An Enzyme-Linked Immunosorbent Spot Assay Measuring Borrelia burgdorferi B31-Specific Interferon Gamma-Secreting T Cells Cannot Discriminate Active Lyme Neuroborreliosis from Past Lyme Borreliosis: a Prospective Study in the Netherlands.

Two-tier serology testing is most frequently used for the diagnosis of Lyme borreliosis (LB); however, a positive result is no proof of active disease. To establish a diagnosis of active LB, better diagnostics are needed. Tests investigating the cellular immune system are available, but studies evaluating the utility of these tests on well-defined patient populations are lacking. Therefore, we investigated the utility of an enzyme-linked immunosorbent spot (ELISpot) assay to diagnose active Lyme neuroborreliosis. Peripheral blood mononuclear cells (PBMCs) of various study groups were stimulated by using Borrelia burgdorferi strain B31 and various recombinant antigens, and subsequently, the number of Borrelia-specific interferon gamma (IFN-γ)-secreting T cells was measured. We included 33 active and 37 treated Lyme neuroborreliosis patients, 28 healthy individuals treated for an early manifestation of LB in the past, and 145 untreated healthy individuals. The median numbers of B. burgdorferi B31-specific IFN-γ-secreting T cells/2.5 × 105 PBMCs did not differ between active Lyme neuroborreliosis patients (6.0; interquartile range [IQR], 0.5 to 14.0), treated Lyme neuroborreliosis patients (4.5; IQR, 2.0 to 18.6), and treated healthy individuals (7.4; IQR, 2.3 to 14.9) (P = 1.000); however, the median number of B. burgdorferi B31-specific IFN-γ-secreting T cells/2.5 × 105 PBMCs among untreated healthy individuals was lower (2.0; IQR, 0.5 to 3.9) (P ≤ 0.016). We conclude that the Borrelia ELISpot assay, measuring the number of B. burgdorferi B31-specific IFN-γ-secreting T cells/2.5 × 105 PBMCs, correlates with exposure to the Borrelia bacterium but cannot be used for the diagnosis of active Lyme neuroborreliosis.

Interferon-γ release assays for the diagnosis of active tuberculosis: a systematic review and meta-analysis.

Interferon-γ release assays (IGRAs) are now established for the immunodiagnosis of latent infection with Mycobacterium tuberculosis in many countries. However, the role of IGRAs for the diagnosis of active tuberculosis (TB) remains unclear. Following preferred reporting items for systematic reviews and meta-analyses (PRISMA) and quality assessment of diagnostic accuracy studies (QUADAS) guidelines, we searched PubMed, EMBASE and Cochrane databases to identify studies published in January 2001-November 2009 that evaluated the evidence of using QuantiFERON-TB® Gold in-tube (QFT-G-IT) and T-SPOT.TB® directly on blood or extrasanguinous specimens for the diagnosis of active TB. The literature search yielded 844 studies and 27 met the inclusion criteria. In blood and extrasanguinous fluids, the pooled sensitivity for the diagnosis of active TB was 80% (95% CI 75-84%) and 48% (95% CI 39-58%) for QFT-G-IT, and 81% (95% CI 78-84%) and 88% (confirmed and unconfirmed cases) (95% CI 82-92%) for T-SPOT.TB®, respectively. In blood and extrasanguinous fluids, the pooled specificity was 79% (95% CI 75-82%) and 82% (95% CI 70-91%) for QFT-G-IT, and 59% (95% CI 56-62%) and 82% (95% CI 78-86%) for T-SPOT.TB®, respectively. Although the diagnostic sensitivities of both IGRAs were higher than that of tuberculin skin tests, it was still not high enough to use as a rule out test for TB. Positive evidence for the use of IGRAs in compartments other than blood will require more independent and carefully designed prospective studies.

The risk of tuberculosis related to tumour necrosis factor antagonist therapies: a TBNET consensus statement.

