Evaluation of interferon-γ release assay in the diagnosis of osteoarticular tuberculosis

The aim of this study was to assess the value of interferon-γ (IFN-γ) release assay (IGRA) (T-SPOT.TB) for patients with suspected osteoarticular tuberculosis (TB) in comparison with conventional and molecular methods. Of 145 patients with suspected osteoarticular TB, recruited from Beijing Chest Hospital between July 2011 and June 2012, 86 (59.3%)had osteoarticular TB (26 with culture-confirmed TB, 60 with probable TB), 24 (16.6%) were not having active TB. The remaining 17 (11.7%) inconclusive TB and 18 (12.4%) possible TB were excluded from final analysis. In addition to conventional tests and molecular method, T-SPOT.TB assay using peripheral blood mononuclear cells to examine IFN-γ response to early secretory antigenic target 6 and culture filtrate protein 10 was also performed. The sensitivity and specificity for T-SPOT.TB assay were 94.2% and 70.8%, respectively. A statistically significant difference in sensitivity was found between T-SPOT.TB assay (94.2%) and other tests (acid-fast bacilli smear (19.7%), culture (34.2%), real-time PCR (36.8%); P < 0.01, respectively). These results suggested that the IGRA assay could provide useful aids in the diagnosis of osteoarticular TB.     

Analytical performances of the Diazyme ADA assay on the Cobas® 6000 system

ObjectiveTo evaluate the analytical performance of the Diazyme ADA assay on the Cobas^® 6000 system for pleural fluid samples analysis.Design and methodsImprecision, linearity, calibration curve stability, interference, and correlation studies were completed.ResultsThe Diazyme ADA assay demonstrated excellent precision (CV < 4%) over the analytical measurement range (0.5-117 U/L). Bilirubin above 50 μmol/L and haemoglobin above 177 μmol/L interfered with the test, inducing a negative and a positive interference respectively. The Diazyme ADA assay correlated well with the Giusti method (r² = 0.93) but exhibited a negative bias (~ ?30%).ConclusionsThe Diazyme ADA assay on the Cobas^® 6000 system represents a rapid, accurate, precise and reliable method for determination of ADA activity in pleural fluid samples.     

QuantiFERON®-CMV assay for the assessment of cytomegalovirus cell-mediated immunity

Cytomegalovirus (CMV) infection has historically been a major complication among immunocompromised patients, such as solid-organ and stem-cell transplant recipients and patients with advanced HIV infection. While the introduction of antiretroviral therapy has almost eradicated CMV infection in HIV-infected patients, CMV disease remains a significant problem in transplant recipients once antiviral prophylaxis is discontinued. QuantiFERON(?)-CMV allows the assessment of cellular immunity against CMV by detecting the production of IFN-γ following in vitro stimulation with CMV antigens. Preliminary studies have shown a correlation between a lack of detectable cell-mediated immunity measured by the QuantiFERON-CMV assay and a higher incidence of CMV infection and disease in immunocompromised patients. Measurement of cell-mediated immunity against CMV appears to be a promising strategy to identify patients at highest risk for the development of CMV disease and, therefore, to individualize preventive strategies for CMV in transplant recipients.     

Diagnosis of extrapulmonary tuberculosis using the Xpert(®) MTB/RIF assay

Evaluation of: Tortoli E, Russo C, Piersimoni C et al. Clinical validation of Xpert MTB/RIF for the diagnosis of extrapulmonary tuberculosis. Eur. Respir. J. doi:10.1183/09031936.00176311 (2012) (Epub ahead of print). The Xpert? MTB/RIF assay has been CE-marked for rapid molecular diagnosis of TB in Europe and has been endorsed by the WHO as a replacement for sputum smear microscopy for diagnosis of pulmonary TB in low- and middle-income countries. However, few data are available to inform recommendations for use of the assay for testing nonsputum clinical samples when investigating suspected extrapulmonary TB (EPTB). We review and discuss the findings of Tortoli and colleagues, who evaluated the assay used for this purpose in a large study of adults and children in Italy. They provide a per-sample analysis of 268 diagnoses of EPTB at a range of anatomic sites (sensitivity: 81.3%; 95% CI: 76.2-85.8) and data for 1206 samples in which EPTB was excluded (specificity: 99.8%; 95% CI: 99.4-100). We discuss how this paper forms an important addition to the growing body of literature demonstrating the utility of Xpert MTB/RIF for EPTB diagnosis when applied to diverse types of clinical samples     

Prospective evaluation of the Meridian Illumigene™ loop-mediated amplification assay and the Gen Probe ProGastro™ Cd polymerase chain reaction assay for the direct detection of toxigenic Clostridium difficile from fecal samples

