Shy-Shin Chang

Multiplex PCR System for Rapid Detection of Pathogens in Patients with Presumed Sepsis – A Systemic Review and Meta-Analysis

Background Blood culture is viewed as the golden standard for the diagnosis of sepsis but suffers from low sensitivity and long turnaround time. LightCycler SeptiFast (LC-SF) is a real-time multiplex polymerase chain reaction test able to detect 25 common pathogens responsible for bloodstream infections within hours. We aim to assess the accuracy of LC-SF by systematically reviewing the published studies. Method Related literature on Medline, Embase, and Cochrane databases was searched up to October 2012 for studies utilizing LC-SF to diagnose suspected sepsis and that provided sufficient data to construct two-by-two tables. Results A total of 34 studies enrolling 6012 patients of suspected sepsis were included. The overall sensitivity and specificity for LC-SF to detect bacteremia or fungemia was 0·75 (95% CI: 0·65–0·83) and 0·92 (95%CI:0·90–0·95), respectively. LC-SF had a high positive likelihood ratio (10·10) and a moderate negative likelihood ratio (0·27). Specifically, LC-SF had a sensitivity of 0·80 (95%CI: 0·70–0·88) and a specificity of 0·95(95%CI: 0·93–0·97) for the bacteremia outcome, and a sensitivity of 0·61 (95%CI: 0·48–0·72) and a specificity of 0·99 (95%CI: 0·99–0·99) for the fungemia outcome. High heterogeneity was found in the bacteremia outcome subgroup but not in the fungemia outcome subgroup. Conclusion LC-SF is of high rule-in value for early detection of septic patients. In a population with low pretest probability, LC-SF test can still provide valuable information for ruling out bacteremia or fungemia.

Comparison of the Test Characteristics of Procalcitonin to C-Reactive Protein and Leukocytosis for the Detection of Serious Bacterial Infections in Children Presenting With Fever Without Source: A Systematic Review and Meta-analysis

STUDY OBJECTIVE We determine the usefulness of the procalcitonin for early identification of young children at risk for severe bacterial infection among those presenting with fever without source. METHODS The design was a systematic review and meta-analysis of diagnostic studies. Data sources were searches of MEDLINE and EMBASE in April 2011. Included were diagnostic studies that evaluated the diagnostic value of procalcitonin alone or compared with other laboratory markers, such as C-reactive protein or leukocyte count, to detect severe bacterial infection in children with fever without source who were aged between 7 days and 36 months. RESULTS Eight studies were included (1,883 patients) for procalcitonin analysis, 6 (1,265 patients) for C-reactive protein analysis, and 7 (1,649 patients) for leukocyte analysis. The markers differed in their ability to predict serious bacterial infection: procalcitonin (odds ratio [OR] 10.6; 95% confidence interval [CI] 6.9 to 16.0), C-reactive protein (OR 9.83; 95% CI 7.05 to 13.7), and leukocytosis (OR 4.26; 95% CI 3.22 to 5.63). The random-effect model was used for procalcitonin analysis because heterogeneity across studies existed. Overall sensitivity was 0.83 (95% CI 0.70 to 0.91) for procalcitonin, 0.74 (95% CI 0.65 to 0.82) for C-reactive protein, and 0.58 (95% CI 0.49 to 0.67) for leukocyte count. Overall specificity was 0.69 (95% CI 0.59 to 0.85) for procalcitonin, 0.76 (95% CI 0.70 to 0.81) for C-reactive protein, and 0.73 (95% CI 0.67 to 0.77) for leukocyte count. CONCLUSION Procalcitonin performs better than leukocyte count and C-reactive protein for detecting serious bacterial infection among children with fever without source. Considering the poor pooled positive likelihood ratio and acceptable pooled negative likelihood ratio, procalcitonin is better for ruling out serious bacterial infection than for ruling it in. Existing studies do not define how best to combine procalcitonin with other clinical information.

