Anti-Kp 90 lga Antibodies in the Diagnosis of Active Tuberculosis*

Anti-Kp 90 lga Antibodies in the Diagnosis of Active Tuberculosis* Sevtap Ankan, MD; Serdar Tuncer, MD; Durdal Us, PhD; Serhat Unal, MD; and $emsettin Usta9elebi, PhD Background: Accurate diagnosis of active tuberculosis (TB) has been difficult historically, yet a great demand persists for a rapid and reliable diagnostic method. Detection of Mycobacterium tuberculosis anti-kp 90 IgA antibodies is one of the more novel techniques. Study objectives: To evaluate the diagnostic value of a recently developed enzyme-linked immunosorbent assay (ELISA) test, which detects IgA antibodies against M tuberculosis Kp 90 antigen, and to compare the results with conventional diagnosis and the polymerase chain reaction (PCR) method. Participants: Serum, ethylenediaminetetraacetic acid (EDTA)-blood, and body fluid samples were obtained from 51 patients with active TB and 71 control subjects. The clinical diagnosis of TB was supported by a positive culture (n = 6), detection of acid-fast bacilli on smear (n = 35), or both (n = 10). Measurements and results: IgA antibodies were detected in sera and/or body fluid samples from 82% of patients with TB and 10% of controls. M tuberculosis DNA was detected in body fluid sample of96% and blood sample of 49% of patients with TB by PCR. None of the blood and 5.6% of the body fluid specimens from controls were PCR-positive. Conclusions: Anti-Kp 90 IgA antibodies were detected using ELISA in 78% of serum and 69% of body fluids from patients with TB, therefore, this test is promising for the diagnosis of active TB and appears to be more reliable, particularly for body fluid samples. (CHEST 1998; 114:1253-1257) Key words: antigen Kp 90; enzyme-linked immunosorbent assay; IgA; polymerase chain reaction; tuberculosis Abbreviations: A60 = an tigen 60; AFB = acid-fast bacilli; BCG = Calmette-Guerin bacillus; BF = body fluid; CSF =cerebrospinal fluid; EDTA = ethylenediaminetetraacetic acid; ELISA= enzyme-linked immunosorbent assay; PB = peripheral blood; PCR = polymerase chain reaction; PPD = purified protein derivative; TB = tuberculosis The incidence of Mycobacterium tuberculosis infection has increased in many parts of the world, including Turkey, in recent years. 1 2 Diagnosis of tuberculosis (TB ), especially in its early stages, is difficult because the clinical features are nonspecific and the usual diagnostic procedures show low sensitivity, are time-consuming, or both. If the global assessment of clinical, microbiological, radiologic, and pathologic findings is suggestive of TB, antituberculous chemotherapy is often empirically initiated before the culture results are available. 3 Faster, more sensitive and specific methods of diagnosing TB are needed, and there has been tremendous progress in recent years to develop rapid and reliable *From the Hacettepe University School of Medicine, Ankara, Turkey. Manuscript received September 3, 1997; revision accepted May 5, 1998. Correspondence to: Diirdal Us, PhD, Hacettepe University School of Medicine, Department of Clinical Microbiology and Microbiology, 06100 Ankara, Turkey laboratory procedures. Apart from the routine diagnostic methods (mycobacterial culture and direct examination of smears and skin tests) polymerase chain reaction (PCR) and serodiagnosis are recently applied techniques for which the reliability has not been established.4.5 The present study was undertaken to estimate the value of detecting anti-kp 90 IgA antibodies using enzyme-linked immunosorbent assay (ELISA) in the diagnosis of active TB. MATERIALS AND METHODS Study Groups and Clinical Samples Patients: The study included a total of 51 patients (34 males and 17 females) with a mean age of 44.4 years (range, 17 to 74 years) who were diagnosed as having active TB in the Hacettepe Un iversity Department of Medicine Section of Infectious Diseases over a 33-month period from January 1994 to September 1996. Mycobacterial culture was performed from the clinical specimens of all patients enrolled in the study. The diagnosis of CHEST I 114 I 5 I NOVEMBER, 1998 1253

