Monica Losi

Use in routine clinical practice of two commercial blood tests for diagnosis of infection with Mycobacterium tuberculosis: a prospective study

BACKGROUND Two commercial blood assays for the diagnosis of latent tuberculosis infection--T-SPOT.TB and QuantiFERON-TB Gold--have been separately compared with the tuberculin skin test. Our aim was to compare the efficacy of all three tests in the same population sample. METHODS We did a prospective study in 393 consecutively enrolled patients who were tested simultaneously with T-SPOT.TB and QuantiFERON-TB Gold because of suspected latent or active tuberculosis. 318 patients also had results available for a tuberculin skin test. FINDINGS Overall agreement with the skin test was similar (T-SPOT.TB kappa=0.508, QuantiFERON-TB Gold kappa=0.460), but fewer BCG-vaccinated individuals were identified as positive by the two blood assays than by the tuberculin skin test (p=0.003 for T-SPOT.TB and p<0.0001 for QuantiFERON-TB Gold). Indeterminate results were significantly more frequent with QuantiFERON-TB Gold (11%, 43 of 383) than with T-SPOT.TB (3%, 12 of 383; p<0.0001) and were associated with immunosuppressive treatments for both tests. Age younger than 5 years was significantly associated with indeterminate results with QuantiFERON-TB Gold (p=0.003), but not with T-SPOT.TB. Overall, T-SPOT.TB produced significantly more positive results (38%, n=144, vs 26%, n=100, with QuantiFERON-TB Gold; p<0.0001), and close contacts of patients with active tuberculosis were more likely to be positive with T-SPOT.TB than with QuantiFERON-TB Gold (p=0.0010). INTERPRETATION T-SPOT.TB and QuantiFERON-TB Gold have higher specificity than the tuberculin skin test. Rates of indeterminate and positive results, however, differ between the blood tests, suggesting that they might provide different results in routine clinical practice.

Interferon-γ release assays for the diagnosis of latent Mycobacterium tuberculosis infection: a systematic review and meta-analysis

We conducted a systematic review and meta-analysis to compare the accuracy of the QuantiFERON-TB® Gold In-Tube (QFT-G-IT) and the T-SPOT®.TB assays with the tuberculin skin test (TST) for the diagnosis of latent Mycobacterium tuberculosis infection (LTBI). The Medline, Embase and Cochrane databases were explored for relevant articles in November 2009. Specificities, and negative (NPV) and positive (PPV) predictive values of interferon-&ggr; release assays (IGRAs) and the TST, and the exposure gradient influences on test results among bacille Calmette–Guérin (BCG) vaccinees were evaluated. Specificity of IGRAs varied 98–100%. In immunocompetent adults, NPV for progression to tuberculosis within 2 yrs were 97.8% for T-SPOT®.TB and 99.8% for QFT-G-IT. When test performance of an immunodiagnostic test was not restricted to prior positivity of another test, progression rates to tuberculosis among IGRA-positive individuals followed for 19–24 months varied 8–15%, exceeding those reported for the TST (2–3%). In multivariate analyses, the odd ratios for TST positivity following BCG vaccination varied 3–25, whereas IGRA results remained uninfluenced and IGRA positivity was clearly associated with exposure to contagious tuberculosis cases. IGRAs may have a relative advantage over the TST in detecting LTBI and allow the exclusion of M. tuberculosis infection with higher reliability.

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.

Performance of tests for latent tuberculosis in different groups of immunocompromised patients.