Rapid Differentiation of Influenza A Virus Subtypes and Genetic Screening for Virus Variants by High-Resolution Melting Analysis

ABSTRACT We assessed the use of high-resolution melting (HRM) analysis for the rapid identification of influenza A virus subtypes and the detection of newly emerging virus variants. The viral matrix gene was amplified by LightCycler real-time reverse transcription-PCR (RT-PCR) in the presence of the LCGreen I fluorescent dye. Upon optimization of the assay conditions, all the major influenza A virus subtypes, including H1N1, H3N2, H5N1, H7N3, and H9N2, were amplifiable by this method and had a PCR product length of 179 bp. Real-time RT-PCR of in vitro-transcribed H3N2 RNA revealed a standard curve for quantification with a linear range (correlation coefficient = 0.9935) across at least 8 log units of RNA concentrations and a detection limit of 103 copies of viral RNA. We performed HRM analysis of the PCR products with the HR-1 instrument and used the melting profiles as molecular fingerprints for virus subtyping. The virus subtypes were identified from the high-resolution derivative plot obtained by heteroduplex formation between the PCR products of the viral isolates tested and those of the reference viral isolates. The melting profiles were consistent with minimal interassay variability. Hence, an HRM database and a working protocol were established for the identification of these five influenza A virus subtypes. When this protocol was used to test 21 clinical influenza A virus isolates, the results were comparable to those obtained by RT-PCR with hemagglutinin-specific primer sets. Sequence variants of the clinical isolates (n = 4) were also revealed by our HRM analytical scheme. This assay requires no multiplexing or hybridization probes and provides a new approach for influenza A virus subtyping and genetic screening of virus variants in a clinical virology laboratory.

Use of serum procalcitonin to detect bacterial infection in patients with autoimmune diseases: A systematic review and meta‐analysis

OBJECTIVE To systematically review evidence of the accuracy of the procalcitonin test for diagnosis of bacterial infection in patients with autoimmune disease. METHODS The major databases Medline, EMBase, and the Cochrane Library were searched for studies published between January 1966 and October 2011 that evaluated procalcitonin, alone or in comparison with other laboratory markers such as C-reactive protein (CRP), as a diagnostic marker for bacterial infection in patients with autoimmune disease and provided sufficient data to permit construction of 2 × 2 tables. RESULTS Nine studies were included in the final meta-analysis. The area under the summary receiver operating characteristic curve values were 0.91 (95% confidence interval [95% CI] 0.88-0.93) for procalcitonin and 0.81 (95% CI 0.78-0.84) for CRP. In general, testing for procalcitonin was highly specific for identifying infectious complications, although it was not as sensitive as testing for CRP. Pooled sensitivity was 0.75 (95% CI 0.63-0.84) for procalcitonin tests and 0.77 (95% CI 0.67-0.85) for CRP tests. Pooled specificity was 0.90 (95% CI 0.85-0.93) for procalcitonin tests and 0.56 (95% CI 0.25-0.83) for CRP tests. The positive likelihood ratio for procalcitonin (7.28 [95% CI 5.10-10.38]) was sufficiently high to qualify procalcitonin testing as a rule-in diagnostic tool, while the negative likelihood ratio (0.28 [95% CI 0.18-0.40]) was not sufficiently low to qualify procalcitonin testing as a reliable rule-out diagnostic tool. CONCLUSION Procalcitonin has higher diagnostic value than CRP for the detection of bacterial sepsis in patients with autoimmune disease, and the test for procalcitonin is more specific than sensitive. A procalcitonin test is not recommended to be used in isolation as a rule-out tool.

Comparison of clinical characteristics and performance of pneumonia severity score and CURB-65 among younger adults, elderly and very old subjects

Background Age-related alterations in the clinical characteristics and performance of severity scoring systems for community-acquired pneumonia (CAP) are unknown. Methods Consecutive patients with CAP presenting to the emergency department were prospectively studied. Patients were classified as younger adults (age 18–64 years), elderly (age 65–84 years) and very old subjects (age ≥85 years). Clinical characteristics, complications, outcomes and validity of the pneumonia severity index (PSI) and CURB-65 categories were compared across these three age categories. Results Analysis involved 348 (35.3%) younger adult patients, 438 (44.3%) elderly patients and 201 (20.0%) very old patients. Compared with younger adults, elderly and very old patients had a higher burden of comorbidities and a higher incidence of CAP-related complications. The 30-day mortality rate was 5.2% in younger adults, 7.1% in elderly patients and 9.5% in very old patients. The area under the ROC curve (AUCs) for PSI were 0.87 (95% CI 0.77 to 0.97), 0.85 (95% CI 0.803 to 0.897) and 0.69 (95% CI 0.597 to 0.787) and the AUCs for CURB-65 were 0.80 (95% CI 0.67 to 0.93), 0.73 (95% CI 0.65 to 0.82) and 0.60 (95% CI 0.47 to 0.73) in the younger adult, elderly and very old patients, respectively. A modified PSI or CURB-65 excluding the age variable increased the AUC in most age categories. There was no significant effect of age on 30-day mortality after adjusting for other PSI or CURB-65 variables. Conclusion Elderly patients with CAP have more atypical clinical manifestations and worse outcomes. The underperformance of the PSI in elderly patients may be due to the inappropriate weight given to the age variable. A modification of the cut-off point for PSI or CURB-65 to define severe pneumonia may improve the score performance in elderly patients.