active TB was established by the isolation of M tuberculosis on Middlebrook 7H10 medium (n = 6), the demonstration of acidfast bacilli (AFB) on direct examination of the clinical specimens (n = 35), or the accomplishment of positive results by both methods (n = 10). The patients diagnosed by histopathologic findings and/or response to antituberculous therapy were excluded from the comparative laboratory analysis in case of the lack of mycobacterium culture. Of 51 patients included in the study, 23 (including 3 AIDS patients) and 19 were diagnosed to have pulmonary TB and TB peritonitis, respectively. The remaining 9 patients had TB at other sites: 4 pericarditis, 2 arthritis, 2 psoas abscess, and 1 meningitis. Serum, ethylenediaminetetraacetic acid (EDTA) blood, and one body fluid (BF) sample (related to the site of infection) were obtained from each patient with TB included in the study. The samples were acquired prior to antituberculous therapy. The purified protein derivative (PPD) skin test results were interpreted as positive if the diameter of the induration was > 10 mm, which was the case for all but six patients. Follow-up Studies: Isoniazid, rifampicin, ethambutol, and pyrazinamide combination therapy was administered to patients with TB for 2 months, followed by 4 months of isoniazid and rifampicin treatment. Sera and EDTA-blood samples of 14 patients with TB could be obtained 2 to 4 months after the initiation, and 7 samples of the same patients could be obtained at the end of antituberculous therapy. Control Subjects: During the study period, 71 immunocompetent subjects ( 43 males and 28 females; age range, 17 to 84 years; mean age, 45.5 years) who were in control for underlying disorders other than TB, were considered as the control group. The underlying disorders comprising the control subjects were as follows: congestive heart failure (24), empyema (9), chronic obstructive pulmonary disease (9), chronic hepatic failure (7), acute bacterial meningitis (7), chronic renal failure (4), nephrotic syndrome (2), rheumatoid arthritis (2), familial Mediterranean fever (2), septic arthritis (2), mesenteric vascular occlusion (1), peritonitis (1), and staphylococcal abscess (1). One serum and one BF sample from each control subject were tested in the study. BF samples obtained from the control subjects were as follows: pleural fluid (32), peritoneal fluid (17), pericardia! fluid (10), cerebrospinal fluid (CSF, 7), arthrocentesis fluid (4), and pus (1). Of 71 control subjects, the PPD skin test was positive in 26. Mycobacterial Culture and Direct Microscopic Examination: Body fluid samples obtained from patients and the control group were cultured on Middlebrook (Middlebrook 7H10) medium after concentration procedures. 6 Direct microscopic examination of the pellet was performed by Ziehl-Neelsen staining. IgA Measurement: Sera and BF samples were examined by ELISA for the presence of IgA antibodies against mycobacterial Kp 90 immunocrossreactive antigenic compound. Commercially available kits were supplied by Kreatech (Amsterdam, The Netherlands). The Kp 90 antigen, prepared by the manufacturer from Calmette-Guerin bacillus (BCG), is the pellet obtained by centrifuging sonicated, French-pressed, broken bacilli at 90,000g for 2 hat 4 C 7 Two microliters of each sample was used, and the test was performed according to the manufacturer's instructions. The results were interpreted spectrophotometrically by measuring the optical density at 450-nm wavelength. PCR Sample Preparation: For BF samples, 0.5 ml (0.2 ml for arthrocentesis material) of each specimen was incubated with an equal volume of l M NaOH for 15 min. After centrifugation, the pellet was washed twice with TE buffer (10 mm Tris-HCI, ph 7.5, 1 mm EDTA), followed by suspension in 100 ILL of distilled water. Ten milliliters of lysis buffer containing 100 mm NaCI, 10 mm Tris-HCl (ph 8), 25 mm EDTA, and 0.5% sodium dodecyl sulfate was added to 5 ml of peripheral blood (PB). The suspension was incubated on a rotary shaker for 30 min and