BACKGROUND Immunocompromised persons infected with Mycobacterium tuberculosis (MTB) have increased risk of tuberculosis (TB) reactivation, but their management is hampered by the occurrence of false-negative results of the tuberculin skin test (TST). The T-cell interferon (IFN)-gamma release blood assays T-SPOT.TB (TS.TB) [Oxford Immunotec; Abingdon, UK] and QuantiFERON-TB Gold In-Tube (QFT-IT) [Cellestis Ltd; Carnegie, VIC, Australia] might improve diagnostic accuracy for latent TB infection (LTBI) in high-risk persons, although their performance in different groups of immunocompromised patients is largely unknown. METHODS AND RESULTS Over a 1-year period, we prospectively enrolled patients in three different immunosuppressed groups, as follows: 120 liver transplantation candidates (LTCs); 116 chronically HIV-infected persons; and 95 patients with hematologic malignancies (HMs). TST, TS.TB, and QFT-IT were simultaneously performed, their results were compared, and intertest agreement was evaluated. Overall, TST provided fewer positive results (10.9%) than TS.TB (18.4%; p < 0.001) and QFT-IT (15.1%; p = 0.033). Significantly fewer HIV-infected individuals had at least one positive test (9.5%) compared with LTCs (35.8%; p < 0.001) and patients with HMs (29.5%; p < 0.001). Diagnostic agreement between tests was moderate (kappa = 0.40 to 0.65) and decreased in the HIV-infected group when the results of the TS.TB were compared with either TST (kappa = 0.16) or QFT-IT (kappa = 0.19). Indeterminate blood test results due to low positive control values were significantly more frequent with QFT-IT (7.2%) than with TS.TB (0.6%; p < 0.001). CONCLUSIONS Blood tests identified significantly more patients as being infected with MTB than TST, although diagnostic agreement varied across groups. Based on these results, we recommend tailoring application of the new blood IFN-gamma assays for LTBI in different high-risk groups and advise caution in their current use in immunosuppressed patients.

Bronchoalveolar lavage enzyme-linked immunospot for a rapid diagnosis of tuberculosis: A Tuberculosis Network European Trialsgroup study

RATIONALE The rapid diagnosis of pulmonary tuberculosis (TB) is difficult when acid fast bacilli (AFB) cannot be detected in sputum smears. OBJECTIVES Following a proof of principle study, we examined in routine clinical practice whether individuals with sputum AFB smear-negative TB can be discriminated from those with latent TB infection by local immunodiagnosis with a Mycobacterium tuberculosis-specific enzyme-linked immunospot (ELISpot) assay. METHODS Subjects suspected of having active TB who were unable to produce sputum or with AFB-negative sputum smears were prospectively enrolled at Tuberculosis Network European Trialsgroup centers in Europe. ELISpot with early-secretory-antigenic-target-6 and culture-filtrate-protein-10 peptides was performed on peripheral blood mononuclear cells (PBMCs) and bronchoalveolar lavage mononuclear cells (BALMCs). M. tuberculosis-specific nucleic acid amplification (NAAT) was performed on bronchoalveolar lavage fluid. MEASUREMENTS AND MAIN RESULTS Seventy-one of 347 (20.4%) patients had active TB. Out of 276 patients who had an alternative diagnosis, 127 (46.0%) were considered to be latently infected with M. tuberculosis by a positive PBMC ELISpot result. The sensitivity and specificity of BALMC ELISpot for the diagnosis of active pulmonary TB were 91 and 80%, respectively. The BALMC ELISpot (diagnostic odds ratio [OR], 40.4) was superior to PBMC ELISpot (OR, 10.0), tuberculin skin test (OR, 7.8), and M. tuberculosis specific NAAT (OR, 12.4) to diagnose sputum AFB smear-negative TB. In contrast to PBMC ELISpot and tuberculin skin test, the BALMC ELISpot was not influenced by previous history of TB. CONCLUSIONS Bronchoalveolar lavage ELISpot is an important advancement to rapidly distinguish sputum AFB smear-negative TB from latent TB infection in routine clinical practice.