Diagnostic value of procalcitonin for bacterial infection in elderly patients – a systemic review and meta-analysis

To summarise evidence for the diagnostic accuracy of procalcitonin (PCT) tests for identifying systemic bacterial infections in elderly patients.

Procalcitonin as a Biomarker for Bacterial Infections in Patients With Liver Cirrhosis in the Emergency Department

OBJECTIVES The objective was to determine the diagnostic accuracy of procalcitonin measurement for bacterial infections in patients with all causes of liver cirrhosis. METHODS The authors conducted a cross-sectional study of 98 patients with cirrhosis treated in the emergency department (ED) of Chang-Gung Memorial Hospital, Taiwan. Serum procalcitonin levels and other clinical information were obtained concurrently. Patients were assigned to a sepsis or nonsepsis group after the medical records were reviewed by two emergency physicians blinded to the study. Receiver operating characteristic (ROC) curve analysis was conducted to determine the sensitivity, specificity, likelihood ratio, and suggested cutoff values. The diagnostic accuracy of the C-reactive protein (CRP) level was also determined for comparison. RESULTS A total of 98 patients were enrolled for analysis in 1 year. Twenty-seven patients (27.6%) were assigned to the sepsis group. Eleven patients (11.2%) had positive blood cultures. The areas under the ROC curves for procalcitonin and CRP in predicting sepsis were 0.89 (95% confidence interval [CI] = 0.77 to 0.92) and 0.81 (95% CI = 0.72 to 0.89), respectively (p = 0.11). The cutoff that maximized Youdens index was 0.49 ng/mL for procalcitonin and 24.7 mg/L for CRP. At these cutoffs, the sensitivity and specificity were 81.5 and 87.3% for procalcitonin and 80.0 and 80.3% for CRP. These results suggest that procalcitonin measurement shows at least an equivalent diagnostic accuracy to CRP measurement. CONCLUSIONS Procalcitonin provided satisfactory diagnostic accuracy in differentiating bacterial infections in patients with all causes of liver cirrhosis in the ED. A cutoff value of 0.5 ng/mL is suggested for clinical use.

Broad-Range Ribosomal RNA Real-Time PCR after Removal of DNA from Reagents: Melting Profiles for Clinically Important Bacteria

With the ability to exponentially amplify regions of DNA, two different PCR-based strategies have been developed for nonculture diagnosis of bacteremia. The first approach targets the species-specific genes for amplification (1), whereas the second approach involves universal PCR amplification of conserved bacterial DNA sequences, such as the 16S rRNA, the 23S rRNA, and the 16S-23S rRNA interspace regions (2)(3)(4). Although universal PCR is not able to distinguish bacteria to the species level, numerous studies have shown that this method provides valuable information complementary to the results of time-consuming and subjective phenotypic tests used in detection of bacterial infection (5) and can be used to differentiate bacterial from viral or other infections (6)(7). In clinical applications, real-time PCR for broad-range amplification of bacterial DNA sequences could offer additional benefits: it is less labor-intensive, less time-consuming, and reduces the risk of PCR carryover contamination. A major obstacle for broad-range PCR amplification is the presence of bacterial DNA in the Taq DNA polymerase and real-time PCR master mixture (8)(9)(10). The contaminating DNA is effectively amplified, giving rise to false-positive results. Efforts have been taken to eliminate contaminating DNA, including the use of Sau 3AI restriction endonuclease (11), DNase I (12)(13), and ultraviolet irradiation (7)(8). Although these strategies may be effective for conventional PCR, the study by Corless et al. (14) indicated that the contamination issue could not be avoided without affecting sensitivity when TaqMan probe-based real-time PCR is performed. Therefore, an appropriate method for removal of contaminating bacterial DNA and subsequent real-time amplification is required. In this study, we address this issue and report optimized conditions for broad-range amplification of the bacterial 16S rRNA gene by real-time PCR using SYBR Green I dye (15) as the fluorescent signal. Bacterial …