Anti-tumour necrosis factor (TNF) monoclonal antibodies or soluble TNF receptors have become an invaluable treatment against chronic inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease and psoriasis. Individuals who are treated with TNF antagonists are at an increased risk of reactivating latent infections, especially tuberculosis (TB). Following TNF antagonist therapy, the relative risk for TB is increased up to 25 times, depending on the clinical setting and the TNF antagonist used. Interferon-γ release assays or, as an alternative in individuals without a history of bacille Calmette-Guérin vaccination, tuberculin skin testing is recommended to screen all adult candidates for TNF antagonist treatment for the presence of latent infection with Mycobacterium tuberculosis. Moreover, paediatric practice suggests concomitant use of both the tuberculin skin test and an interferon-γ release assay, as there are insufficient data in children to recommend one test over the other. Consequently, targeted preventive chemotherapy is highly recommended for all individuals with persistent M. tuberculosis-specific immune responses undergoing TNF antagonist therapy as it significantly reduces the risk of progression to TB. This TBNET consensus statement summarises current knowledge and expert opinions and provides evidence-based recommendations to reduce the TB risk among candidates for TNF antagonist therapy.

LTBI: latent tuberculosis infection or lasting immune responses to M. tuberculosis? A TBNET consensus statement.

Tuberculosis control relies on the identification and preventive treatment of individuals who are latently infected with Mycobacterium tuberculosis. However, direct identification of latent tuberculosis infection is not possible. The diagnostic tests used to identify individuals latently infected with M. tuberculosis, the in vivo tuberculin skin test and the ex vivo interferon-gamma release assays (IGRAs), are designed to identify an adaptive immune response against, but not necessarily a latent infection with, M. tuberculosis. The proportion of individuals who truly remain infected with M. tuberculosis after tuberculin skin test or IGRA conversion is unknown. It is also uncertain how long adaptive immune responses towards mycobacterial antigens persist in the absence of live mycobacteria. Clinical management and public healthcare policies for preventive chemotherapy against tuberculosis could be improved, if we were to gain a better understanding on M. tuberculosis latency and reactivation. This statement by the TBNET summarises knowledge and limitations of the currently available tests used in adults and children for the diagnosis of latent tuberculosis infection. In summary, the main issue regarding testing is to restrict it to those who are known to be at higher risk of developing tuberculosis and who are willing to accept preventive chemotherapy.

Interferon-gamma release assays during follow-up of tuberculin skin test-positive contacts.

SETTING: Following a large-scale contact investigation, individuals with a positive tuberculin skin test (TST) result were offered preventive tuberculosis treatment. OBJECTIVE: To investigate the effect of isoniazid (INH) treatment and the effect of time on interferon gamma release assay (IGRA) results during follow-up. DESIGN: TST-positive subjects (n = 122) detected during the large-scale contact investigation were included in the study. Blood was obtained every 6 months over 2 years to perform both tests. RESULTS: Preventive INH treatment was completed by 36 of the 122 (29.5%) subjects, 71 (58.2%) were followed up with 6-monthly X-ray screening and 15 (12.3%) did not complete INH treatment. The overall percentage of individuals with a positive result remained stable during the 2 years, at approximately 45-50%, but individual responses varied over time. The majority of initially low IGRA results remained below the cut-off value, initially high IGRA results remained positive, while initially intermediate IGRA results were followed by more dynamic patterns. CONCLUSION: This study showed a highly variable pattern of IGRA responses over time and suggests limited value for their use during follow-up of latently infected individuals. However, the significance of different kinetic patterns observed among subjects with intermediate initial IGRA results warrants further study.

[A patient with Legionella pneumophila serogroup-3 pneumonia, detected by PCR]. / Een patient met een pneumonie door Legionella pneumophila serogroep 3, vastgesteld door PCR.

A 59-year-old man was hospitalised because of dyspnoea, productive cough, fever, chills and malaise. Severe community-acquired pneumonia was diagnosed. Legionella urinary antigen testing, which can only detect serogroup 1, and the first culture ofa bronchoalveolar lavage (BAL) fluid sample were negative for Legionella. However, L. pneumophila DNA was detected by PCR in the BAL washing sample. Eventually, L. pneumophila serogroup 3 was isolated from this specimen by repeated culture. Although, in The Netherlands, legionellosis is caused by L. pneumophila serogroup 1 in more than 90% of all cases, this case demonstrates that a negative result of urinary antigen testing does not necessarily exclude this diagnosis. It is therefore advocated to expand the diagnostics to a Legionella PCR on respiratory material of patients with clinical signs of Legionella pneumonia in whom the urinary antigen test is negative.

Early inhibition of activated fibrinolysis predicts microbial infection, shock and mortality in febrile medical patients.

