Diagnosis of Coxiella burnetii infection: comparison of a whole blood interferon-gamma production assay and a Coxiella ELISPOT.

Diagnosis of ongoing or past infection with Coxiella burnetii, the causative agent of Q fever, relies heavily on serology: the measurement of C. burnetii-specific antibodies, reflecting the host's humoral immune response. However, cell-mediated immune responses play an important, probably even more relevant, role in infections caused by the intracellular C. burnetii bacterium. Recent studies have investigated interferon-gamma (IFN-γ) based assays, including a whole-blood IFN-γ production assay and a Coxiella enzyme-linked immunospot (Coxiella ELISPOT), as potential diagnostic tools for Q fever diagnosis. Both are in-house developed assays using stimulating antigens of different origin. The main objective of this study was to compare the test performance of the IFN-γ production assay and the Coxiella ELISPOT for detecting a cellular immune response to C. burnetii in Q fever patients, and to assess the correlation between both assays. To that end, both tests were performed in a well-defined patient group of chronic Q fever patients (n = 16) and a group of healthy seronegative individuals (n = 17). Among patients, both the Coxiella ELISPOT and the IFN-γ production assay detected positive response in 14/16. Among controls, none were positive in the Coxiella ELISPOT, whereas the IFN-γ production assay detected positive results in 1/17 and 3/17, when using Henzerling and Nine Mile as stimulating antigens, respectively. These results suggest the Coxiella ELISPOT has a somewhat higher specificity than the IFN-γ production assay when Nine Mile is used as antigen stimulus. The assays showed moderate correlation: the Spearman correlation coefficient r ranged between 0.37-0.60, depending on the antigens used. Further investigation of the diagnostic potential for C. burnetii infection of both assays is warranted.

Sarcoidosis and Purified Protein Derivative reactivity.

BACKGROUND: The possible association between (tuberculous and nontuberculous) mycobacterial infections and sarcoidosis is still a matter of dispute. Using diagnostic tests for specific T-cell responses, this association can be investigated in an innovative manner. OBJECTIVE: To measure the T-cell responsiveness to the purified protein derivative (PPD) antigen in blood and broncho-alveolar lavage (BAL) fluid in patients with sarcoidosis and patients with other causes of interstitial lung disease. It was hypothesized that if a mycobacterial infection of the lung is of importance for the development of sarcoidosis, T-cell responsiveness towards the PPD antigen would be increased in patients with sarcoidosis when compared to patients with other causes of interstitial lung disease. METHODS: A single-center study was conducted which included patients with and without sarcoidosis. Venous blood was collected and BAL was performed for, inter alia, Interferon Gamma Release Assay´s (IGRA) with different stimulating antigens, including PPD, ESAT-6, CFP-10 and, as a control, Epstein-Barr virus (EBV). RESULTS: A total of 118 patients were included. There is no difference between PPD reactivity in BAL fluid in patients with or without sarcoidosis. In patients without sarcoidosis, ELISpot PPD in blood shows more reactivity compared to patients with sarcoidosis, although this difference is not significant. ELISpot EBV and TB results are not significant different between both groups. CONCLUSION: These results provide no evidence for the involvement of different mycobacteria in the pathogenesis of sarcoidosis.

Added value of use of a purified protein derivative-based enzyme-linked immunosorbent spot assay for patients with Mycobacterium bovis BCG infection after intravesical BCG instillations.

In this case series, we describe four cases in which the use of gamma interferon release assays with purified protein derivative (PPD) as a stimulating antigen was able to demonstrate PPD-specific immune activation. This may help to improve the adequate diagnosis of (systemic) Mycobacterium bovis BCG infections after intravesical BCG instillations for bladder carcinoma.

Developing a new clinical tool for diagnosing chronic Q fever: the Coxiella ELISPOT.

Definitively establishing a clinical diagnosis of chronic Q fever remains challenging, as the diagnostic performance of both conventional serological tests and PCR is limited. Given the importance of an early diagnosis of chronic Q fever, there is a need for a reliable diagnostic test. We developed an enzyme-linked immunospot assay to measure Coxiella burnetii (C. burnetii)-specific T-cell responses (Coxiella ELISPOT) to both phase I and phase II antigens and tested convalescent Q fever patients (without chronic disease, n = 9) and patients with an established diagnosis of chronic Q fever (n = 3). The Coxiella ELISPOT adequately identified convalescent Q fever patients from healthy controls by demonstrating C. burnetii-specific T-cell interferon-γ production to both phase I and phase II antigens. Compared to convalescent Q fever patients, chronic Q fever patients showed a distinct Coxiella ELISPOT profile characterized by a much higher spot count for both phase I and phase II (18-fold for phase II, 8-fold higher for phase I) and a consistent shift towards more phase I reactivity. The diagnostic potential of the Coxiella ELISPOT is promising and warrants further investigation.

Variation in T-SPOT.TB spot interpretation between independent observers from different laboratories.

