Pietro Roversi

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.

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.

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

Repeated tuberculin testing does not induce false positive ELISPOT results

The Enzyme Linked ImmunoSpot (ELISPOT) is a new rapid T cell based blood test (otherwise known as an interferon-γ assay) for the diagnosis of latent tuberculosis infection.1–3 The commercially available form of the assay, T-SPOT® TB (Oxford Immunotec, Abingdon, UK) has European regulatory approval as an in vitro diagnostic test and is increasingly being used in clinical practice. The test is based on the enumeration of interferon-γ producing T cells which are specific for two highly antigenic proteins, early secretory antigenic target-6 (ESAT-6) and culture filtrate protein 10 (CFP-10).1 These proteins are expressed by Mycobacterium tuberculosis but are absent from M bovis BCG vaccine. Hence, the test does not give false positive results in BCG vaccinated individuals.1–3 ESAT-6 and CFP-10 are, however, contained within tuberculin purified protein derivative (PPD). Since ELISPOT is a highly sensitive method for measuring even low numbers of antigen specific T cells,4 concerns have been raised as to whether repeated tuberculin skin tests might induce T cell responses to these specific antigens, resulting in false …

Sarcoidosis: challenging diagnostic aspects of an old disease

Over the past few years, there have been substantial advances in our understanding of sarcoidosis immunopathogenesis. Conversely, the etiology of the disease remains obscure for a number of reasons, including heterogeneity of clinical manifestations, often overlapping with other disorders, and insensitive and nonspecific diagnostic tests. While no cause has been definitely confirmed, there is increasing evidence that one or more infectious agents may cause the disease, although the organism may no longer be viable. Here we present 2 cases, in which sarcoidosis preceded tuberculosis and non-Hodgkin lymphoma. Development of new lesions in a patient with chronic/remitting sarcoidosis should be looked at with suspicion and promptly investigated in order to rule out an alternative/concomitant diagnosis. In such cases, tissue confirmation from the most accessible site, and bone marrow biopsy-if lymphoma is in the differential diagnosis-should be performed. In conclusion, we strongly advise that physicians be ready to reconsider the diagnosis of sarcoidosis in the presence of atypical manifestations or persistent/progressive disease despite conventional therapy.

Coronary artery disease concomitant with chronic obstructive pulmonary disease

Numerous epidemiologic studies have linked the presence of chronic obstructive pulmonary disease (COPD) to coronary artery disease (CAD). However, prevalence, pathological processes, clinical manifestations and therapy are still debated, as progress towards uncovering the link between these two disorders has been hindered by the complex nature of multimorbidity.

Diagnosis of occult tuberculosis in hematological malignancy by enumeration of antigen-specific T cells.

Diagnosis of occult tuberculosis in hematological malignancy by enumeration of antigen-specific T cells

Role of the quantiferon-TB test in ruling out pleural tuberculosis: a multi-centre study.

Diagnosing pleural tuberculosis (plTB) might be difficult due to limited sensitivity of conventional microbiology tools. As M. tuberculosis (MTB)-specific T cells are recruited into pleural space in plTB, their detection may provide useful clinical information. To this aim, in addition to standard diagnostic tests, we used the QuantiFERON-TB Gold In-Tube (QFT-IT) test in blood and pleural effusion (PE) samples from 48 patients with clinical suspicion of plTB, 18 (37.5%) of whom had confirmed plTB. Four of them (22.2%) tested positive with a nucleic acid amplification test for MTB. The tuberculin skin test was positive in most confirmed plTB cases (88.9%). Positive QFT-IT tests were significantly more frequent in patients with confirmed plTB, as compared to patients with an alternative diagnosis, both in blood (77.7 vs 36.6%, p=0.006) and in PE samples (83.3% vs 46.6%, p=0.02). In addition, both blood and PE MTB-stimulated IFN-γ levels were significantly higher in plTB patients (p=0.03 and p=0.0049 vs non-plTB, respectively). In blood samples, QFT-IT had 77.8% sensitivity and 63.3% specificity, resulting in 56.0% positive (PPV) and 82.6% negative (NPV) predictive values. On PE, QFT-IT sensitivity was 83.3% and specificity 53.3% (PPV 51.7% and NPV 84.2%). The optimal AUC-derived cut-off for MTB-stimulated pleural IFN-γ level was 3.01 IU/mL (77.8% sensitivity, 80% specificity, PPV 68.4% and NPV 82.8%). These data suggest that QFT-IT might have a role in ruling out plTB in clinical practice.