To evaluate the contribution of an imbalance between coagulation activation and fibinolysis activation and inhibition to morbidity and mortality in sepsis, we determined in medical hospitalized patients at inclusion (day 0) for fever (temperature above 38.0 degrees C axillary or 38.3 degrees C rectally), and daily thereafter for two days, circulating thrombin-antithrombin III (TAT) complexes, plasmin-alpha2-antiplasmin (PAP) complexes (day 0 only), tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1) and interleukin (IL)-6, the latter as a marker of the inflammatory host response. Study variables were 1) positive microbiological results for specimens from local sites associated with a clinical infection, positive blood cultures (including parasitemia) or both, within 7 days after inclusion, 2) development of shock, i.e. systolic blood pressure <90 mmHg or a reduction of 40 mmHg from baseline within 7 days after inclusion, and 3) death related to febrile illness within 28 days after inclusion. The peak plasma levels of TAT complexes were elevated in 44% and the PAP complexes in all patients. The t-PA and PAI-1 levels were elevated in 74 and 94% of patients, respectively. Values for TAT and PAP did not differ among subgroups, while peak t-PA and IL-6 levels were higher in patients with positive microbiological results, developing shock or ultimately dying than in those without the complications (p<0.005). Peak PAI-1 levels were elevated in patients developing shock and ultimate death versus those with an uncomplicated course (p <0.05). Peak IL-6 related to PAI-1 and t-PA levels, which interrelated. Patients with elevated TAT levels had increased plasma levels of IL-6, PAP, PAI-1 and t-PA versus those with normal TAT (p <0.05). Our data indicate that inhibition of activated fibrinolysis, which may partly depend on both cytokinemia and activation of coagulation, predicts microbial infection, septic shock and mortality of febrile medical patients. This suggests an early pathogenic role of inhibition of activated fibrinolysis in the downhill course of serious microbial infection.

Prediction of mortality in febrile medical patients: How useful are systemic inflammatory response syndrome and sepsis criteria?

STUDY OBJECTIVES: The aim was to evaluate demographic, clinical, and laboratory variables in febrile patients, with or without a microbiologically confirmed infection, for prediction of death, in comparison to the systemic inflammatory response syndrome (SIRS) and its criteria, such as abnormal temperature, tachycardia, tachypnea, and abnormal WBC count, and to sepsis, that includes SIRS and an infection. DESIGN: A prospective cohort study. SETTING: Department of internal medicine at a university hospital. PATIENTS: In 300 consecutive, hospitalized medical patients with new onset of fever, demographic, clinical, and laboratory variables were obtained during the 2 days after inclusion, while microbiological results for a follow-up period of 7 days were collected. Patients were followed up for survival or death, up to a maximum of 28 days after inclusion. MEASUREMENTS AND RESULTS: Of all patients, 95% had SIRS, 44% had sepsis with a microbiologically confirmed infection, and 9% died. A model with a set of variables all significantly (p<0.01) contributing to the prediction of mortality was derived. The set included the presence of hospital-acquired fever, the peak respiratory rate, the nadir score on the Glasgow coma scale, and the nadir albumin plasma level within the first 2 days after inclusion. This set of variables predicted mortality for febrile patients with microbiologically confirmed infection even better. The predictive values for mortality of SIRS and sepsis were less than that of our set of variables. CONCLUSIONS: In comparison to SIRS and sepsis, the new set of variables predicted mortality better for all patients with fever and also for those with microbiologically confirmed infection only. This type of effort may help in refining definitions of SIRS and sepsis, based on prognostically important demographic, clinical, and laboratory variables that are easily obtainable at the bedside.

The clinical host response to microbial infection in medical patients with fever.