T-SPOT.TB is a specific assay for the diagnosis of tuberculosis. The assay needs to be performed with freshly isolated cells, and interpretation requires training. T-SPOT.TB has been used in various clinical-epidemiological settings, but so far no studies have evaluated the effect of interobserver variation in test reading. Our aim was to evaluate variation between different observers in reading T-SPOT.TB results. The study was nested within an ongoing cohort study, in which part of the T-SPOT.TB had been performed with frozen material. Culture plates were read visually by four different observers from two laboratories and by two automated readers. Of 313 T-SPOT.TB assays, 235 were performed with fresh cells and 78 were performed with frozen cells. No significant difference was found between results obtained with fresh cells and those obtained with frozen cells. The percentage of positive results varied between readers by maximally 15%; five/six raters were within a 6% difference in positive results. Analysis of the observed interrater differences showed that some individuals systematically counted more spots than others did. Because test interpretation includes subtraction of background values, this systematic variance had little influence on interindividual differences. The test result as positive or negative varied between independent raters, mainly due to samples with values around the cutoff. This warrants further study regarding determinants affecting the reading of T-SPOT.TB.

Follow-up study of tuberculosis-exposed supermarket customers with negative tuberculin skin test results in association with positive gamma interferon release assay results.

We report a follow-up study of 29 subjects with negative tuberculin skin test (TST) results in association with positive gamma interferon release assay (IGRA) results, mainly due to responses to CFP-10 in the T-SPOT.TB assay, during a contact investigation. One year later, 12/29 subjects (41%) had converted to positive TST results in association with negative IGRA results.

Comparison of two interferon-gamma assays and tuberculin skin test for tracing tuberculosis contacts.

BACKGROUND: The tuberculin skin test (TST) has low specificity. QuantiFERON-TB Gold (QFT-G) and T-SPOT.TB are based on interferon (IFN)-gamma responses to Mycobacterium tuberculosis-specific antigens. A novel in-tube format of QFT-G (QFT-GIT) offers logistical advantages. OBJECTIVE: To compare TST, QFT-GIT, and T-SPOT.TB in bacillus Calmette-Guérin unvaccinated contacts and correlate results with measures of recent exposure. METHODS: When a supermarket employee with smear-positive tuberculosis had infected most close contacts, a contact investigation among more than 20,000 customers was performed. We recruited subjects randomly on the day of TST administration (n = 469) and subjects with TST of more than 0 mm on the day of TST reading (n = 316). QFT-GIT and T-SPOT.TB were performed. Demographic data and measures of exposure were collected. TST results were analyzed at a cutoff of 10 or 15 mm. Blood tests were interpreted following the manufacturers' criteria and by varying cutoff levels. RESULTS: Among 785 study participants, TST results were associated with age, whereas positive IFN-gamma responses were significantly associated with cumulative shopping time, most markedly for QFT-GIT. Among participants with a TST of 15 mm or greater, sensitivity of QFT-GIT and T-SPOT.TB was 42.2 and 51.3%, respectively. Interassay agreement was 89.6% (kappa = 0.59). By varying cutoff values, agreement between the IFN-gamma assays was optimal at 93.6% (kappa = 0.71) using a cutoff of 0.20 IU/ml for QFT-GIT and 13 spots for T-SPOT.TB. CONCLUSIONS: Blood test results were associated with exposure, whereas the TST was not. A possible lack of sensitivity of IFN-gamma assays in detecting individuals with TST of 15 mm or greater, despite negative bacillus Calmette-Guérin vaccination status, warrants further investigation into alternative cutoff values.

Azithromycin inhibits interleukin-6 but not fibrinogen production in hepatocytes infected with cytomegalovirus and chlamydia pneumoniae.

Chlamydia pneumoniae and cytomegalovirus (CMV) have been associated with the development of atherosclerosis. Inflammatory stimuli initiate the biosynthesis of fibrinogen, interleukin (IL)-6 and plasminogen activator inhibitor (PAI)-1 in the liver. Chronic infection may perpetuate the inflammatory status. We hypothesized that infection of human hepatocytes with the intracellular pathogens C pneumoniae and CMV accelerates biosynthesis of fibrinogen, IL-6, and PAI-1 but that this biosynthesis can be reduced with the use of azithromycin. HepG2 human hepatocytes were infected with C pneumoniae and CMV in vitro in the presence of 0, 0.016, 0.125, or 1 microg/mL azithromycin. We measured IL-6, PAI-1, and fibrinogen after 24, 48, 72, and 96 hours. C pneumoniae-infected hepatocytes produce IL-6 (2667 +/- 309 pg/mL vs 137 +/- 120 pg/mL in uninfected cells after 96 hours. Incubation with 0.016 microg/mL azithromycin decreased IL-6 levels to a mean of 1516 +/- 402 pg/mL, and incubation with 0.125 and 1 microg/mL azithromycin decreased IL-6 to 871 +/- 364 and 752 +/- 403 pg/mL, respectively. C pneumoniae-induced IL-6 production was time- and dose-dependent. The interaction of C pneumoniae with azithromycin treatment was significant, indicating an inhibitory effect of azithromycin on C pneumoniae-induced IL-6 production. CMV infection did not lead to IL-6 production by hepatocytes. C pneumoniae and CMV infection did not induce any changes in PAI-1 production. Fibrinogen production was increased by CMV infection after 72 hours (838 +/- 88 ng/mL; P <.01) and after 96 hours by infection with both C pneumoniae and CMV (765 +/- 100 and 846 +/- 123 ng/mL, respectively; P <.05). Azithromycin did not suppress CMV- or C pneumoniae-induced fibrinogen production. Moreover, we could not confirm an antiinflammatory effect of azithromycin in experiments with cross-titrations of azithromycin against either IL-1 or IL-6 (P >.05). Azithromycin reduces C pneumoniae-induced IL-6 production, but not fibrinogen production, by human hepatocytes. This is a result of the antimicrobial properties of azithromycin and not a direct antiinflammatory effect.