Usefulness of natriuretic peptide for the diagnosis of Kawasaki disease: a systematic review and meta-analysis

Objective To examine the diagnostic value of serum B-type natriuretic peptide (BNP) in acute Kawasaki disease (KD). Design Systematic review and meta-analysis. Data sources A systematic literature search strategy was designed and carried out using MEDLINE, EMBASE and the Cochrane Library from inception to December 2013. We also performed manual screening of the bibliographies of primary studies and review articles, and contacted authors for additional data. Study eligibility criteria We included all BNP and NT-pro (N-terminal prohormone) BNP assay studies that compared paediatric patients with KD to patients with febrile illness unrelated to KD. We excluded case reports, case series, review articles, editorials, congress abstracts, clinical guidelines and all studies that compared healthy controls. Primary and secondary outcome measures The performance characteristics of BNP were summarised using forest plots, hierarchical summary receiver operating characteristic (ROC) curves and bivariate random effects models. Results We found six eligible studies including 279 cases of patients with KD and 203 febrile controls. Six studies examined NT-proBNP and one examined BNP. In general, NT-proBNP is a specific and moderately sensitive test for identifying KD. The pooled sensitivity was 0.89 (95% CI 0.78 to 0.95) and the pooled specificity was 0.72 (95% CI 0.58 to 0.82). The area under the summary ROC curve was 0.87 (95% CI 0.83 to 0.89). The positive likelihood ratio (LR+ 3.20, 95% CI 2.10 to 4.80) was sufficiently high to be qualified as a rule-in diagnostic tool in the context of high pre-test probability and compatible clinical symptoms. A high degree of heterogeneity was found using the Cochran Q statistic. Conclusions Current evidence suggests that NT-proBNP may be used as a diagnostic tool for KD. NT-proBNP has high diagnostic value for identifying KD in patients with protracted undifferentiated febrile illness. Prospective large cohort studies are needed to help determine best cut-off values and further clarify the role of NT-proBNP in the diagnosis process of KD.

Inflammatory markers in cord blood or maternal serum for early detection of neonatal sepsis—a systemic review and meta-analysis

Objective:To perform a quantitative review of the evidence on the diagnostic value of inflammatory markers in maternal serum or umbilical cord blood for the diagnosis of early-onset neonatal sepsis (EONS).Study Design:We searched multiple databases for studies published through March 2013 that evaluated the diagnostic performance of procalcitonin (PCT), C-reactive protein (CRP) and interleukin-6 (IL-6), and leukocyte count (white blood cell, WBC) in either umbilical cord blood or maternal serum for diagnosis of EONS. We summarized test performance characteristics with the use of forest plots, hierarchical summary receiver operating characteristic curves and bivariate random effects models.Result:Our search identified 3874 citations, of which 15 studies evaluating 2178 episodes of suspected neonatal infection were included for analysis. IL-6 in cord blood with a pooled-positive likelihood ratio (LR+) of 9.47 (95% confidence interval: 3.86 to 23.3), PCT in cord blood with a LR+ of 5.72 (1.56 to 21.0) and IL-6 in maternal serum with a LR+ of 5.47 (2.10 to 14.2) can be qualified as a valid rule-in test. IL-6 in cord blood with a LR− of 0.10 (0.05 to 0.21) and PCT in cord blood with a LR− of 0.20 (0.12–0.37) can be qualified as a useful rule-out test. Either CRP or WBC was inadequate for diagnosis of EONS.Conclusion:For cord blood sample, IL-6 or PCT can be used as reliable rule-in and rule-out tool. For maternal serum, only IL-6 appeared to be sufficient for rule-in diagnosis. An interventional study may be needed to answer whether the addition of these tests will improve the outcome of patients with EONS.