centrifuged at 1,500g for 15 min. After discarding the supernatant, the same procedure was repeated once more. The pellet was suspended in 2 ml of TE buffer and filtered through disposable filters of 5-ILm pore width (Vestar; San Dimas, CA). The suspension was centrifuged at 10,000g for 2 min, and the pellet was washed with distilled water twice and suspended with 50 ILL of distilled water. Final suspensions of BF and PB-EDTA samples were incubated in boiled water for 10 min after the sample-preparation steps. Five microliters of the suspension supernatant was used for amplification. In order to determine the presence of reaction inhibition, M tuberculosis ATCC 25177 (1000 colony forming units) was added to one aliquot of each sample, followed by the sample-preparation steps, as mentioned above. The samples that were detected to have enzymatic inhibition were prepared as follows: 200 ILL of body fluid and a lyzed and filtered blood pellet were incubated in 5 0 0 - diges ~ L L tion buffer (100 mm NaCI, 10 mm Tris-HCI [ph 8], 25 mm EDTA, 0.5% sodium dodecyl sulfate, 0.2 mglml proteinase-k) at 65 C overnight. Phenol-chloroform extraction and precipitation procedures were done and DNA was suspended in 20 ILL of distilled water. Amplification: Primers specific to the 123-base pair fragment of the repetitive insertion element IS6110, in the chromosome of the M tuberculosis complex, were used in amplification reactions." Amplifications were carried out in 50-ILL volumes of reaction mixture containing 50 mm KCI, 10 mm Tris-HCl (ph 9), 1% Triton X-100, 2 mm MgC1 2, 50 mm of each dntp, 20 pmol of each primer, l unit of Taq polymerase (Prom ega Biotec; Madison, WI), and a 5-ILL DNA sample. Forty cycles of denaturation at 94 C for 30 s and annealing-extension at 68 C for 90s were performed in an automated thermal cycler (Stuart Scientific; Staffordshire, UK). The reaction mixture without DNA was used as negative control. A boiled suspension of 1000 colony forming units of M tuberculosis ATCC 25177 in TE buffer was used for positive amplification control. Amplification Product Detection and Interpretation of the Results: The presence of 123-base pair amplification products was analyzed by electrophoresis of a 10-ILL amplified mixture on 2% agarose gel. Gels were stained with ethidium bromide (0.1 mglml) and photographed using an ultraviolet transilluminator. If amplification bands were obtained from both duplicates, the result was interpreted as positive. If the band was visualized only for the inhibitor control, the result was accepted to be negative. Statistical Analysis: The x 2 test (if the expected value was less than 5, the Fisher exact test was used) was used for analysis of the differences between the results of the control group and patients with TB. The correlation between IgA and PCR results was tested with the McNemar l test. Statistical tests were performed with the InStat package program (Version 2.02; GraphPad Software; San Diego, CA). A p value of< 0.05 was considered a significant difference. RESULTS PCR and IgA results with respect to the study group, origin of the specimen, culture, and direct examination results are shown in Table 1. IgA antibodies were detected in sera, BF samples, or both from 42 (82%) patients. All samples obtained from the three AIDS patients were IgA-positive. In each patient, the IgA positivity of the serum was not 1254 Clinical Investigations

Table I-Rate of M tuberculosis Positivity by PCR and IgA Test With Respect to Clinical Samples, Positive Culture, and Direct Microscopic Examination Results Compared With the Controls Conventional Methods* PCR IgA Clinical form (n) AFB,% Culture,% BFt,% PB t,% BFt,% Serumt,% Pulmonary TB (23) 21 (91 ) 9 (39) 23 (100) 10 (43) 17 (74) 19 (83) Peritonitis (19) 16 (84) 4 (21) 18 (95) 9 (50) 16 (84) 16 (84) Pericarditis ( 4) 4 1 4 3 1 3 Arthritis (2) 2 2 2 1 1 Meningitis ( l) 0 1 1 1 0 1 Psoas abscess (2) 2 0 1 0 0 0 Total patients (51 ) 45 (88.2) 16 (31.3) 4 9 (96) 25 (49) 35 (68.6) 40 (78.4) Controls (71 ) 0 0 4 (5.6) 0 2 (2.8) 7 (9.8) *Conventional methods were