Performance of commercial blood tests for the diagnosis of latent tuberculosis infection in children and adolescents

BACKGROUND. The accurate diagnosis of latent tuberculosis infection reduces the risk of progression to severe disseminated disease. However, in young children, a major limitation of the standard tuberculin skin test is that false-negative results cannot be detected. The new interferon-γ release assays QuantiFERON-TB Gold (Cellestis Carnegie Victoria, Australia), QuantiFERON-TB In-Tube (Cellestis), and T-SPOT.TB (Oxford Immunotec, Abingdon, United Kingdom) show promise of greater accuracy, but they may also be affected by impaired cellular immunity, resulting in indeterminate results (ie, insufficient response in positive-control wells). OBJECTIVE. To evaluate the impact of age on the performance of interferon-γ release assays when used in a routine hospital setting among children tested for suspected active or latent TB infection. METHODS. We retrospectively studied 496 children 0 to 19 years of age who had been tested with the tuberculin skin test and at least 1 interferon-γ release assay: 181 with QuantiFERON-TB Gold and 315 with QuantiFERON-TB In-Tube. In 154 of the children, paired interferon-γ release assay testing was available: 87 with QuantiFERON-TB Gold/T-SPOT.TB and 67 with QuantiFERON-TB In-Tube/T-SPOT.TB. RESULTS. Compared with T-SPOT.TB, the rates of indeterminate results were significantly higher for both QuantiFERON-TB Gold and QuantiFERON-TB In-Tube. QuantiFERON-TB Gold and QuantiFERON-TB In-Tube also gave indeterminate results more frequently in children <4 years of age than in those ≥4 years of age. Indeterminate results were associated with younger age for both QuantiFERON-TB Gold and QuantiFERON-TB In-Tube but not for T-SPOT.TB. Considering age as a binary variable (<4 and ≥4 years of age), a significantly higher concentration of phytohaemagglutinin-produced interferon-γ was observed in older children with both QuantiFERON-TB Gold and QuantiFERON-TB In-Tube. CONCLUSIONS. Different blood tests for the diagnosis of latent tuberculosis infection in children seem to perform differently, because both QuantiFERON-TB tests were more likely than T-SPOT.TB to give indeterminate results in children <4 years of age.

Triggering receptor expressed on myeloid cells: role in the diagnosis of lung infections

The triggering receptor expressed on myeloid cells (TREM)‐1 is a recently described molecule, which plays an important role in myeloid cell-activated inflammatory responses. TREM‐1 is expressed on blood neutrophils and monocytes, and also on alveolar macrophages, thus suggesting a potential role in lung inflammatory responses against infections. To investigate the differential expression of TREM‐1 in lung infections, its levels were assessed in bronchoalveolar lavage specimens from patients with community-acquired pneumonia or tuberculosis. TREM‐1 was also investigated in patients with interstitial lung diseases, as a model of noninfectious inflammatory disease of the lung. TREM‐1 expression was significantly increased in lung neutrophils and in lung macrophages of patients with pneumonia (n=7; 387.9±61.4 and 660.5±18.3, respectively) compared with patients with pulmonary tuberculosis (n=7; 59.2±13.1 and 80.6±291.2) and patients with interstitial lung diseases (n=10; 91.8±23.3 and 123.9±22.8). In contrast, TREM‐1 expression on peripheral blood neutrophils was no different among the three groups. In conclusion, these data suggest that triggering receptor expressed on myeloid cells‐1 is selectively expressed in the lungs of patients with pneumonia caused by extracellular bacteria and not in patients with tuberculosis, providing a potential marker for differential diagnosis.

