Rapid diagnosis of pulmonary TB by BAL enzyme-linked immunospot assay in an immunocompromised host

Immunocompromised patients with acid-fast bacilli (AFB) smear-negative active pulmonary tuberculosis (pTB) often present with nonspecific clinical symptoms and findings. T-cell interferon-γ release assays (TIGRA) performed on whole blood (using ELISA) or peripheral blood mononuclear cells (using enzyme-linked immunospot assay (ELISPOT)) are more sensitive for the diagnosis of Mycobacterium tuberculosis (MTB) infection than the tuberculin skin test (TST), but cannot distinguish active from latent MTB infection. The present authors report a 38-yr-old female presenting with a 3-week history of malaise, dyspnoea, fevers and coughing, who had received immunosuppressive therapies over 8 months for mixed connective tissue disease. Chest radiograph and thoracic computed tomography showed ground glass opacities in both lower lobes. The TST-induration was 0 mm and AFBs or MTB nucleic acid was not detected on sputum and bronchial secretions. However, TIGRAs performed on peripheral blood cells were reactive. A high frequency of MTB-specific T-cells compatible with the immunodiagnosis of active pTB was detected among bronchoalveolar lavage cells using ELISPOT. Antituberculous therapy was initiated 18 days before MTB was discovered on sputum cultures. Detection of Mycobacterium tuberculosis-specific T-cells in the bronchoalveolar lavage using enzyme-linked immunospot assay is a promising tool for the diagnosis of active pulmonary tuberculosis in immunocompromised patients with negative acid-fast bacilli smears.

R eactivation of latent Mycobacterium tuberculosis (MTB) infection (LTBI) is associated with significant morbidity and mortality, especially in immunocompromised individuals [1,2]. However, the diagnosis of active pulmonary tuberculosis (pTB) can be challenging in the immunocompromised host because clinical appearance and findings, such as typical radiological patterns, may be absent [3]. Additionally, in ,65% of immunocompromised patients with active pTB, the result of the tuberculin skin test (TST) is falsely negative [4]. When detection of acid-fast bacilli (AFB) in sputum or bronchial secretions is not possible and MTB DNA cannot be detected by nucleic acid amplification, the diagnosis of pTB by identification of MTB in liquid or solid culture media is delayed for several weeks [5]. In ,20% of patients with pTB, MTB cannot be recovered from cultures of bronchial secretions at all [6].
Recently, enumeration of MTB-specific T-cells from the bronchoalveolar lavage (BAL) was shown to be a promising tool for the rapid detection of patients with smear-negative pTB [7]. However, it was unclear whether the immunodiagnosis of active pTB could be also established by BAL enzyme-linked immunospot assay (ELISPOT) in immunocompromised hosts.