Clostridium difficile is the most common and important cause of toxigenic colitis in the health care setting. Laboratory diagnostics have included bacterial culture with further identification of toxigenic stains, or more commonly, direct detection of preformed toxin in stool samples using biological or immunochemistry assays. Recently, molecular amplification assays for the direct detection of toxin-encoding genes have become available commercially. We prospectively evaluated 2 FDA-cleared molecular amplification tests, the Illumigene C. difficile and the ProGastro Cd PCR assay, for the direct detection of toxigenic C. difficile from fecal samples. Of 446 samples tested, 418 produced matching amplification results, 88 positive and 330 negative, and 13 resolved with repeat testing. Toxigenic culture and direct cytotoxin testing were used to resolve the remaining 15 discordant samples. Overall, each assay performed well and correctly identified 97% of positive samples.     

Diagnostic performance of whole-blood interferon-γ assay and enzyme-linked immunospot assay for active tuberculosis'

The aim of this study was to compare the diagnostic performance of 2 interferon-γ release assays, an enzyme-linked immunospot assay (T-SPOT.TB; Oxford Immunotec Ltd., Oxford, UK) and the QuantiFERON-TB Gold in-Tube assay (QFT-GIT; Cellestis Ltd., Carnegie, Australia), in patients with suspected active tuberculosis (TB). From October 2009 to October 2011, a total of 200 patients with suspected TB were enrolled. Clinical and microbiological characteristics of the patients were collected and blood samples were obtained for T-SPOT.TB and QFT-GIT assays. Among the 200 subjects, 98 (49%) had culture-confirmed TB, 18 (9%) had probable TB, and the remaining 84 (42%) subjects did not have TB. The sensitivity, specificity, positive predictive value, and negative predictive value for active TB diagnosis by the T SPOT. TB were 83%, 71%, 81%, and 75%, respectively. For QFT-GIT, the sensitivity, specificity, positive predictive value, and negative predictive value for active TB diagnosis were 66%, 76%, 80%, and 62%, respectively. The QFT-GIT assay resulted in more indeterminate and false-negative results than the T-SPOT.TB assay, especially in immunocompromised patients. In conclusion, T-SPOT.TB had a higher sensitivity and resulted in fewer indeterminate results than the QFT-GIT assay for diagnosing active TB.     

Selected performance characteristics of the Roche Elecsys® testosterone II assay on the Modular analytics E 170 analyzer

BackgroundSerum testosterone measurements have utility in diagnosis of clinical conditions characterized by both increased and decreased testosterone concentrations. Studies have indicated that testosterone immunoassays may give inaccurate results for women and children. We evaluated the performance of a second generation testosterone immunoassay from Roche Diagnostics.MethodsTesto II performed on a Modular Analytics E 170 analyzer is an automated random access electrochemiluminometric assay. We evaluated limit of blank (LoB), imprecision, linearity, interference, and method comparison with liquid chromatography-tandem mass spectrometry assay (LC-MS/MS). Method comparison included the current generation Roche testosterone assay (Testo I) and the Access 2 testosterone chemiluminometric assay (Beckman Coulter). Results for men and women were analyzed for analytic concordance. The relative % differences of immunoassay compared to LC-MS/MS results were evaluated.ResultsThe LoB was 0.07 nmol/l. Total imprecision was < 6%. The assay was linear from 0.2 to 46.6 nmol/l. Negative interference was observed for lipemia at concentrations > 22.5 g/l. Analytic concordance showed improved specificity for women. Comparison of results to LC-MS/MS indicated comparable performance with other immunoassays for men and improved performance for women, boys, and girls with mean differences of 0.5%, ? 0.7%, and 24.4%, respectively.ConclusionsThe Roche Testo II assay demonstrated excellent precision. Comparison to 2 other automated immunoassays showed comparable performance for men and improved performance for women and children. However, challenges still exist for quantifying testosterone concentrations < 10.4 nmol/l for men and < 1.7 nmol/l for women and children by immunoassay.     