STUDY OBJECTIVES: Predictors among demographic, clinical, and laboratory variables for a microbial (nonviral/nonchlamydial) infection in hospitalized medical patients with new onset of fever (temperature > or =38.0 degrees C axillary or > or =38.3 degrees C rectal) were analyzed and compared with the criteria for the systemic inflammatory response syndrome (SIRS), including an abnormal body temperature and WBC count, tachypnea and tachycardia, and sepsis, defined as SIRS and the presence of a clinical infection. DESIGN: A prospective cohort study. SETTING: Department of internal medicine at a university hospital. PATIENTS: In 300 hospitalized medical patients with new onset of fever, demographic, clinical, and laboratory variables were obtained during the first 2 days after inclusion, and peak and nadir values, when appropriate, were taken. Microbiologic results for 7 days were collected. Clinical information was used to decide on the presence of a clinical infection. MEASUREMENTS AND RESULTS: One hundred thirty-three of 300 patients (44%) had a microbial infection: 26% suffered from local microbial infection only, 9% from bacteremia only, and 9% had bloodstream plus local microbial infections. Patients with a microbial infection had a higher World Health Organization performance score at home (p<0.05), higher peak body temperature (p<0.001), higher nadir and peak WBC counts (p<0.05), lower nadir platelet count (p<0.01), higher peak alanine and aspartate aminotransferases (p<0.01), and lower nadir albumin (p<0.001) levels in blood during the first 2 days after inclusion than those without infection. Using multivariate techniques, predictors for microbial infection or bacteremia alone, independent of age, sex, underlying disease, and clinical infection, were peak temperature, peak WBC count, and nadir platelet count and albumin level. In contrast, conventional SIRS/sepsis definitions and criteria predicted microbial infection less well, mainly because tachypnea and tachycardia were of no predictive value. CONCLUSIONS: In febrile medical patients, microbial infection can be predicted with use of easily obtained clinical and laboratory variables, including peak temperature, peak WBC count, and nadir platelet count and albumin level within the first 2 days. The new model predicted microbial infection better than conventional SIRS/sepsis criteria. This may help to improve the clinical recognition of the systemic host response to microbial infection and to refine SIRS/sepsis definitions.

A refugee with a supraclavicular lymph node. An uncommon first presentation of carcinoma of the prostate.

In this case report, we describe a patient with an uncommon presentation of disseminated prostate carcinoma. Initial presentation could mimic tuberculosis or hematological malignancy. The literature is reviewed for this kind of presentation. Furthermore, the value of immuno-histochemical stains in relation to prostate carcinoma is discussed.

Improving the timeframe between blood collection and interferon gamma release assay using T-Cell Xtend®.

UNLABELLED: Vacutainer CPT tubes require blood samples for TSPOT.TB to be processed within 8 h. In this study we evaluated the ability of T-Cell Xtend to maintain the number and function of lymphocytes after 24 and 48 h of blood storage, giving similar test results as in freshly isolated specimens. METHODS: Whole blood specimens from 59 individuals were collected in Vacutainer CPT tubes (CPT) and lithium heparin (LH) tubes. CPT tubes were processed within 8 h. T-Cell Xtend was added to LH tubes after 24 or 48 h. We also left LH tubes untreated for 48 h. Total number of white blood cells (WBC) and proportions of lymphocytes and granulocytes were determined in the isolated Peripheral Blood Mononuclear Cells (PBMC). We also evaluated the performance of T-Cell Xtend in the TSPOT.TB assay. RESULTS: PBMC yields from T-Cell Xtend treated LH samples did not differ from PBMC yields from CPT tubes, but T-Cell Xtend had a pronounced effect on the proportions of lymphocytes and granulocytes. The mean lymphocyte percentage in PBMCs isolated from fresh CPT blood was 84.31 ± 1.14% (at t = 48 h), but was decreased to 52.72 ± 3.34% (p < 0.05) in untreated LH blood (at t = 48 h). This effect was neutralized by T-Cell Xtend (85.44 ± 0.74%). We observed a similar but opposite effect on granulocytes: The mean proportion in untreated LH blood was increased to 40.9 ± 3.67% (p < 0.001) compared to CPT blood (8.26 ± 0.89%). Treatment of LH samples with T-Cell Xtend (48 h) restored the proportion of granulocytes to 8.47 ± 0.61%. Enumeration of spots in the TSPOT.TB assay demonstrated good agreement between CPT and T-Cell Xtend results, even after 48 h. CONCLUSIONS: T-Cell Xtend efficiently removes granulocytes from PBMC suspensions and increases the proportion of lymphocytes. TSPOT.TB results from T-Cell Xtend treated blood samples are at least comparable to the results obtained from the current CPT method. Use of standard lithium heparin blood combined with T-Cell Xtend allows up to 48 h storage of blood samples for batched processing and may further decrease the rate of indeterminate TSPOT.TB results.

Diagnostic potential of an enzyme-linked immunospot assay in tuberculous pericarditis.