Risk Assessment of Tuberculosis in Immunocompromised Patients. A TBNET Study

RATIONALE In the absence of active tuberculosis, a positive tuberculin skin test (TST) or interferon-γ release assay (IGRA) result defines latent infection with Mycobacterium tuberculosis, although test results may vary depending on immunodeficiency. OBJECTIVES This study compared the performance of TST and IGRAs in five different groups of immunocompromised patients, and evaluated their ability to identify those at risk for development of tuberculosis. METHODS Immunocompromised patients with HIV infection, chronic renal failure, rheumatoid arthritis, solid-organ or stem-cell transplantation, and healthy control subjects were evaluated head-to-head by the TST, QuantiFERON-TB-Gold in-tube test (ELISA), and T-SPOT.TB test (enzyme-linked immunospot) at 17 centers in 11 European countries. Development of tuberculosis was assessed during follow-up. MEASUREMENTS AND MAIN RESULTS Frequencies of positive test results varied from 8.7 to 15.9% in HIV infection (n = 768), 25.3 to 30.6% in chronic renal failure (n = 270), 25.0% to 37.2% in rheumatoid arthritis (n = 199), 9.0 to 20.0% in solid-organ transplant recipients (n = 197), 0% to 5.8% in stem-cell transplant recipients (n = 103), and 11.2 to 15.2% in immunocompetent control subjects (n = 211). Eleven patients (10 with HIV infection and one solid-organ transplant recipient) developed tuberculosis during a median follow-up of 1.8 (interquartile range, 0.2-3.0) years. Six of the 11 patients had a negative or indeterminate test result in all three tests at the time of screening. Tuberculosis incidence was generally low, but higher in HIV-infected individuals with a positive TST (3.25 cases per 100 person-years) than with a positive ELISA (1.31 cases per 100 person-years) or enzyme-linked immunospot result (1.78 cases per 100 person-years). No cases of tuberculosis occurred in patients who received preventive chemotherapy. CONCLUSIONS Among immunocompromised patients evaluated in this study, progression toward tuberculosis was highest in HIV-infected individuals and was poorly predicted by TST or IGRAs. Clinical trial registered with www.clinicaltrials.gov (NCT 00707317).

A multicentre evaluation of the accuracy and performance of IP-10 for the diagnosis of infection with M. tuberculosis

IP-10 has potential as a diagnostic marker for infection with Mycobacterium tuberculosis, with comparable accuracy to QuantiFERON-TB Gold In-Tube test (QFT-IT). The aims were to assess the sensitivity and specificity of IP-10, and to evaluate the impact of co-morbidity on IP-10 and QFT-IT. 168 cases with active TB, 101 healthy controls and 175 non-TB patients were included. IP-10 and IFN-γ were measured in plasma of QFT-IT stimulated whole blood and analyzed using previously determined algorithms. A subgroup of 48 patients and 70 healthy controls was tested in parallel with T-SPOT.TB IP-10 and QFT-IT had comparable accuracy. Sensitivity was 81% and 84% with a specificity of 97% and 100%, respectively. Combining IP-10 and QFT-IT improved sensitivity to 87% (p < 0.0005), with a specificity of 97%. T-SPOT.TB was more sensitive than QFT-IT, but not IP-10. Among non-TB patients IP-10 had a higher rate of positive responders (35% vs 27%, p < 0.02) and for both tests a positive response was associated with relevant risk factors. IFN-γ but not IP-10 responses to mitogen stimulation were reduced in patients with TB and non-TB infection. This study confirms and validates previous findings and adds substance to IP-10 as a novel diagnostic marker for infection with M. tuberculosis. IP-10 appeared less influenced by infections other than TB; further studies are needed to test the clinical impact of these findings.

Early diagnosis of subclinical multidrug-resistant tuberculosis.

