Ronan McMullan

A Prospective Clinical Trial of a Real-Time Polymerase Chain Reaction Assay for the Diagnosis of Candidemia in Nonneutropenic, Critically Ill Adults

BACKGROUND Invasive Candida infection among nonneutropenic, critically ill adults is a clinical problem that has received increasing attention in recent years. Poor performance of extant diagnostic modalities has promoted risk-based, preemptive prescribing in view of the poor outcomes associated with inadequate or delayed antifungal therapy; this risks unnecessary overtreatment. A rapid, reliable diagnostic test could have a substantial impact on therapeutic practice in this patient population. METHODS Three TaqMan-based real-time polymerase chain reaction assays were developed that are capable of detecting the main medically important Candida species, categorized according to the likelihood of fluconazole susceptibility. Assay 1 detected Candida albicans, Candida parapsilosis, Candida tropicalis, and Candida dubliniensis. Assays 2 and 3 detected Candida glabrata and Candida krusei, respectively. The clinical performance of these assays, applied to serum, was evaluated in a prospective trial of nonneutropenic adults in a single intensive care unit. RESULTS In all, 527 specimens were obtained from 157 participants. All 3 assays were run in parallel for each specimen; they could be completed within 1 working day. Of these, 23 specimens were obtained from 23 participants categorized as having proven Candida infection at the time of sampling. If a single episode of Candida famata candidemia was excluded, the estimated clinical sensitivity, specificity, and positive and negative predictive values of the assays in this trial were 90.9%, 100%, 100% and 99.8%, respectively. CONCLUSIONS These data suggest that the described assays perform well in this population for enhancing the diagnosis of candidemia. The extent to which they may affect clinical outcomes, prescribing practice, and cost-effectiveness of care remains to be ascertained.

Improving molecular detection of Candida DNA in whole blood: comparison of seven fungal DNA extraction protocols using real-time PCR

The limitations of classical diagnostic methods for invasive Candida infections have led to the development of molecular techniques such as real-time PCR to improve diagnosis. However, the detection of low titres of Candida DNA in blood from patients with candidaemia requires the use of extraction methods that efficiently lyse yeast cells and recover small amounts of DNA suitable for amplification. In this study, a Candida-specific real-time PCR assay was used to detect Candida albicans DNA in inoculated whole blood specimens extracted using seven different extraction protocols. The yield and quality of total nucleic acids were estimated using UV absorbance, and specific recovery of C. albicans genomic DNA was estimated quantitatively in comparison with a reference (Qiagen kit/lyticase) method currently in use in our laboratory. The extraction protocols were also compared with respect to sensitivity, cost and time required for completion. The TaqMan PCR assay used to amplify the DNA extracts achieved high levels of specificity, sensitivity and reproducibility. Of the seven extraction protocols evaluated, only the MasterPure yeast DNA extraction reagent kit gave significantly higher total nucleic acid yields than the reference method, although nucleic acid purity was highest using either the reference or YeaStar genomic DNA kit methods. More importantly, the YeaStar method enabled C. albicans DNA to be detected with highest sensitivity over the entire range of copy numbers evaluated, and appears to be an optimal method for extracting Candida DNA from whole blood.

Sepsis: the LightCycler SeptiFast Test MGRADE®, SepsiTest™ and IRIDICA BAC BSI assay for rapidly identifying bloodstream bacteria and fungi – a systematic review and economic evaluation

