REVISED MANUSCRIPT-CVI00321-09 (Revision #1) 1 The immunologic paradox in the diagnosis of 2 tuberculous meningitis 3 Sung-Han Kim and Yang Soo Kim 4 Department of Infectious Diseases, Asan Medical Center, University of Ulsan College 5 of Medicine, Seoul, Republic of Korea

6 Corresponding author and reprints: Yang Soo Kim, MD, Department of Infectious 7 Disease, Asan Medical Center, University of Ulsan College of Medicine, 388-1 8 Poonganp dong, Songpa-gu, Seoul, 138-736, Republic of Korea (e-mail: 9 yskim@www.amc.seoul.kr). 10 Tel: 82-2-3010-3305 FAX: 82-3010-6970 11 12 Words counts of abstracts- 45 13 Words counts of text- 1080 14 Number of Figure- 1 15 Short title- immunologic paradox in TB meningitis 16 * No author received financial support. 17 * There are no potential conflicts of interest for any authors. 18

Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.00321-09 CVI Accepts, published online ahead of print on 21 October 2009

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Abstract 19 20 We report a patient with microbiologically documented tuberculous meningitis 21

showing that the therapeutic paradox, a therapy-induced switch to a neutrophil-22 predominant situation in the differential cell count of cerebrospinal fluid, had a 23 correlation with a immunologic paradox, a increased Mycobacterium tuberculosis-24 specific IFN-γ-producing T cell response. 25 26 27 28 29 30 31 32 33 34 35 36 37

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Case report 38 A 34-year-old female patient presented with a 1 week history of general malaise, 39

headache, and fever of 38.5oC. Examination of the cerebrospinal fluid (CSF) on the first 40

day revealed a lymphocytic pleocytosis (WBC 130/mm3; lymphocytes 75% and 41

polymorphonuclear cells 7%), increased protein (134 mg/dL), decreased glucose (32 42 mg/dL; ratio of glucose concentration in CSF to that in serum 0.3), and high adenosine 43 deaminase levels (12 IU/L). Microscopic examination of the CSF for acid-fast bacilli 44 was negative. Serological test for human immunodeficiency virus was negative. A brain 45 MRI showed suspicious tuberculous granulomas. A chest X-ray was normal. A 46 tuberculin skin test with 2 tuberculin units became negative (induration, 0 mm). From 47 the time of admission, she was treated with isoniazid, rifampin, ethambutol, and 48 pyrazinamide. Dexamethasone was given on the first day and tapered off over 4 weeks. 49 After initiation of antituberculous therapy her symptoms began gradually to improve. 50 Follow-up examinations of the CSF on day 14 and day 28 revealed a pleocytosis (WBC 51 120/mm

3; lymphocyte 49% and polymorphonuclear cells 42% and WBC 42/mm

3; 52

lymphocyte 82% and polymorphonuclear cells 4%, respectively), normal protein (52 53 mg/dL and 43 mg/dL, respectively), and decreased glucose levels (30 mg/dL and 41 54 mg/dL, respectively). Later, the CSF sample taken on day 0 grew M. tuberculosis 4 55

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weeks later, and an anti-tuberculous susceptibility test revealed that the M. tuberculosis 56 was susceptible to all drugs tested. On day 0, day 14, and day 28, we performed 57 enzyme-linked immunospot (ELISPOT) assays to detect interferon-γ-secreting T-cells in 58 peripheral blood mononuclear cells (PBMC) and cerebrospinal fluid mononuclear cells 59 (CSF-MC), stimulated by two antigens, early secretory antigenic target-6 and culture 60 filtrate protein-10. The ELISPOT assays (T-SPOT.TB, Oxford Immunotec, Abingdon, 61 UK) were performed as described in a previous study (6). Briefly, PBMC were 62 immediately (within 30 minutes) separated from 8-mL samples of peripheral venous 63 blood. Concurrent with venous sampling, 5 to 10-mL samples of CSF were obtained, 64 and CSF-MC was separated from the CSF within 30 minutes of sampling. The cells 65 were suspended in AIM-V media (GIBCO, Rockville, MD, USA) at a concentration of 66 2.5 × 10

6 cells/mL PBMC and 2.5 × 10

6 cells/mL CSF-MC. The prepared PBMC and 67

CSF-MC were plated (2.5 × 105 cells/well) on plates pre-coated with anti-human IFN- γ 68

antibody and cultured for 18 hours. Spots were then counted using an automated 69 microscope (ELiSpot 04 HR; Autoimmune Diagnostika GmbH, Strassberg, Germany). 70 The detailed results of the ELISPOT assays are shown in the Figure. The frequencies of 71 IFN-γ-secreting T-cells in CSF-MC increased 2 weeks after anti-tuberculous therapy 72 despite of clinical improvement and then slightly decreased 4 weeks after anti-73