CASE REPORT
In January 2007, a 38-yr-old nonsmoking female presented to an outside hospital, with a 3-week history of severe malaise, progressive dyspnoea, recurrent fevers and a dry cough. Approximately 15 yrs ago, the patient fled her home country, Sri Lanka, and, following an odyssey, she was then granted asylum in Germany. The patient denied a history of TB or known TB contacts.
However, 18 months prior to admission, she gradually developed myalgias of the upper arms radiating into both hands, accompanied by recurrent swelling of the fingers, pain in the upper spine, morning stiffness and symmetric arthralgias of the knees. The diagnosis of seropositive rheumatoid arthritis was performed after finding the rheumatic factor in her serum, and treatment with systemic corticosteroids (20 mg of prednisolone equivalent) was initiated with good clinical response. Unfortunately, 8 months prior to admission, after tapering the corticosteroids to a dose of 5 mg?day -1 , the symptoms reappeared and weekly treatment with 15 mg of the folate antimetabolite methotrexate was started in order to spare corticosteroids.
The physical examination on admission revealed oedema of the hands, feet, lips and cheeks. The patient described sudden onset of cold fingers and toes in association with sharply demarcated colour changes of the skin, which was diagnosed as Raynaud's phenomenon. Increasing hyposensitivity of the finger and toe tips was also described. Upon passive movement, the patient complained of painful ankle, hand and knee joints. There were painful purple nodules on both tibiae and calves.
Screening for HIV, hepatitis B and C, Epstein-Barr virus, and Borrelia burgdorferi was negative. An anti-nuclear antibody with a titre of 1:20,480 (speckled-type) was found. Antibodies against recombinant A, C and 68 kD-ribonucleoprotein were detected. The diagnosis of mixed connective tissue disease (MCTD) was performed and treatment with prednisolone at a dosage of 20 mg was reinitiated without continuing methotrexate.
Despite these interventions, the patient suffered from ongoing fever up to 39uC. Several blood cultures remained sterile and, despite extensive radiological imaging, no focus for her febrile illness could be found. A T-cell interferon (IFN)-c release assay (TIGRA; QuantiFERON-TB Gold in-tube test TM ; Cellestis Ltd, Carnegie, Australia) on the peripheral blood was strongly positive with 3.12 IU?mL -1 (cut-off value ,0.35 IU?mL -1 ), suggesting latent infection with MTB, past TB or active TB. As the antigens used for the TIGRA, early antigenic target (ESAT)-6 and culture filtrate protein (CFP)-10 may cross-react with antigens form Mycobacterium leprae [8,9], a skin biopsy from the lesions on the lower leg was performed, which revealed a diagnosis of erythema nodosum. PCR for Mycobacterium leprae from the lesions was negative.
After 4 weeks in hospital, the patient was transferred to the present authors' clinic (Medical Clinic, Research Center Borstel, Borstel, Germany) for further diagnosis. Following admission, a high-resolution computed tomography scan of the thorax showed no pleural effusions, adenopathy, cavitations, consolidations or tree-in-bud phenomena. Symmetrical ground glass opacity predominantly in the lower lung segments, as in nonspecific interstitial pneumonitis, compatible with the diagnosis of MCTD, was demonstrated ( fig. 1) [10,11].
AFBs were not seen on three separate morning sputa. MTB DNA could not be detected in sputum of the patient by MTBspecific nucleic acid amplification technique (BDProbeTec TM ET assay; Becton Dickinson, Sparks, MD, USA). Subsequently, bronchoscopy with BAL and transbronchial biopsies was performed. The BAL differential count revealed a lymphocytic alveolitis with 77% alveolar macrophages, 18% lymphocytes, 4% neutrophils and 0.6% eosinophils. Neither AFBs nor MTB DNA could be detected on the BAL. Histopathological examination of the biopsies showed lymphocyte and neutrophil cell infiltrates without demonstration of granulomas.
Upon receipt of these results, treatment with rifampin, ethambuthol and pyrazinamid was added for quadruple antituberculous therapy. After 18 days, MTB was discovered on a sputum culture from the patient.
In TB, antigen-specific T-cells clonally expand and are recruited to the site of infection [24][25][26]. The IFN-c release measured by MTB-specific ELISPOT can be directly related to the number of antigen-specific mononuclear cells in biological specimens. Thus, it was recently shown that MTB-specific IFNc-secreting T-cells are concentrated among mononuclear cells in the BAL, in pleural effusion, ascites and cerebrospinal fluid of patients with smear-negative pulmonary or extrapulmonary tuberculosis [7,[27][28][29]. Enumerating MTB-specific T-cells among cells from the pleural effusion by ELISPOT also proved to be efficient for the diagnosis of active pleural TB in an immunocompromised patient [30].
In a prospective pilot study, MTB-specific ELISPOT was evaluated for the diagnosis of active pTB in patients with suspected pTB in whom AFBs were not detectable from three consecutive sputum smears [7]. ESAT-6 and CFP-10 peptidespecific responses were only detectable in BALMCs in the 12 patients with active pTB in the study by JAFARI et al. [7], while these responses were absent in 25 patients with LTBI or a history of TB but without current reactivation or pulmonary infiltrates of other origin.
In the present report the first evidence is provided that enumerating of Mycobacterium tuberculosis-specific T-cells by enzyme-linked immunospot assay among bronchoalveolar lavage mononuclear cells may be a useful diagnostic tool for the rapid detection of active tuberculosis in immunocompromised individuals. Large prospective multicentre trials, similar to the one conducted currently by the Tuberculosis Network European Trials group, are needed to validate the clinical utility of the Mycobacterium tuberculosis-specific bronchoalveolar lavage enzyme-linked immunospot assay and to compare its diagnostic performance with a Mycobacterium tuberculosisspecific nucleic acid amplification testing for the rapid diagnosis of pulmonary tuberculosis in patients with smearnegative acid-fast bacilli sputum findings.

DIAGNOSIS OF PULMONARY TB BY BAL-ELISPOT
A. STRASSBURG ET AL.