BACKGROUND Sepsis can lead to multiple organ failure and death. Timely and appropriate treatment can reduce in-hospital mortality and morbidity. OBJECTIVES To determine the clinical effectiveness and cost-effectiveness of three tests [LightCycler SeptiFast Test MGRADE(®) (Roche Diagnostics, Risch-Rotkreuz, Switzerland); SepsiTest(TM) (Molzym Molecular Diagnostics, Bremen, Germany); and the IRIDICA BAC BSI assay (Abbott Diagnostics, Lake Forest, IL, USA)] for the rapid identification of bloodstream bacteria and fungi in patients with suspected sepsis compared with standard practice (blood culture with or without matrix-absorbed laser desorption/ionisation time-of-flight mass spectrometry). DATA SOURCES Thirteen electronic databases (including MEDLINE, EMBASE and The Cochrane Library) were searched from January 2006 to May 2015 and supplemented by hand-searching relevant articles. REVIEW METHODS A systematic review and meta-analysis of effectiveness studies were conducted. A review of published economic analyses was undertaken and a de novo health economic model was constructed. A decision tree was used to estimate the costs and quality-adjusted life-years (QALYs) associated with each test; all other parameters were estimated from published sources. The model was populated with evidence from the systematic review or individual studies, if this was considered more appropriate (base case 1). In a secondary analysis, estimates (based on experience and opinion) from seven clinicians regarding the benefits of earlier test results were sought (base case 2). A NHS and Personal Social Services perspective was taken, and costs and benefits were discounted at 3.5% per annum. Scenario analyses were used to assess uncertainty. RESULTS For the review of diagnostic test accuracy, 62 studies of varying methodological quality were included. A meta-analysis of 54 studies comparing SeptiFast with blood culture found that SeptiFast had an estimated summary specificity of 0.86 [95% credible interval (CrI) 0.84 to 0.89] and sensitivity of 0.65 (95% CrI 0.60 to 0.71). Four studies comparing SepsiTest with blood culture found that SepsiTest had an estimated summary specificity of 0.86 (95% CrI 0.78 to 0.92) and sensitivity of 0.48 (95% CrI 0.21 to 0.74), and four studies comparing IRIDICA with blood culture found that IRIDICA had an estimated summary specificity of 0.84 (95% CrI 0.71 to 0.92) and sensitivity of 0.81 (95% CrI 0.69 to 0.90). Owing to the deficiencies in study quality for all interventions, diagnostic accuracy data should be treated with caution. No randomised clinical trial evidence was identified that indicated that any of the tests significantly improved key patient outcomes, such as mortality or duration in an intensive care unit or hospital. Base case 1 estimated that none of the three tests provided a benefit to patients compared with standard practice and thus all tests were dominated. In contrast, in base case 2 it was estimated that all cost per QALY-gained values were below £20,000; the IRIDICA BAC BSI assay had the highest estimated incremental net benefit, but results from base case 2 should be treated with caution as these are not evidence based. LIMITATIONS Robust data to accurately assess the clinical effectiveness and cost-effectiveness of the interventions are currently unavailable. CONCLUSIONS The clinical effectiveness and cost-effectiveness of the interventions cannot be reliably determined with the current evidence base. Appropriate studies, which allow information from the tests to be implemented in clinical practice, are required. STUDY REGISTRATION This study is registered as PROSPERO CRD42015016724. FUNDING The National Institute for Health Research Health Technology Assessment programme.

Accuracy of LightCycler® SeptiFast for the detection and identification of pathogens in the blood of patients with suspected sepsis: a systematic review protocol

Background There is growing interest in the potential utility of molecular diagnostics in improving the detection of life-threatening infection (sepsis). LightCycler® SeptiFast is a multipathogen probe-based real-time PCR system targeting DNA sequences of bacteria and fungi present in blood samples within a few hours. We report here the protocol of the first systematic review of published clinical diagnostic accuracy studies of this technology when compared with blood culture in the setting of suspected sepsis. Methods/design Data sources: the Cochrane Database of Systematic Reviews, the Database of Abstracts of Reviews of Effects (DARE), the Health Technology Assessment Database (HTA), the NHS Economic Evaluation Database (NHSEED), The Cochrane Library, MEDLINE, EMBASE, ISI Web of Science, BIOSIS Previews, MEDION and the Aggressive Research Intelligence Facility Database (ARIF). Study selection: diagnostic accuracy studies that compare the real-time PCR technology with standard culture results performed on a patients blood sample during the management of sepsis. Data extraction: three reviewers, working independently, will determine the level of evidence, methodological quality and a standard data set relating to demographics and diagnostic accuracy metrics for each study. Statistical analysis/data synthesis: heterogeneity of studies will be investigated using a coupled forest plot of sensitivity and specificity and a scatter plot in Receiver Operator Characteristic (ROC) space. Bivariate model method will be used to estimate summary sensitivity and specificity. The authors will investigate reporting biases using funnel plots based on effective sample size and regression tests of asymmetry. Subgroup analyses are planned for adults, children and infection setting (hospital vs community) if sufficient data are uncovered. Dissemination Recommendations will be made to the Department of Health (as part of an open-access HTA report) as to whether the real-time PCR technology has sufficient clinical diagnostic accuracy potential to move forward to efficacy testing during the provision of routine clinical care. Registration PROSPERO—NIHR Prospective Register of Systematic Reviews (CRD42011001289).

Rapid detection of health-care-associated bloodstream infection in critical care using multipathogen real-time polymerase chain reaction technology: a diagnostic accuracy study and systematic review.