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tuberculous therapy. However, the frequencies of IFN-γ-secreting T-cells in PBMC 74 slightly decreased 2 weeks after anti-tuberculous therapy and then increased 4 weeks 75 after anti-tuberculous therapy. 76

77 78 The diagnosis of tuberculous meningitis (TBM) is challenging. Therefore, if TBM is 79

seriously suspected, many physicians usually begin empirical anti-tuberculous therapy 80 and reconsider the diagnosis a few weeks after treatment commences (3). In this 81 problematic clinical situation, a phenomenon known as the “therapeutic paradox”, 82 revealing a therapy-induced switch to a neutrophil-predominant situation in the 83 differential cell count of CSF, has been regarded by some authors as pathognomonic of 84 TBM (5,12).

It has been postulated that this phenomenon arises because of a 85

hypersensitivity reaction related to the release of tubercular proteins during anti-86 tuberculous therapy (2,4). However, to our knowledge, there has been no report 87 showing that this hypersensitive reaction has a correlation with in vitro cell-mediated 88 immunity such as a Mycobacterium tuberculosis-specific IFN-γ-producing T cell 89 response. In this report we characterize an “immunologic paradox” in a patient with 90 microbiologically documented TBM. 91

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We used the term of “immunologic paradox” as a phenomenon revealing a therapy-92 induced increase of M. tuberculosis-speicific T cell response in the CSF or peripheral 93 blood. Arias-Bouda et al. reported that an initial increase in antibody levels was 94 observed in the early phase of treatment for 36% of all tuberculosis patients (1). Nicol et 95 al. also showed an initial increased ELISPOT response to ESAT-6 during the first month 96 of treatment, followed by a progressive decreased ELISPOT response to both ESAT-6 97 and CFP-10 (10). We assume that this is another representation of the immunologic 98 paradox. These phenomena could be explained by an intense stimulation of the humoral 99 and cell-mediated immune responses by antigens released from killed bacteria (1,2). 100 Several reports on the therapeutic paradox in TBM have been described. Sütalas et al. 101

showed that the therapeutic paradox developed in one-third of patients with TBM, and 102 clinical deterioration was found in half of such patients (12). Garcia-Monco et al. also 103 reported a patient who developed the therapeutic paradox without clinical deterioration 104 (5). However, they did not characterize any association between shifted responses of 105 polymorphonuclear dominance and increased cell-mediated immune responses to M. 106 tuberculosis antigens. In this case report, we clearly show that the therapeutic paradox 107 was associated with the immunologic paradox of increased cell-mediated immune 108 responses to M. tuberculosis-specific antigens. Interestingly, the immunologic paradox 109

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shown by CSF-MC preceded that exhibited by PBMC in our patient. This is plausible in 110 view of our previous finding that M. tuberculosis-specific T-cells are more 111 compartmentalized to the CSF or peritoneal fluid than to the circulating blood in 112 patients with TBM or TB peritonitis (6,7). However, further studies are needed to 113 determine the proportion of patients with TBM who show immunologic paradoxes in 114 CSF-MC or PBMC. It also remains to be determined whether these immunologic 115 responses a few weeks after commencement of treatment could assist in differentiating 116 TBM from other viral or bacterial meningitides. 117

Interleukin (IL)-8, a neutrophil attracting chemokine, is known to be made by a variety 118 of leukocyte populations following stimulation by M. tuberculosis (9). It is interesting 119 issue that biomarkers such as IL-8 can predict patients with TB meningitis who will 120 subsequently develop the therapeutic paradox after anti-tuberculous therapy. Indeed, 121 one study reported that IL-8 is elevated in tuberculous pleural effusions (11). NK cells 122 provide a first line defense against many infections by lysis of infected cells as well as 123 by secretion of antiviral cytokines such as IFN-γ (8). So, IFN-γ-producing spots in the 124 ELISPOT assay do not measure CD4+ or CD8+ T cells directly since other IFN-γ cells, 125 such as NK or non-cytotoxic cells, also contribute to the IFN-γ-producing spots. So, 126 further studies are needed on these issues. 127

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In conclusion, our study suggests that appearance of the immunologic paradox in 128 repeated spinal punctures or serial blood samples in a patient with suspected TBM may 129 give a promising clue to the presence of the most diagnostically difficult form of 130 tuberculosis. 131