used only for diagnosis of active TB. The results were not comparatively evaluated \ \ ~ PCR t h or IgA. BF and!or sputum samples were used for direct examination and culture. f McNemar X 2 = 11.529 (p < 0.05). t McNemar x 2 = 10.316 (p < 0.05). Pleural fluid samples of control subjects whose sera were positive also. always associated with a positive result in that patient's BF sample. The antibodies could be detected in sera but not BF samples of seven patients. The samples displaying the mentioned discrepancy were pleural fluid (3 samples), pericardia! fluid (2), CSF (1), and peritoneal fluid (1). In contrast, BF samples but not sera of two patients were found to comprise IgA antibodies. The antibodies were positive in one pleural and one peritoneal fluid sample, each obtained from an individual patient. When the results of both sera and body fluids were considered, anti-kp 90 IgA ELISA was positive in 82% of patients who were clinically diagnosed as having TB. However, there were control subjects presenting with positive results. Out of 71, IgA ELISA results were found positive for 7 (10%) sera belonging to the patients with congestive heart failure (4), empyema (2 ), and chronic hepatic failure (1). IgA was detected in both pleural fluid sample and serum, in one patient with empyema and one patient with congestive heart failure. There was a significant difference (p < 0.05) between patients with TB and the control group in terms of positivity of PCR or IgA results. The specificity of anti-kp 90 IgA ELISA was found to be 90%, however, specificity of the method increased to 97% if only body fluids were concerned. M tuberculosis DNA was detected in blood-edt A and/or at least one BF sample of 49 (96%) patients. The Specificity of M tuberculosis PCR was found to be 94%. IgA and PCR results were not always correlated in terms of evidence of active TB. Both methods gave a positive result in 33 (65%) of the patients. However, IgA antibodies, but not M tuberculosis DNA, could be detected in two samples, one being a p eritoneal fluid sample. The correlation between PCR and IgA results in patients with TB was found to be statistically significant (McNemar x 2 = 11.529, p < o.o5). The blood PCR test was positive in 43% (10 of 23) and 54% (15 of 28) of patients with pulmonary and extrapulmonary TB, respectively. PB-PCR and serum IgA results were correlated for most but not all patients. IgA antibodies were detected in 21 of 25 (84%) PCR-positive blood samples. Among 26 blood-pcr-negative samples, 14 (54%) were IgApositive. A statistically significant correlation was detected between the PB-PCR and serum IgA results (McNemar X 2 = 10.316, p < 0.05). None of the control patients had positive PB-PCR results. M tuberculosis DNA was detected in BF samples of four control patients (three pleural and one peritoneal fluid sample) by PCR. There was no direct correlation between IgA or PCR positivity and the PPD skin test. Of the IgA-positive seven and PCR-positive four control patients, only two and one, respectively, scored positively on the PPD skin test. EDTA-blood and serum samples were obtained from 14 patients, 2 to 4 months after the initiation of antituberculous therapy. Of these, 11 had IgA antibodies and 8 had positive PB-PCR results at the time of diagnosis. Of the samples taken from these patients after the initiation of therapy, seven and five were IgA- and PCR-positive, respectively. At the end of therapy, all five blood samples yielded a negative PCR result; however, serum of one patient was still IgA-positive. DISCUSSION Because the conventional diagnostic methods are low in sensitivity, time consuming, or both, presumptive diagnosis of mycobacterial diseases is usually based on clinical findings such as persistent cough, fever, and weight loss. Although tuberculin skin tests and radiologic findings may help, the diagnosis must CHEST I 114 I 5 I NOVEMBER, 1998 1255