BACKGROUND There is growing interest in the potential utility of real-time polymerase chain reaction (PCR) in diagnosing bloodstream infection by detecting pathogen deoxyribonucleic acid (DNA) in blood samples within a few hours. SeptiFast (Roche Diagnostics GmBH, Mannheim, Germany) is a multipathogen probe-based system targeting ribosomal DNA sequences of bacteria and fungi. It detects and identifies the commonest pathogens causing bloodstream infection. As background to this study, we report a systematic review of Phase III diagnostic accuracy studies of SeptiFast, which reveals uncertainty about its likely clinical utility based on widespread evidence of deficiencies in study design and reporting with a high risk of bias. OBJECTIVE Determine the accuracy of SeptiFast real-time PCR for the detection of health-care-associated bloodstream infection, against standard microbiological culture. DESIGN Prospective multicentre Phase III clinical diagnostic accuracy study using the standards for the reporting of diagnostic accuracy studies criteria. SETTING Critical care departments within NHS hospitals in the north-west of England. PARTICIPANTS Adult patients requiring blood culture (BC) when developing new signs of systemic inflammation. MAIN OUTCOME MEASURES SeptiFast real-time PCR results at species/genus level compared with microbiological culture in association with independent adjudication of infection. Metrics of diagnostic accuracy were derived including sensitivity, specificity, likelihood ratios and predictive values, with their 95% confidence intervals (CIs). Latent class analysis was used to explore the diagnostic performance of culture as a reference standard. RESULTS Of 1006 new patient episodes of systemic inflammation in 853 patients, 922 (92%) met the inclusion criteria and provided sufficient information for analysis. Index test assay failure occurred on 69 (7%) occasions. Adult patients had been exposed to a median of 8 days (interquartile range 4-16 days) of hospital care, had high levels of organ support activities and recent antibiotic exposure. SeptiFast real-time PCR, when compared with culture-proven bloodstream infection at species/genus level, had better specificity (85.8%, 95% CI 83.3% to 88.1%) than sensitivity (50%, 95% CI 39.1% to 60.8%). When compared with pooled diagnostic metrics derived from our systematic review, our clinical study revealed lower test accuracy of SeptiFast real-time PCR, mainly as a result of low diagnostic sensitivity. There was a low prevalence of BC-proven pathogens in these patients (9.2%, 95% CI 7.4% to 11.2%) such that the post-test probabilities of both a positive (26.3%, 95% CI 19.8% to 33.7%) and a negative SeptiFast test (5.6%, 95% CI 4.1% to 7.4%) indicate the potential limitations of this technology in the diagnosis of bloodstream infection. However, latent class analysis indicates that BC has a low sensitivity, questioning its relevance as a reference test in this setting. Using this analysis approach, the sensitivity of the SeptiFast test was low but also appeared significantly better than BC. Blood samples identified as positive by either culture or SeptiFast real-time PCR were associated with a high probability (> 95%) of infection, indicating higher diagnostic rule-in utility than was apparent using conventional analyses of diagnostic accuracy. CONCLUSION SeptiFast real-time PCR on blood samples may have rapid rule-in utility for the diagnosis of health-care-associated bloodstream infection but the lack of sensitivity is a significant limiting factor. Innovations aimed at improved diagnostic sensitivity of real-time PCR in this setting are urgently required. Future work recommendations include technology developments to improve the efficiency of pathogen DNA extraction and the capacity to detect a much broader range of pathogens and drug resistance genes and the application of new statistical approaches able to more reliably assess test performance in situation where the reference standard (e.g. blood culture in the setting of high antimicrobial use) is prone to error. STUDY REGISTRATION The systematic review is registered as PROSPERO CRD42011001289. FUNDING The National Institute for Health Research Health Technology Assessment programme. Professor Daniel McAuley and Professor Gavin D Perkins contributed to the systematic review through their funded roles as codirectors of the Intensive Care Foundation (UK).

Genotypic subgrouping of clinical isolates of Candida albicans and Candida dubliniensis by 25S intron analysis

Aims: To determine the frequency, distribution and association of genotypes of Candida albicans and C. dubliniensis in invasive and noninvasive clinical isolates.

Comparison of API20C with molecular identification of Candida spp isolated from bloodstream infections

A study was carried out to compare the API20C technology with polymerase chain reaction amplification and direct sequencing of the short internal transcribed spacer region 2 (ITS2) for the identification of 58 isolates of invasive candida species obtained from patients with bloodstream infections over the seven year period 1994 to 2000. Overall, there was only one disagreement between the phenotypic and genotypic identification, where the API scheme identified the isolate as C albicans but the molecular method identified it as C dubliniensis. This study demonstrated that the API20C method is useful in the identification of Candida spp isolated from blood culture and that molecular methods do not enhance identifications made using the API20C scheme. However, for correct reporting of C dubliniensis, an emerging bloodborne pathogen, it is recommended that all isolates identified as C albicans by the API20C scheme are further examined phenotypically and/or genotypically.