Context Immunosuppressed people with tuberculosis may have false-negative results on a tuberculin skin test. Could T-cell-based blood tests help diagnose tuberculosis in these people? Contribution This case report describes an asymptomatic man taking azathioprine for Crohn disease. His wife had tuberculosis, and he had a negative result on a tuberculin skin test but a positive result on an enzyme-linked immunospot (ELISPOT) assay, a rapid blood test that detects T cells specific for antigens expressed by Mycobacterium tuberculosis. Bronchoalveolar lavage and culture confirmed multi-drug-resistant tuberculosis. Implications The ability of the ELISPOT assay to help diagnose tuberculosis in immunosuppressed patients with negative tuberculin test results warrants testing in large prospective studies. The Editors The current tool for detecting Mycobacterium tuberculosis infection in asymptomatic exposed contacts is the century-old tuberculin skin test, which has numerous drawbacks (1). Recently developed T-cellbased tests offer a new approach for detecting asymptomatic M. tuberculosis infection (2-4). One of these, the ex vivo enzyme-linked immunospot (ELISPOT) assay for interferon- detects T cells that are specific for antigens expressed by M. tuberculosis but absent from M. bovis bacille Calmette-Gurin (2). Two studies in adults suggest that the ELISPOT assay has a high sensitivity (range, 92% to 96%) in patients with culture-confirmed tuberculosis, including patients with HIV co-infection (2, 5). In recent tuberculosis contacts, the assay correlates significantly more closely with M. tuberculosis exposure than does the tuberculin skin test and, unlike the skin test, is independent of vaccination status (6, 7). Thus, it seems to have a higher sensitivity and specificity than the tuberculin skin test for detecting latent tuberculosis infection (7). We have not known whether these features of the ELISPOT assay would actually lead to improved detection and management of people with M. tuberculosis infection in clinical practice. We report what we believe to be the first clinical application of the ELISPOT assay to an important and difficult problem: the evaluation of a person receiving immunosuppressive therapy who was recently exposed to tuberculosis. Case Report A 24-year-old female illegal immigrant from Moldova delivered a healthy baby at the University Hospital of Modena, Modena, Italy. Although she was noted to be thin and to have a persistent cough, chest radiography was delayed until 1 week after delivery. Radiography and high-resolution computed tomography of the lungs strongly suggested active pulmonary tuberculosis (Figure 1, parts A and B). When informed of her suspected diagnosis, the woman related that she had had a fever and cough for 4 months, but that anxiety about her status as an illegal immigrant had prevented her from seeking medical attention sooner. Ten years earlier, she had been treated for pulmonary tuberculosis with 2 unspecified oral drugs for about 2 months. Three sputum samples were strongly positive (3+) (8) for acid-fast bacilli on ZiehlNeelsen staining, and results of HIV serologic testing were negative. Standard 4-drug antituberculosis therapy was started. Three weeks later, the sputum specimens grew M. tuberculosis complex resistant to isoniazid and rifampin. After switching to a 5-drug regimen (pyrazinamide, moxifloxacin, ethambutol, streptomycin, and clofazimine), the woman progressively improved. Figure 1. Imaging studies of the patients. A. B. C. D. The womans closest contact was her 41-year-old husband. He was taking long-term immunosuppressive therapy (azathioprine, 150 mg/d) for inactive Crohn disease. He had no symptoms, and his physical examination findings were normal. His complete blood count and differential leukocyte count were normal. We offered the husband testing with the tuberculin skin test and ELISPOT assay because we recognized that the tuberculin skin test might yield a false-negative result (poor sensitivity) because of the immunosuppressive therapy. If infected, the man would be at high risk for progression to active multidrug-resistant tuberculosis with its attendant high morbidity and mortality. The man gave informed consent, and the Modena Ethics Committee (institutional review board) approved the testing. The tuberculin skin test was administered by the Mantoux method using 5 IU (0.1 mL) of purified protein derivativeSiebert (PPD-S, Biocine Test PPD, Sclavo, Siena, Italy). The maximum diameter of cutaneous induration was measured with a ruler and recorded 72 hours after inoculation; 5 mm was used as the cutoff for a positive test result. This is the lowest threshold for a positive tuberculin skin test result and is used for recently exposed contacts of persons with infectious tuberculosis (9). Immediately after administration of the tuberculin skin test, a venous blood sample was taken, and the ELISPOT assay was performed by using antigens highly