Tuberculous pericarditis is a rare disease in developed countries. The diagnosis is difficult to set since there are no robust rapid tests, and culture of pericardial fluid for Mycobacterium tuberculosis is often negative. T-SPOT.TB, an enzyme-linked immunospot (ELISPOT) test, measures the gamma interferon response of lymphocytes against tuberculosis antigens and can be performed on blood and body fluids. We describe a patient with tuberculous pericarditis for which the diagnosis was rapidly set by positive T-SPOT.TB results, which were confirmed by isolation of Mycobacterium tuberculosis in pericardial fluid culture. We performed a literature search to assess the diagnostic potential of ELISPOT testing in tuberculous pericarditis. The limited data on this subject indicate that T-SPOT.TB aids in diagnosing active tuberculosis (TB) infection and results in a more rapid decision to start antituberculosis treatment. Enumerating TB-specific lymphocytes and testing blood/compartmental fluid simultaneously can provide useful information on active tuberculous pericarditis.

Rapid diagnosis of CNS tuberculosis by a T-cell interferon-gamma release assay on cerebrospinal fluid mononuclear cells.

Central nervous system tuberculosis remains a clinical diagnostic challenge. The ex vivo Mycobacterium tuberculosis-specific enzyme-linked immunospot assay (ELISPOT) is a novel assay for the rapid detection of M. tuberculosis-specific T-lymphocytes in the peripheral blood. However, when performed on peripheral blood, this assay cannot distinguish between active tuberculosis or latent tuberculosis infection. On the assumption that M. tuberculosis-specific T-lymphocytes migrate to sites of infection, we were able to demonstrate high levels of M. tuberculosis-specific cells by ELISPOT in the cerebrospinal fluid of a patient with tuberculous meningitis and intracerebral tuberculoma four weeks before cerebrospinal fluid culture became positive for M. tuberculosis by culture.

Exclusion of active Mycobacterium tuberculosis complex infection with the T-SPOT.TB assay.

The present brief report describes four cases with mycobacterial infection and negative T-SPOT.TB tests. This test proved to be a useful tool to help rule out the diagnosis of active Mycobacterium tuberculosis infection and could therefore prevent unnecessary or inappropriate therapy.

Use of a T-cell interferon-gamma release assay for the diagnosis of tuberculous pleurisy.

The diagnosis of pleural tuberculosis (plTB) by the analysis of pleural effusions (PEs) with standard diagnostic tools is difficult. In routine clinical practice, the present authors evaluated the performance of a commercially available Mycobacterium tuberculosis (MTB)-specific enzyme-linked immunospot assay on peripheral blood mononuclear cells (PBMCs) and pleural effusion mononuclear cells (PEMCs) in patients with suspect plTB. The T-SPOT.TB test (Oxford Immunotec Ltd, Abingdon, UK) was performed on PBMCs and PEMCs in 20 patients with a clinical and radiological suspect of plTB and in 21 control subjects with a diagnosis of PE of nontuberculous origin at four centres participating in the European Tuberculosis Network. In total, 18 (90%) out of 20 patients with plTB tested T-SPOT.TB-positive on PBMCs and 19 (95%) out of 20 on PEMCs. Among controls, T-SPOT.TB was positive in seven out of 21 (33%) patients when performed on PBMCs (these patients were assumed to be latently infected with MTB) and five (23%) out of 21 when performed on PEMCs. Sensitivity and specificity of T-SPOT.TB for the diagnosis of active plTB when performed on PEMCs were 95 and 76%, respectively. Enumerating Mycobacterium tuberculosis-specific T-cells in pleural effusion mononuclear cells by ELISPOT is feasible in routine clinical practice and may be useful for a rapid and accurate diagnosis of pleural tuberculosis.

Effect of tuberculin skin testing on a Mycobacterium tuberculosis-specific interferon-gamma assay.

Recently, interferon-gamma release assays (IGRA) for specific diagnosis of Mycobacterium tuberculosis infection have become available. In recent UK tuberculosis (TB) guidelines, it has been advised to screen for latent M. tuberculosis infection using the tuberculin skin test (TST), followed by IGRA if the TST is positive. Since TST can boost immune responses to tuberculin, the present authors evaluated whether TST administration affects the result of QuantiFERON-TB Gold in-tube (QFT-GIT), a whole blood-based IGRA. QFT-GIT was performed on the day of TST administration and the day of reading in 15 TST-negative subjects, 46 TST-positive subjects with recent or remote exposure to M. tuberculosis and five cured TB patients. No systematic boosting of QFT-GIT responses from negative to positive was observed. Only in a few TST-positive persons did TST enhance pre-existing QFT-GIT responses. Screening for latent Mycobacterium tuberculosis infection using tuberculin skin testing followed by interferon-gamma release assays on the day of reading is a reliable approach, as the specificity of QuantiFERON-TB Gold in-tube is not affected by prior tuberculin skin test administration.