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.

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.

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.

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.

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.

A possible role for Epstein-Barr virus in the pathogenesis of pleural effusion.

A high percentage of pleural effusions remain unexplained despite an intensive diagnostic workup. Epstein-Barr virus (EBV) infections occur worldwide and affect the majority of the population. The present study investigated the prevalence and clinical relevance of EBV in pleural effusions. A prospective study was performed in which 60 consecutive patients with pleural effusion were enrolled. Real-time quantitative EBV-PCR was performed on pleural fluid and serum. Pleural fluid was further evaluated using standard biochemical, cytological and microbiological procedures. Demographic data, medical history and medication were recorded. A total of 24 (40%), from 60 pleural fluids tested, were positive in the EBV-PCR. Median EBV-DNA levels for positive samples was 454 genome equivalents (geq).mL-1 (range 36-163,446 geq.mL-1). A total of 20 (59%) out of 34 unexplained pleural effusions were EBV-PCR positive. Serological analysis of all patients with a positive PCR revealed a previous infection. Patients with a positive EBV-PCR on pleural fluid were more likely to have a positive EBV-PCR on serum than patients with a negative PCR on pleural fluid. Epstein-Barr virus reactivation in pleural fluid is a frequent event and the absence of an alternative diagnosis to explain the nature of the effusion in the majority of cases suggests an aetiological role for Epstein-Barr virus in the development of pleural effusion.

[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.

Circulating inflammatory mediators predict shock and mortality in febrile patients with microbial infection.

The host response to microbial infection is associated with the release of inflammatory mediators. We hypothesized that the type and degree of the systemic response as reflected by levels of circulating mediators predict morbidity and mortality, according to the invasiveness of microbial infection. We prospectively studied 133 medical patients with fever and culture-proven microbial infection. For 3 days after inclusion, the circulating levels of activated complement C3a, interleukin (IL)-6, and secretory phospholipase A(2) (sPLA(2)) were determined daily. Based on results of microbiological studies performed for up to 7 days, patients were classified as having local infections (Group 1, n = 80 positive local cultures or specific stains for fungal or tuberculous infections) or bacteremia (Group 2, n = 52 plus 1 patient with malaria parasitemia). Outcome was assessed as the development of septic shock and as mortality up to 28 days after inclusion. Fifteen patients (11%) developed septic shock and overall mortality was 18% (n = 24). Bacteremia was associated with shock and shock predisposed to death. Circulating mediator levels were generally higher in Group 2 than in Group 1. Circulating levels of IL-6 and sPLA(2) were higher in patients developing septic shock and in nonsurvivors, particularly in Group 1. High C3a was particularly associated with nonsurvival in Group 2. In Group 1, the area under the curve (AUC) of the receiver operating characteristic (ROC) curve for the peak sPLA(2) for shock development was 0.79 (P < 0.05). The AUC of the ROC curve of the peak IL-6 and sPLA(2) for mortality was 0.69 and 0.68 (P < 0.05), respectively. In Group 2, the AUC of the ROC for peak C3a predicting mortality was 0.73 (P < 0.05). In conclusion, in medical patients with fever and microbial infection, the systemic inflammatory host response predicts shock and death, at an early stage, dependent on the invasiveness of microbial infection. The results suggest a differential pathogenetic role of complement activation on the one hand and release of cytokine and lipid mediators on the other in bacteremic and local microbial infections, respectively. They may partly explain the failure of strategies blocking proinflammatory cytokines or sPLA(2) in human sepsis and may extend the basis for attempts to inhibit complement activation at an early stage in patients at risk of dying from invasive microbial infections.

Circulating inflammatory mediators in patients with fever: predicting bloodstream infection.

