not sufficient to exclude the diagnosis of Q fever endocarditis. Micro-immunofluorescence or repeat complement fixation testing is recommended when Q fever endocarditis is suspected clinically.

Evaluation of a broadly reactive nucleic acid sequence based amplification assay for the detection of noroviruses in faecal material.

A recently described nucleic acid sequence based amplification (NASBA) assay for the detection of genogroup I (GI) and genogroup II (GII) norovirus RNA in faecal samples was evaluated against a reverse transcription polymerase chain reaction (RT-PCR). Both assays were used to screen a panel of 38 faecal samples known to contain 17 different norovirus strains and 131 clinical samples collected from 60 gastroenteritis outbreaks of unknown aetiology. The NASBA assay detected 13 out of the 17 strains of norovirus in the characterised panel, failing to detect a single GII strain and three GI strains. There was 90% agreement between the two assays used to detect norovirus in clinical samples from outbreaks. NASBA detected norovirus RNA in all 64 samples positive by RT-PCR and also detected norovirus RNA in additional 13 samples that were negative by RT-PCR. The sensitivity and specificity of NASBA was 100% and 80%, respectively, compared to RT-PCR results. The norovirus NASBA assay was shown to be highly sensitive and specific, and its ease of use and rapid turnaround time makes it a favourable alternative to RT-PCR for the investigation of norovirus outbreaks.

Risk factors, clinical features and genotype distribution of diagnosed hepatitis C virus infections: a pilot for a sentinel laboratory-based surveillance.

Hepatitis C is a global public health problem. A cross-sectional survey was undertaken to determine the frequency of reported risk factors and possible transmission routes in individuals in whom HCV antibody (anti-HCV) was newly detected. Seven public health laboratories in England and Wales reported persons with positive anti-HCV tests over a three-month period (1st November 1996-31st January 1997). A questionnaire was then sent to the clinician or general practitioner (GP) who requested the test. A total of 320 laboratory reports were received from participating laboratories and 221 (69%) questionnaires were received from clinicians and GPs. Of those patients from whom a questionnaire was received (median age 36 years; males 72.9%, females 23.1%), 86% had one or more risk factors for infection reported by the clinician/GP. Injecting drug use (68%) was the main risk factor reported. Reasons for testing included being in a known risk group (65%), liver disease (19%) and blood donation (1.4%). Of the total responders, 67% were asymptomatic, and of those that had had liver function tests 50% were abnormal. The most prevalent HCV genotypes were 3a and 1a. Risk factors for HCV infection can be identified using a simple postal questionnaire to clinicians/GPs who request patient screening.

An outbreak of viral gastroenteritis following environmental contamination at a concert hall.

In January 1999, an outbreak of viral gastroenteritis affected more than 300 people who attended a metropolitan concert hall over a 5-day period. Norwalk-like virus (NLV) was confirmed in faecal samples by reverse transcription polymerase chain reaction assay. The index case was a concert attendee who vomited in the auditorium and adjacent male toilet. Gastrointestinal illness occurred among members of 8/15 school parties who attended the following day. Children who sat on the same level of the auditorium as the index case were much more likely to be ill than those seated elsewhere (relative risk 7.1, 95% confidence interval 5.4-9.2. P < 0.001). The majority of other reported cases had not been present on the evening of the vomiting incident. Disinfection procedure was poor and the disinfectant used contained no sodium hypochlorite. Transmission most likely occurred through direct contact with contaminated fomites. The outbreak has implications for disinfection procedures following vomiting incidents at public venues.

Guidelines for laboratory monitoring of treatment of persistent virus infections.

A growing number of antiviral agents are available for treatment of persistent viral infections. This has increased the requirement for virology laboratories to undertake sophisticated assays for monitoring the efficacy of treatment and identifying drug failure at an early stage. The consensus guidelines within this article address the laboratory requirements for monitoring treatment of the herpes viruses, HIV-1, Hepatitis B and Hepatitis C.

Molecular-based strategies for assessment of CMV infection and disease in immunosuppressed transplant recipients.

OBJECTIVES: Molecular assays are now considered to be the "gold standard" for assessment of human cytomegalovirus (CMV) infection and disease in those at risk from severe associated clinical manifestations. There is, however little consistency in the methods used in different centres. This study was undertaken to compare different qualitative molecular-based approaches for assessment of CMV activation from latency in samples from immunosuppressed transplant recipients. METHODS: Nucleic acid amplification techniques based on the polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA) were undertaken for the assessment of CMV replication and associated disease in immunosuppressed transplant recipients. Samples from 32 transplant recipients were tested during this study using three molecular-based strategies: (1) detection of CMV DNA in whole blood extracts (positive after a single round of PCR considered "high-level" positive, N = 55); (2) detection of cell-free CMV DNA in plasma (two methods, N = 55 for each); and (3) detection of late pp67 CMV mRNA after NASBA (N = 51). Results using a commercial pp65 antigenemia assay were available for comparison from 40 samples. RESULTS: Seven samples were positive for CMV by all methods and 36 were negative by all methods undertaken. The other 12 samples gave discordant results using different molecular methods. The correlation between whole blood "high-level" PCR, NASBA for pp67 mRNA and antigenemia results was generally good. Results presented show that plasma PCR results do not always correlate with methods utilizing whole blood as the substrate and that inhibitors in these samples could be problematic. Whole blood PCR gave more positive results than the other assays but use of a nested assay on whole blood or plasma led to detection of CMV in individuals who had no other indicators of virus replication and who did not develop associated disease (low specificity). Although the number of confirmed CMV disease episodes was low in this study, the problems of low positive predictive value for sensitive, qualitative PCR assays was clearly demonstrated. CONCLUSION: Assays based on qualitative detection of viral nucleic acid may provide information useful for management of CMV but caution is necessary when making comparisons between results using different molecular strategies. It remains to be proven in large, comparative clinical studies in which the approach and method give the best balance between sensitivity, specificity and clinical relevance for different patient groups.