specific for M. tuberculosis complex (2). The antigens were recombinant early secretory antigenic target-6 (ESAT-6), recombinant culture filtrate protein-10 (CFP-10), and peptide pools derived from these antigens. The tuberculin skin test result was negative (induration was 4 mm), whereas the ELISPOT assay result was positive (Figure 2). Because of the positive ELISPOT assay result, the man underwent radiography and high-resolution computed tomography of the chest. Chest radiography showed poorly defined nonspecific shadowing in the periphery of the upper zone of the right lung (Figure 1, part C). High-resolution computed tomography of the chest showed several small foci of consolidation, one with very early cavitation (Figure 1, part D). Fiberoptic bronchoscopy with bronchoalveolar lavage of the anterior segment of the right upper lobe was performed. After lavage fluid revealed acid-fast bacilli, the patient was prescribed the same 5 antituberculosis drugs as his wife. Mycobacterium tuberculosis complex was isolated from lavage fluid cultures 5 weeks later. The drug resistance pattern was the same as that of his wifes isolate, and molecular strain typing (DNA fingerprinting) using IS6110 restriction fragment length polymorphism analysis (10) indicated that his isolate was identical to that of his wife. Three months later, the ELISPOT assay and the tuberculin skin test were repeated and, once again, the skin test result was negative (4-mm induration) and the ELISPOT assay result was positive. The patient remained asymptomatic and his chest radiograph showed improvement. Figure 2. Photomicrographs of enzyme-linked immunosorbent (ELISPOT) assay results from the blood sample of the husband of the index patient. Mycobacterium tuberculosis A B C D Discussion Clinical application of this novel T-cellbased test to evaluate a recent tuberculosis contact resulted in the early diagnosis and prompt treatment of subclinical, active, multidrug-resistant pulmonary tuberculosis in an asymptomatic person with a negative tuberculin skin test result. In addition to providing direct benefit to the husband, early diagnosis probably prevented secondary transmission of this M. tuberculosis strain in the community. There are 2 possible explanations for why the ELISPOT assay and not the tuberculin skin test could detect the presence of early subclinical multidrug-resistant tuberculosis. First, the ELISPOT assay may in fact have higher sensitivity than the tuberculin skin test for detecting asymptomatic M. tuberculosis infection, as suggested by previous studies (5-7, 11). Second, the ELISPOT assay may be less susceptible than the tuberculin skin test to false-negative results in iatrogenically immunosuppressed persons, as has already been shown in persons with HIV-associated immunosuppression (5). A large and increasing number of patients are receiving medications that cause mild to moderate immunosuppression and, as in the case reported here, many have impaired delayed-type hypersensitivity responses and false-negative skin test results. Screening for asymptomatic M. tuberculosis infection is especially important in patients starting therapy with antitumor necrosis factor- agents (for example, infliximab) (12). Reactivation tuberculosis is a major adverse effect of this potent new therapy (12), but diagnosing M. tuberculosis infection in these patients is especially difficult because most are already receiving immunosuppressive agents (13). Although the ELISPOT assay is a sensitive and specific test for M. tuberculosis infection, it does not distinguish between active tuberculosis disease and latent tuberculosis infection. This distinction is determined by the results of clinical assessment and chest radiography. When, as in the case reported here, there are no symptoms or signs of active disease, ELISPOT assay results may help clinicians interpret results of chest radiography. Because of the high specificity of the ELISPOT assay, nonspecific abnormalities on chest radiography in tuberculosis contacts with positive ELISPOT assay results are highly suggestive of active pulmonary tuberculosis, whereas a normal chest radiograph is consistent with latent tuberculosis infection if no clinical features of extrapulmonary tuberculosis are present. In contrast, the high sensitivity of the ELISPOT assay means that nonspecific chest radiographic abnormalities in persons with negative ELISPOT assay results are unlikely to be due to tuberculosis. We have shown that this novel T-cellbased test detected early, active multidrug-resistant tuberculosis in the absence of symptoms and in the context of a negative tuberculin skin test result. We believe that our report demonstrates for the first time the potential of the ELISPOT assay for positively affecting clinical outcomes. The ELISPOT assay is being used to screen all the hospital contacts of the source case described in this report in order to help prevent a nosocomial outbreak of multidrug-resist