The systemic host response to microbial infection involves clinical signs and symptoms of infection, including fever and elevated white blood cell (WBC) counts. In addition, inflammatory mediators are released, including activated complement product C3a, interleukin 6 (IL-6), and the acute-phase reactant secretory phospholipase A(2) (sPLA(2)). To compare the value of the latter with the former in predicting (the degree of) microbial infection at the bedside, we determined clinical variables and took blood samples daily for 3 consecutive days in 300 patients with a new fever (>38.0 degrees C rectally or >38.3 degrees C axillary). Microbiological culture results for 7 days after inclusion were collected. Patients were divided into clinical and microbial categories: those without and with a clinical focus of infection and those with negative cultures, with positive local cultures or specific stains for fungal (n = 13) or tuberculous infections (n = 1), and with positive blood cultures, including one patient with malaria parasitemia. The area under the curve (AUC) of the receiver operating characteristic (ROC) for prediction of positive cultures was 0.60 (P < 0.005) for peak temperature and 0.59 (P < 0.01) for peak WBC count, 0.60 (P < 0.005) for peak C3a, 0.63 (P < 0.001) for peak IL-6, and 0.61 (P < 0.001) for peak sPLA(2). The AUC under the ROC curve for prediction of positive blood cultures was 0.68 (P < 0.001) for peak temperature and 0.56 for peak WBC count (P < 0.05). The AUC for peak C3a was 0.69, that for peak IL-6 was 0.70, and that for sPLA(2) was 0.67 (for all, P < 0.001). The degree of microbial invasion is thus a major determinant of the clinical and inflammatory host response in patients with fever. Moreover, circulating inflammatory mediators such as C3a and IL-6 may help to predict positive blood cultures, together with clinical signs and symptoms of the host response to microbial infection, even before culture results are available. This may help in the designing of entry criteria for therapeutic intervention studies.

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 shock in febrile medical patients with a clinical infection.

OBJECTIVE: Shock in the course of fever is likely caused by septic shock. Because septic shock carries a high mortality rate, early recognition could benefit the patient. We tried to predict the development of shock in medical patients with fever and a clinical infection, on the basis of clinical and microbiological information, and to evaluate the role therein of systemic inflammatory response syndrome (SIRS) criteria: abnormal body temperature, tachycardia, tachypnea, and abnormal white blood cell counts. DESIGN: Prospective observational study. SETTING: Department of Internal Medicine at a university hospital. PATIENTS: Patients were 212 consecutive medical patients with newly onset fever (temperature, >38.0 degrees C axillary or >38.3 degrees C rectally) and a clinical source of infection. MEASUREMENTS AND MAIN RESULTS: Of the 212 patients enrolled, 14 developed shock (i.e., a decrease in systolic arterial blood pressure of >40 mm Hg) during a maximum follow-up period of 7 days after inclusion. In univariate analyses, advanced age, prior urogenital disease, an abdominal source, nosocomial infections, and bacteremia predisposed patients to shock (p < .05). For clinical variables, obtained daily for 2 days after inclusion, a low performance (p < .001), the peak respiratory rate (p < .05), the peak heart rate (p < .05), the nadir score on the Glasgow Coma Scale (p < .005), the peak and nadir white blood cell counts (p < .005), and the nadir albumin (p < .01) and peak creatinine concentrations in blood (p < .001) predicted shock development. In multivariate analysis, the presence of bacteremia, the peak respiratory rate, the nadir Glasgow Coma Scale score, and the peak white blood cell count positively and the peak erythrocyte sedimentation rate negatively contributed to prediction of shock development. In contrast, SIRS had less predictive value, mainly because of lack of predictive value of peak heart rate and temperature in multivariate models. CONCLUSION: In febrile medical patients with a clinical infection, the development of shock involves an interaction between circulating microbial products and the host response, which can be recognized clinically by variables easily obtained at the bedside and partly different from the set used to define SIRS.

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.

Prediction of microbial infection and mortality in medical patients with fever: plasma procalcitonin, neutrophilic elastase-alpha1-antitrypsin, and lactoferrin compared with clinical variables.

Fever suggests the likelihood of severe microbial infection. Abnormal temperature, tachycardia, tachypnea, and abnormal white blood cell counts define the systemic inflammatory response syndrome (SIRS). In 300 hospitalized medical patients with fever, we determined clinical variables and procalcitonin, elastase-alpha1-antitrypsin, and lactoferrin levels in plasma. Of the patients, 71% had clinical infection (by clinical judgment) and 44% had microbial infection (by microbiological testing). SIRS occurred in 95%, and the 28-day mortality rate was 9%. The sensitivity for predicting microbial infection, bacteremia, and mortality was less but the specificity was greater for supranormal procalcitonin, elastase-alpha1-antitrypsin, and lactoferrin levels than for SIRS. The area under the receiver operating characteristic curve (AUC) for microbial infection was higher for procalcitonin and elastase-alpha1-antitrypsin levels than for clinical variables and lactoferrin level. The AUC for bacteremia was also higher for inflammatory factors (>0.70; P < .001) than for clinical variables. The AUC for mortality (P < .05) was 0.79 for the respiratory rate, 0.69 for elastase-alpha1-antitrypsin level, 0.65 for heart rate, 0.61 for procalcitonin level, and 0.60 for white blood cell count. In febrile medical patients, plasma procalcitonin and elastase-alpha1-antitrypsin levels may predict microbial infection and bacteremia better than (and mortality as well as) do clinical symptoms.

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.

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.