Resource utilisation, length of hospital stay, and pattern of investigation during acute medical hospital admission

Objectives: To describe the patient demographic characteristics and organisational factors that influence length of stay (LOS) among emergency medical admissions. Also, to describe differences in investigation practice among consultant physicians and to examine the impact of these on LOS. Design: Prospective observational study. Setting: General medicine department of a teaching hospital in Belfast, UK. Participants: Data were recorded for patients who were admitted as emergencies and reviewed on the post-take ward rounds (PTWR) attended by the investigation coordinator. Outcome measures: Non-laboratory investigations requested, LOS, and diagnosis on discharge. Results: Of 830 episodes evaluated, the median LOS was 7 days (interquartile range 3–12 days); this was significantly longer for admissions on Fridays (p = 0.0011) and for patients managed on medical wards (p<0.0001). There was a positive correlation between patient age and LOS (r = 0.32, p<0.0001). Chest radiographs (p = 0.002) and echocardiography (p = 0.015) were associated with a prolonged LOS; no investigations were associated with a shortened LOS. Diagnoses of congestive heart failure, respiratory disease, and cancer were associated with a longer LOS; a diagnosis of angina was associated with a shorter LOS. Considerable variation in investigation ordering, but no difference in LOS, was observed between consultants. High use of a given medical test did not correlate with high use of other tests. Conclusion: A systematic means of dealing with the NHS resource crisis should include an improved organisational strategy as well as social care provision. A more unified approach to investigation practice should also have a sparing effect on resources.

Diagnostic accuracy of pulmonary host inflammatory mediators in the exclusion of ventilator-acquired pneumonia

Background Excessive use of empirical antibiotics is common in critically ill patients. Rapid biomarker-based exclusion of infection may improve antibiotic stewardship in ventilator-acquired pneumonia (VAP). However, successful validation of the usefulness of potential markers in this setting is exceptionally rare. Objectives We sought to validate the capacity for specific host inflammatory mediators to exclude pneumonia in patients with suspected VAP. Methods A prospective, multicentre, validation study of patients with suspected VAP was conducted in 12 intensive care units. VAP was confirmed following bronchoscopy by culture of a potential pathogen in bronchoalveolar lavage fluid (BALF) at >104 colony forming units per millilitre (cfu/mL). Interleukin-1 beta (IL-1β), IL-8, matrix metalloproteinase-8 (MMP-8), MMP-9 and human neutrophil elastase (HNE) were quantified in BALF. Diagnostic utility was determined for biomarkers individually and in combination. Results Paired BALF culture and biomarker results were available for 150 patients. 53 patients (35%) had VAP and 97 (65%) patients formed the non-VAP group. All biomarkers were significantly higher in the VAP group (p<0.001). The area under the receiver operator characteristic curve for IL-1β was 0.81; IL-8, 0.74; MMP-8, 0.76; MMP-9, 0.79 and HNE, 0.78. A combination of IL-1β and IL-8, at the optimal cut-point, excluded VAP with a sensitivity of 100%, a specificity of 44.3% and a post-test probability of 0% (95% CI 0% to 9.2%). Conclusions Low BALF IL-1β in combination with IL-8 confidently excludes VAP and could form a rapid biomarker-based rule-out test, with the potential to improve antibiotic stewardship.

Tea tree oil (5%) body wash versus standard care (Johnson's Baby Softwash) to prevent colonization with methicillin-resistant Staphylococcus aureus in critically ill adults: a randomized controlled trial

OBJECTIVES To determine whether the daily use of 5% tea tree oil (TTO) body wash (Novabac 5% Skin Wash) compared with standard care [Johnsons Baby Softwash (JBS)] had a lower incidence of methicillin-resistant Staphylococcus aureus (MRSA) colonization. PATIENTS The study setting was two intensive care units (ICUs; mixed medical, surgical and trauma) in Northern Ireland between October 2007 and July 2009. The study population comprised 391 patients who were randomized to JBS or TTO body wash. METHODS This was a Phase 2/3, prospective, open-label, randomized, controlled trial. TRIAL REGISTRATION ISRCTN65190967. The primary outcome was new MRSA colonization during ICU stay. Secondary outcomes included the incidence of MRSA bacteraemia and maximum increase in sequential organ failure assessment score. RESULTS A total of 445 patients were randomized to the study. After randomization, 54 patients were withdrawn; 30 because of a positive MRSA screen at study entry, 11 due to lack of consent, 11 were inappropriately randomized and 2 had adverse reactions. Thirty-nine (10%) patients developed new MRSA colonization (JBS n = 22, 11.2%; TTO body wash n = 17, 8.7%). The difference in percentage colonized (2.5%, 95% CI - 8.95 to 3.94; P = 0.50) was not significant. The mean maximum increase in sequential organ failure assessment score was not significant (JBS 1.44, SD 1.92; TTO body wash 1.28, SD 1.79; P = 0.85) and no study patients developed MRSA bacteraemia. CONCLUSIONS Compared with JBS, TTO body wash cannot be recommended as an effective means of reducing MRSA colonization.