Validation of a rapid liquid chromatography–tandem mass spectrometric assay for the determination of octreotide plasma concentrations

ObjectivesThe aim of this study was to develop and validate a fast liquid chromatography–tandem mass spectrometric (LC–MSMS) assay to determine circulating concentrations of octreotide (a somatostatin analog used in acromegaly and neuroendocrine tumors) in patients' plasma samples.Design and methods500 μL of heparin–plasma was used to extract the drug on a cation exchanger SPE column, and the eluate was injected into a LC–MSMS system. Reversed phase chromatography was performed on a phenyl grafted column in isocratic mode. Octreotide and internal standard (triptorelin) were identified in positive electrospray ionization mode using ion transitions of m/z 512 > 120 and 890 > 249, respectively.ResultsThe assay was linear in the concentration range of 0.5–25 ng/mL, with intra- and inter-assay imprecisions of < 10.6% and < 12.2%, respectively. The limits of quantification and detection were 0.5 and 0.25 ng/mL. The recovery and ion suppression effect ranged between 79.7 and 84.5% and between − 8.1 and − 21.3%, respectively. A subcutaneous injection of 0.1 mg octreotide induced a time- and patient-dependent surge of peptide concentrations peaking at 2 h.ConclusionThis fast, sensitive, and selective method for quantification of plasma octreotide by LC–MSMS might be used to investigate the pharmacokinetic–pharmacodynamic relationship, with potential contribution to treatment optimization and therapeutic drug monitoring application.     

Performance evaluation of the new ADVIA® Centaur system cyclosporine assay (single-step extraction)

BackgroundCyclosporine (CsA) monitoring is essential for transplant success. We report a performance study of the recently released, fully automated Siemens ADVIA Centaur^® CsA assay.MethodsWhole blood samples from 248 transplant patients were prepared using a new 1-step extraction method. Performance evaluations vs. HPLC–tandem MS (LC-MS/MS), Abbott TDx and AxSYM assays were conducted according to CLSI EP5-A2 and EP9-A2 guidelines.ResultsThe correlation coefficient for LC-MS/MS and ADVIA Centaur was ≥ 0.97 at each site, and for each transplant type. Regression analysis yielded y = 0.94x + 19 for all sites: 95% CI = 0.91–0.96 (slope) and 10–28 (intercept). Absolute and relative bias was minimal for C0 and C2 sampling. Centaur vs. Abbott TDx and AxSYM assays: y = 0.72x + 6, r = 0.98, 95% CI = 0.70–0.73 (slope), 3–9 (intercept); and y = 0.69x + 18, r = 0.97, 95% CI = 0.67–0.71 (slope), 8–27(intercept). Within run CVs were 4.5%–7.1%, total CVs were 5.3%–7.7%.ConclusionsThe ADVIA Centaur assay compared favorably with LC-MS/MS and Abbott assays, displaying good correlation for all transplant types and methods.     

Vitamin D status of county hospital patients assessed by the DiaSorin LIAISON® 25-hydroxyvitamin D assay

BackgroundVitamin D is an essential nutrient, and there is a growing appreciation of its clinical significance. In addition, there has recently been a discussion as to the best method of measuring this analyte in serum: immunoassay, HPLC or LC–MS/MS. Due to the increased interest in vitamin D, there has been an exponential increase in the number of clinicians testing their patients and therefore also in the volume of this test run in clinical laboratories.MethodsVitamin D levels were determined by chemiluminescence immunoassay in a reference laboratory and a subset by LC–MS/MS at San Francisco General Hospital.ResultsWe developed a robust and rapid LC–MS/MS assay to detect 25-hydroxyvitamin D3 and D2 in serum for use in the routine clinical laboratory. Additionally, we determined that 71% of patients served by San Francisco General Hospital have insufficient serum vitamin D levels (≤ 29 ng/ml) and that these levels are significantly associated with parathyroid hormone levels, total calcium concentration, age and ethnicity.ConclusionsThe high degree of vitamin D insufficiency at San Francisco General Hospital may be reflective of the status of the patients served by this county hospital; largely an underserved, multi-ethnic urban population.     

The role of the NxTAG® respiratory pathogen panel assay and other multiplex platforms in clinical practice

Introduction: The advent of nucleic acid amplification tests has significantly improved the aetiologic diagnosis of respiratory infections. However, multiplex real-time polymerase chain reaction (PCR) can be technologically challenging.

Areas covered: This paper reports the results of the main published studies on the NxTAG Respiratory Pathogen Panel (RPP) and discusses the advantages and disadvantages of extensive use of multiplex assays in clinical practice.

Expert commentary: Currently available data seem to indicate that routine use of multiplex assays, including NxTAG RPP Assay, should be recommended only when epidemiological data concerning circulation of viruses and bacteria have to be collected. Their use in clinical practice seems debatable. They have limited sensitivity and specificity at least in the identification of some infectious agents or, as in the case of NxTAG RPP, they have not been evaluated in a sufficient number of patients to allow definitive conclusions. In the future, the clinical relevance of multiplex assays, including NxTAG RPP, could significantly increase, mainly because a number of new antiviral agents effective against several respiratory viruses for which no drug is presently available will be marketed. In addition, it is highly likely that the efficiency of multiplex assays will be significantly improved.