be confirmed by isolation and identification of the etiologic agent. 3 The novel methods for the diagnosis of mycobacterial diseases include the detection of mycobacterial structures such as cell wall components by chromatographic techniques and the detection of bacterial nucleic acid by amplification techniques such as PCR. In recent years, ELISA, proposed to confirm TB etiology by antibody detection, appeared to be a promising diagnostic method with the use of different antigenic components. 9 Until now, several mycobacterial antigens, antigen 5, purified mycobacterial glycolipids, BCG, and antigen 60 (A60) of M tuberculosis were used to develop ELISA. The diagnostic value of anti-a60 IgM, IgG, and IgA antibodies was investigated in patients with pulmonary and extrapulmonary TB in previous studies.10-13 Measurement of IgM antibodies facilitates the diagnosis of current infection or reactivation; however, IgG antibodies cannot differentiate patients with active TB from those who had TB in the preceding 2 years. 9 Because of its secretory function, detection of IgA in body fluids may be a valuable diagnostic clue pmticularly for extrapulmonary TB. In a recent rep01t, anti-kp 90 IgA antibodies were found in sera of 13 of 15 patients with proven active TB.l 4 In another study by Alifano et al, 15 62 of 88 patients with active pulmonary TB, 2 of 47 healthy volunteers, 4 of 28 patients with non-tb lung disease, and one of 12 patients \Vith healed TB were found to be IgA-positive in sera by using the same commercial kit. Our results indicate that IgA antibodies are detectable in 78 and 69% of sera and body fluids obtained from patients vvith active TB, respectively. All samples obtained from the three AIDS patients were IgA-positive. Diagnosis of TB could be confirmed in 82% of patients by detection of IgA antibody in sera, body fluids, or both. IgA antibody was found to be positive in only 10% of control subjects. When the results of both sera and body fluids were taken into account, the sensitivity and specificity of anti-kp 90 IgA ELISA was found to be 82 and 90%, respectively. However, specificity of the method increases to 97% if only body fluids were concerned, indicating that this method may be an alternative for the rapid diagnosis of M tuberculosis infections, particularly for testing body fluids. The results of this study also imply that, in terms of sensitivity, body fluids are as efficient as sera for IgA antibody detection. Descriptions of numerous PCR assays for detecting M tuberculosis in different clinical specimens and settings have been published in previous studies.16-18 Although the sensitivity and specificity varied widely (59 to 100% and 70 to 100%, respectively) depending on the laboratory protocols and clinical settings, amplification-based systems seemed to be feasible and promising for routine use in clinical laboratories. 5 We detected M tuberculosis DNA in body fluids of 96% of our patients, and we found the M tuberculosis PCR specificity to be 94%. PB lymphocyte-based PCR was proposed by Schluger et al 19 for use in the rapid diagnosis of pulmonary TB. In our preliminary PCR studies, M tuberculosis DNA was detected in PB lymphocytes of 3 of 12 patients with TB (data not shown). However, the sample-preparation method was later altered because four additional PB samples in the former trial were found to be positive, probably because the latter protocol could detect both intraand extracellular bacilli. 20 Although we obtained a higher rate of positivity in patients with extrapulmonary TB (54%) compared with pulmonary TB (43%), the difference was not significant (p > 0.05). We did not obtain amplification bands in any of the PB samples of control subjects. The higher s ensitivity (95%) and lower specificity (89%) rates of PB-based PCR in 41 patients with pulmonary TB were reported b y Condos et al. 21 This may be due to a lower detection limit and a potential risk of contamination of the nested-pcr protocol used in the latter study. At the end of antituberculous therapy, all of our cases that we could follow-up were PB-PCR-negative and one remained positive for IgA antibody. This patient responded to therapy clinically. 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