132 133 134

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References 135 1. Arias-Bouda, L. M., S. Kuijper, V. der Werf A, L. N. Nguyen, H. M. Jansen, and 136 A. H. Kolk. 2003. Changes in avidity and level of immunoglobulin G antibodies to 137 Mycobacterium tuberculosis in sera of patients undergoing treatment for pulmonary 138 tuberculosis. Clin. Diagn. Lab. Immunol. 10:702–709. 139 2. Be, N. A., K. S. Kim, W. R. Bishai, and S. K. Jain. 2009. Pathogenesis of central 140 nerve system tuberculosis. Curr. Mol. Med. 9:94-99. 141 3. Donald, P. R. and J. F. Schoeman. 2004. Tuberculous meningitis. N. Engl. J. Med. 142 351:1719-1720. 143 4. Garcia-Monco, J. C. 1999. Central nervous system tuberculosis. Neurol. 144 Clin.17:737-759. 145 5. Garcia-Monco, J. C., E. Ferreira, and M. Gomez-Beldarrain. 2005. The 146 therapeutic paradox in the diagnosis of tuberculous meningitis. Neurology. 65:1991-147 1992. 148 6. Kim, S. H., K. Chu, S. J. Choi, K. H. Song, H. B. Kim, N. J. Kim, S. H. Park, B. 149 W. Yoon, M. D. Oh, and K. W. Choe. 2008. Diagnosis of central nervous system 150 tuberculosis by T-cell-based assays on peripheral blood and cerebrospinal fluid 151 mononuclear cells. Clin. Vaccine. Immunol. 15:1356-1362. 152

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7. Kim, S. H., O. H Cho, S. J. Park, B. D. Ye, H. Sung, M. N. Kim, S. O. Lee, S. H. 153 Choi, J. H. Woo, and Y. S. Kim. 2009. Diagnosis of abdominal tuberculosis by T-cell-154 based assays on peripheral blood and peritoneal fluid mononuclear cells. J. Infect. (in 155 press, 2009 Sep 21 Epub ahead of print) 156 8. Kirwan, S., D. Merriam, N. Barsby, A. McKinnon, and D. N. Burshtyn. 2006. 157 Vaccinia virus modulation of natural killer cell function by direct infection. Virology. 158 30:75-87. 159 9. Lyon, M. J., T. Yoshimura T., and D. N. McMurray. 2004. Interleukin (IL)-8 160 (CXCL8) induces cytokine expression and superoxide formation by guinea pig 161 neutrophils infected with Mycobacterium tuberculosis. Tuberculosis (Edinb). 84:283-162 292. 163 10. Nicol, M. P., D. Pienaar, K. Wood, B. Eley, R. J. Wilkinson, H. Henderson, L. 164 Smith, S. Samodien, and D. Beatty. 2005. Enzyme-linked immunospot assay 165 responses to early secretory antigenic target 6, culture filtrate protein 10, and purified 166 protein derivative among children with tuberculosis: implications for diagnosis and 167 monitoring of therapy. Clin. Infect. Dis. 40:1301-1308. 168 11. Supriya, P., P. Chandrasekaran, and S. D. Das. 2008. Diagnostic ultility of 169 interferon-γ-induced protein of 10 kDa (IP-10) in tuberculous pleurisy. Diagn. 170

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Microbiol. Infect. Dis. 62:186-192. 171 12. Sütlaş, P. N., A. Unal, H. Forta, S. Senol, and D. Kirbaş. 2003. Tuberculous 172 meningitis in adults: review of 61 cases. Infection. 31:387-391. 173

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Figure legend. Evolution of M. tuberculosis-specific T-cell responses in a patient with 174 tuberculous meningitis. The enzyme-linked immunospot assays were performed using 175 2.5 × 10

5 peripheral mononuclear cells (PBMC) or 2.5 × 10

5 mononuclear cells from 176

cerebrospinal fluid (CSF-MC) on day 0, day 14, and day 28 after anti-tuberculous 177 therapy. The diagnosis was confirmed by isolating M. tuberculosis from a culture of 178 cerebrospinal fluid. Data are presented as spot forming cells/2.5 × 10

5 PBMC or CSF-179

MC. TNTC, too numerous to count accurately. 180

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Day 0

Day 28

Day 14

PBMC

PBMC

PBMC

CSF-MC

Positive control ESAT-6 CFP-10 negative control

CSF-MC

Positive control ESAT-6 CFP-10 negative control

CSF-MC

TNTC

TNTC

108 TNTC 63 TNTC0TNTC 0

15 TNTC TNTC TNTC0TNTC 2

TNTC 340 TNTC TNTC TNTC3TNTC 2

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