Diagnostic Accuracy of Intracellular Mycobacterium. tuberculosis Detection for Tuberculous Meningitis

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1 Diagnostic Accuracy of Intracellular Mycobacterium tuberculosis Detection for Tuberculous Meningitis Running title: Laboratory diagnosis of tuberculous Guo-Dong Feng (MD PhD);Ming Shi(MD PhD); Lei Ma(MD PhD); Ping Chen(MD PhD); Bing-Ju Wang(MD);Min Zhang(MD); Xiao-Lin Chang(MD); Xiu-Chu Su(MD); Yi-Ning Yang(BS); Xin-Hong Fan(MD); Wen Dai(BS); Ting-Ting Liu(BS); Ying He(BS); Ting Bian (BS); Li-Xin Duan(MD); Jin-Ge Li(MD); Xiao-Ke Hao (MD PhD); Jia-Yun Liu (MD PhD); Xing Xue(MD PhD); Yun-Zhang Song(MD); Hai-Qin Wu(MD); Guo-Qiang Niu(MD); Li Zhang(MD); Cui-Juan Han(MD); Hong Lin(MD); Zhi-Hui Lin(MD);Jian-Jun Liu(MD); Jian Jian(MD); Jin-She Zhang(MD); Ye Tian(MD); Bai-Yu Zhou(MD); Jing Wang(MD); Chang-Hu Xue(MD); Xiao-Fang Han(MD); Jian-feng Wang(MD); Shou-Lian Wang(MD); Guy E. Thwaites (MD PhD; Gang Zhao(MD PhD) Online Data Supplement E1

2 Table E1. The admission clinical profile, treatment and outcome of patients without TBM, but with either a positive MZN or ESAT-6 stain Test result sex Age (years) Glasgow coma score (/15) Diagnosis Treatment Anti TB drugs Steroids Outcome ESAT-6 + Female MZN and ESAT-6 + Male MZN + Male MZN + Male MZN + Male ESAT-6+ Male 29 8 MZN + Male 31 9 ESAT-6 and MZN + Male 3 12 Cryptococcal Cryptococcal Cryptococcal Cryptococcal Cryptococcal Bacterial Bacterial Bacterial Amphotericin B Flucytosine NO NO Bad Amphotericin B Flucytosine NO NO Bad Amphotericin B Flucytosine NO NO Dead Amphotericin B Flucytosine NO NO Good Amphotericin B Flucytosine NO NO Dead Rocephin NO Yes Good Rocephin Ceftriaxone NO Yes Good Rocephin NO Yes Good E2

3 Table E2. The admission CSF profile of patients without TBM, but with either a positive MZN or ESAT-6 stain Test result CSF cytology white blood /mm 3 Monocyte % Lymphocyte % Neutrophil % CSF chemistry Protien g/l ESAT MZN and ESAT-6 + Glucose mmol/l MZN MZN MZN ESAT MZN ESAT-6 and MZN E3

4 Table E3. Baseline clinical variables associated with a positive modified ZN and ESAT-6 stain in patients with a final diagnosis of TBM Variable MZN positive MZN negative P-value ESAT-6 positive ESAT-6 negative P-value (n=205) (n=75) (n=166) (n=91) Age (years) 32 (2-87) 36 (2-87) (2-82) 33 (2-87) Duration of illness (days) 7 (1-288) 9 (2-251) (1-288) 9 (1-191) >1 day of anti-tb drugs 24 (11.7%) 1 (1.3%) (10.2%) 5 (5.5%) MRC grade (65.0%) 26 (69.4%) 104 ( ( (25.0%) 10 ( ( ( (10.0) 4 (8.2) 16 (9.9 4 (7.3 Chest X-ray appearances of active 28 (13.7%) 3 (4.0%) (12.7%) 4 (4.4) tuberculosis CSF total white cells (/mm 3 ) 95 (0-5420) 24 (0-5300) < (0-5300) 31 (0-1178) <0.001 CSF total neutrophils (/mm 3 ) 3 (0-4688) 0 (0-4585) (0-4585) 0 (0-689) CSF total monocytes (/mm 3 ) 7 (0-488) 4 (0-583) (0-583) 4 (0-371) CSF total lymphocytes (/mm 3 ) 48 (0-995) 18 (0-689) < (0-995) 20 (0-689) <0.001 CSF glucose (mmol/l) 2.7 ( ) 2.4 ( ) ( ) 2.4 ( ) E4

5 CSF protein (g/dl) 0.9 ( ) 0.8 ( ) ( ) 0.8 ( ) 0.07 E5

6 Supplementary figure legends. Figure E1. Example of slides positive for MZN and ESAT-6. CSF MZN and ESAT-6 stain on a patient with cryptococcal. A. The coexistence of acid-fast bacteria (showed in the upper right inset, arrow) with cryptococcus (showed in the lower left inset); B. The coexistence of ESAT-6-positive monocyte (showed in the upper right inset) with cryptococcus (showed in the lower left inset). Insets in (A) and (B) were the magnification views (100x) of boxed areas in their respective panels (40x). ESAT-6 positive CSF monocyte (large arrow) in a patient with culture-confirmed tuberculous (x100). C. Acid-fast bacillus (large arrow) within a CSF neutrophil following MZN stain (x100). D. In all cases, the cells were counterstained with methylene blue. Figure E2. The numbers of intracellular bacteria seen in CSF white cells following the modified Ziehl-Neelsen stain Figure E3. The mean proportions of CSF white cells staining positive for AFB by the modified ZN stain (error bars are 1 standard deviation) Figure E4. The numbers of CSF white cells staining positive for ESAT-6 Figure E5. The mean proportions of CSF white cells staining positive for ESAT-6 (error bars are 1 standard deviation) E6

7 Supplementary figure E1. C D

8 Figure E2. The numbers of intracellular bacteria seen in CSF white cells following the modified Ziehl-Neelsen stain a) Definite TBM (n=37) b) Probable TBM (n=42) c) Possible TBM (n=104) d) Not TBM (n=40)

9 Figure E3. The mean proportions of CSF white cells staining positive for AFB by the modified ZN stain (error bars are 1 standard deviation) a) Definite TBM (n=37) b) Probable TBM (n=42) c) Possible TBM (n=104) d) Not TBM (n=40)

10 Figure E4. The numbers of CSF white cells staining positive for ESAT-6 a) Definite TBM (n=30) b) Probable TBM (n=46) c) Possible TBM (n=90) c) Not TBM (n=40)

11 Figure E5. The mean proportions of CSF white cells staining positive for ESAT-6 (error bars are 1 standard deviation) a) Definite TBM (n=30) b) Probable TBM (n=46) c) Possible TBM (n=90) d) Not TBM (n=40)

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