Interferon-gamma-release assays detect recent tuberculosis re-infection in elderly contacts.

The tuberculin skin test (TST) does not discriminate between recent and remote latent tuberculosis infection (LTBI). This study was carried out to test two interferon-γ (IFN-γ)-based blood assays in recent contacts with high prevalence of remote LTBI. We performed a contact tracing investigation in a nursing home for the elderly, where elderly patients were exposed to a case of pulmonary tuberculosis. TST, QuantiFERON-TB Gold (QFT-G) and T-SPOT.TB (TS.TB) were performed 8 weeks after the end of potential exposure. IFN-γ measurements were recorded and correlation with exposure was evaluated. Twenty-seven (37.5%), 32 (44.4%) and 16 (22.2%) subjects were TST, TS.TB and QFT-G positive, respectively; agreement between TS.TB and QFT-G was good among exposed subjects only (κ=0.915, 0.218 in unexposed, p<0.001). When amounts of IFN-γ were corrected for the number of producing T cells, specific IFN-γ production was significantly different between exposed and unexposed individuals (16.75±5.40 vs 2.33±0.71 IFN-γ IU/1000 SFC, p=0.0001). QFT-G and TS.TB provided discordant results among elderly contacts. Unlike TST, the specific IFN-γ response might discriminate between recent and long-lasting tuberculosis infection.

LA DIAGNOSI DI INFEZIONE TUBERCOLARE LATENTE IN PAZIENTI CON EMOLINFOPATIE MALIGNE: USO DEI TEST IMMUNOLOGICI.

Background. I pazienti affetti da emolinfopatie maligne presentano un aumentato rischio di progressione da infezione tubercolare latente (ITBL) a tubercolosi (TB) attiva dovuto sia alla patologia di base sia a concomitanti trattamenti immunosoppressivi. Il test cutaneo tubercolinico (TCT) presenta una ridotta sensibilità in questi pazienti a causa di una energia cutanea. L’utilizzo dei test basati sulla produzione di interferone-gamma (IFN-γ) dopo stimolazione antigene-specifica potrebbe rappresentare uno strumento diagnostico sia per la diagnosi di ITBL sia di TB attiva in questa popolazione. Scopo dello studio era valutare la sensibilità dei test QuantiFERON-TB Gold-In tube (QFT-G IT, Cellestis,Victoria,Australia) e T-SPOT.TB (TS.TB,Oxford Immunotech,Abingdon,UK) basati sulla produzione “in vitro” di IFN-γ da parte delle cellule-T in risposta alla stimolazione con antigenimicobatterici, ESAT-6 e CFP10, rispetto al TCT in una popolazione di pazienti affetti da emolinfopatia maligna al momento della diagnosi e durante terapia immunosoppressiva. Metodi. TCT, QFT-G e TS.TB sono stati eseguiti simultaneamente nei pazienti con emolinfopatie maligne al momento della diagnosi e durante la terapia. Risultati. Sono stati arruolati 93 pazienti (età media 61.3±14.5 anni): alla diagnosi, 10/93(10.7%), 24/93(25.8%) e 18/93(19.4%) pazienti sono risultati positivi rispettivamente con TCT, TS.TB e QFT-G IT. Il test QFT-G IT mostrava un maggior numero di risultati indeterminati (4.3%) rispetto al TS.TB (0%). Durante trattamento immunosoppressivo, 3/42 (7.1%), 7/42 (16.7%) e 5/42(11.9%) pazienti sono risultati positivi rispettivamente con TCT, TS.TB e QFT-G IT. I test su sangue mostravano un buon livello di concordanza sia al momento della diagnosi (k=0.57) sia durante trattamento immunosoppressivo (k=0.61). Conclusioni. Questi risultati indicano che un numero significativo di pazienti con emolinfopatia maligna può avere una ITBL non diagnosticata dal TCT. Inoltre, questi risultati mostrano che i due test su sangue sono minimamente influenzati dal trattamento immunosoppressivo e potrebbero essere proposti come standard diagnostico per la diagnosi di ITBL in questi pazienti. 031