Screening for Chlamydia trachomatis infection using the BDProbeTec ET Chlamydia trachomatis amplified DNA assay on urine in a GUM clinic setting: a simple, fast and cost-effective alternative.

This study compared the BDProbeTec ET Chlamydia trachomatis amplified DNA assay on urine specimens with culture of genital swabs for the detection of C. trachomatis in patients attending the Department of Genitourinary Medicine (GUM), Cardiff Royal Infirmary. Almost twice as many patients tested positive by BDProbeTec ET than by culture. A similar difference was found for both males and females. The case notes of those patients positive by BDProbeTec ET alone were analysed and a significantly greater number were found to have risk indicators for C. trachomatis infection when compared with age and sex comparable controls, providing clinical validation of our findings. The BDProbeTec ET assay was easy to use, more importantly, the test format features an internal control integral with every sample. The cost per true positive was calculated as comparable with culture. We conclude that the BDProbeTec ET assay is a superior alternative to culture for identifying patients infected with C. trachomatis in the GUM clinic setting.

Consensual validation of clinical practice model practice guidelines.

Clinical Practice Model (CPM) Practice Guidelines facilitate the delivery of consistent, high-quality patient care by clarifying nursing services and supporting the practice and documentation of each step of the nursing process. CPM Practice Guidelines are unique because they are part of an integrated interactive systems thinking framework to support professional practice; they are reviewed and improved routinely using multiple sources of data and are used by hundreds of nurses daily throughout the United States and Canada. Guidelines are developed using a rigorous standardized format and process. Consensual validation is an important part of establishing and maintaining the credibility of these Guidelines.

Management of hospital outbreaks of gastro-enteritis due to small roundstructured viruses.

Small round structured viruses (SRSVs, Norwalk-like viruses, NLVs) are the most common cause of outbreaks of gastro-enteritis in hospitals and also cause outbreaks in other settings such as schools, hotels, nursing homes and cruise ships. Hospital outbreaks often lead to ward closure and major disruption in hospital activity. Outbreaks usually affect both patients and staff, sometimes with attack rates in excess of 50%. For this reason, staff shortages can be severe, particularly if several wards are involved at the same time. SRSVs may be spread by several routes: faecal-oral; vomiting/aerosols; food and water. Viruses may be introduced into the ward environment by any of these routes and then propagated by person-to-person spread. In an outbreak setting, the diagnosis can usually be made rapidly and confidently on clinical and epidemiological grounds, particularly if vomiting is a prominent symptom. By the time an SRSV outbreak has been recognized at ward level, most susceptible individuals will have been exposed to the virus and infection control efforts must prioritize the prevention of spread of infection to other clinical areas bycontainment of infected/exposed individuals (especially the prevention of patient and staff movements to other areas), hand-hygiene and effective environmental decontamination. This report of the Public Health Laboratory Service Viral Gastro-enteritis Working Group reviews the epidemiology of outbreaks of infection due to SRSVs and makes recommendations for their management in the hospital setting. The basic principles which underpin these recommendations will also be applicable to the management of some community-based institutional outbreaks.

Development and application of a PCR-based method including an internal control for diagnosis of congenital cytomegalovirus infection.

Cytomegalovirus (CMV) is the most common cause of congenital infection in the developed world. We have designed and evaluated an assay that includes an internal control for amplification and detection of CMV DNA in amniotic fluid and neonatal urine samples. We present data on the use of this assay in the diagnosis of congenital CMV infection. A total of 145 amniotic and fetal fluid samples were examined by this assay; 83 were from healthy pregnant women and 62 were from women who were being investigated because of concerns over the pregnancy (diagnostic group). CMV DNA was detected in three amniotic fluid samples from the diagnostic group but was not detected in any samples taken from healthy pregnant women. Thirty-nine urine samples were obtained from 19 neonates with suspected congenital infection; CMV DNA was detected in urine from 6 of these patients. The assay provides useful information about CMV infection in the fetus and the neonate; when used in conjunction with other diagnostic tools it will enable mothers and obstetricians to make informed decisions about the management of pregnancies complicated by CMV infection.

A comparison of two rapid whole-blood tests and laboratory serology, in the diagnosis of Helicobacter pylori infection.

OBJECTIVE: To compare two rapid whole-blood serology tests for Helicobacter pylori and a laboratory serology assay against a gold standard. DESIGN: Prospective comparison of tests in 81 patients. SETTING: A hospital rapid access endoscopy clinic. PARTICIPANTS: Dyspeptic patients requiring assessment of H. pylori status. INTERVENTIONS: Measurement of H. pylori antibody status by Quickvue One-step, Helisal, and Premier H. pylori test; 13C urea breath test for H. pylori, and gastric biopsies for histology, culture and rapid urease test. MAIN OUTCOME MEASURE: Sensitivity and specificity of Quickvue One-step, Helisal and Premier tests, compared to a gold standard based on 13C urea breath test, biopsy culture, histology and urease test. RESULTS: The Quickvue assay has significantly greater sensitivity (81%) than Helisal (67%), but without appreciable loss of specificity (86% and 93%, respectively). The Premier laboratory assay is significantly more sensitive than both of the rapid blood tests (96%), with comparable specificity to the Quickvue assay. CONCLUSION: The rapid serology tests used in this study are quick and convenient to use, but do not approach the sensitivity of a laboratory assay in detecting H. pylori status in this group of dyspeptic patients attending an endoscopy clinic.