Accepted Manuscript Diagnostic usefulness of T-cell based assays for tuberculous meningitis in HIVuninfected patients Ki-Ho Park, Mi Suk Lee, Sun-Mi Kim, Su-Jin Park, Yong Pil Chong, Sang-Oh Lee, Sang-Ho Choi, Yang Soo Kim, Jun Hee Woo, Joong Koo Kang, Sang-Ahm Lee, Sung-Han Kim, MD PII:
S0163-4453(16)00029-3
DOI:
10.1016/j.jinf.2016.01.012
Reference:
YJINF 3667
To appear in:
Journal of Infection
Received Date: 30 October 2015 Revised Date:
6 January 2016
Accepted Date: 7 January 2016
Please cite this article as: Park K-H, Lee MS, Kim S-M, Park S-J, Chong YP, Lee S-O, Choi S-H, Kim YS, Woo JH, Kang JK, Lee S-A, Kim S-H, Diagnostic usefulness of T-cell based assays for tuberculous meningitis in HIV-uninfected patients, Journal of Infection (2016), doi: 10.1016/j.jinf.2016.01.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
REVISED MANUSCRIPT#1: YJINF-D-15-00940
2
Diagnostic usefulness of T-cell based assays for tuberculous meningitis in HIV-
3
uninfected patients
4 Ki-Ho Parka, Mi Suk Leea, Sun-Mi Kimb, Su-Jin Parkb, Yong Pil Chongb, Sang-Oh Leeb,
6
Sang-Ho Choib, Yang Soo Kimb, Jun Hee Woob, Joong Koo Kangc, Sang-Ahm Leec, and
7
Sung-Han Kimb,*
RI PT
5
9
a
SC
8
Division of Infectious Diseases, Department of Internal Medicine, Kyung Hee University
Hospital, Kyung Hee University School of Medicine, Seoul, Republic of Korea
11
b
12
Medicine, Seoul, Republic of Korea
13
c
14
Seoul, Republic of Korea
M AN U
10
Department of Infectious Diseases, Asan Medical Center, University of Ulsan College of
TE D
Department of Neurology, Asan Medical Center, University of Ulsan College of Medicine,
15
* Correspondence. Sung-Han Kim, MD, Department of Infectious Diseases, Asan Medical
17
Center, University of Ulsan College of Medicine, 88, Olympic-ro 43-gil, Songpa-gu, Seoul
18
138-736, Republic of Korea. E-mail: [email protected]
20 21
AC C
19
EP
16
Running head: ELISPOT assay for tuberculous meningitis
22
Word count for abstract: 250
23
Word count for text: 3736
24
Number of tables: 4
25
Number of figures: 3
ACCEPTED MANUSCRIPT Number of supplementary tables: 2
27
Number of supplementary figures: 1
AC C
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TE D
M AN U
SC
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26
ACCEPTED MANUSCRIPT Summary
2
Objectives: Early diagnosis and treatment of tuberculous meningitis (TBM) is essential for a
3
positive outcome, but sensitive, specific, and rapid diagnostic tests for TBM are lacking. We
4
evaluated the diagnostic utility of enzyme-linked immunosorbent spot (ELISPOT) assays in
5
HIV-uninfected patients with suspected TBM.
6
Methods: All HIV-uninfected patients with suspected TBM were prospectively enrolled at a
7
tertiary care hospital in an intermediate TB-burden country, during a 6-year period. ELISPOT
8
assays were performed on peripheral blood mononuclear cells (PBMC) and cerebrospinal
9
fluid-mononuclear cells (CSF-MC).
SC
RI PT
1
Results: Of the 276 evaluable patients, 90 (33%) were classified as having TBM (30 definite
11
cases, 19 probable, and 41 possible), and 186 (67%) as having non-TBM. When comparing
12
definite TBM versus non-TBM, the sensitivity and specificity of the PBMC ELISPOT assay
13
(≥6 spots; manufacturer’s recommended cut-off) for diagnosing TBM were 96% (95% CI,
14
82–100) and 58% (95% CI, 50–66), respectively. The CSF-MC ELISPOT assay (≥38 spots;
15
receiver operating characteristic [ROC]-derived cut-off) was a useful rule-in test with
16
specificity of 95% (96% CI, 90–98). Its sensitivity was 68% (95% CI, 45–86), which was
17
superior those of AFB smear microscopy (14%; P < 0.001) and CSF M. tuberculosis PCR
18
(41%; P = 0.07). Combining this assay with M. tuberculosis PCR, clinical score, and both
19
together increased sensitivity to 86%, 91%, and 95%, respectively, while retaining about 95%
20
specificity.
21
Conclusions: The CSF-MC ELISPOT assay appears to be a rapid and accurate rule-in test for
22
the diagnosis of TBM and a useful adjunct for diagnosing TBM in HIV-uninfected patients.
AC C
EP
TE D
M AN U
10
23 24
Keywords: Tuberculosis; Meningitis; Cerebrospinal fluid; Diagnosis.
25 1
ACCEPTED MANUSCRIPT Introduction
27
Tuberculous meningitis (TBM) is the most severe form of tuberculosis, causing substantial
28
morbidity and mortality. Early diagnosis and treatment is essential to save lives and reduce
29
long-term morbidity.1,2 However, TBM is sometimes difficult to diagnose with certainty,
30
especially in its early phase, because the initial clinical features are non-specific and the
31
laboratory tests are insensitive. Cerebrospinal fluid (CSF) acid-fast bacilli (AFB) smear
32
microscopy, although rapid and inexpensive, has very low sensitivity in routine clinical
33
practice (less than 20%).3-5 Culture is the gold standard for diagnosing TBM, but it is not
34
useful for deciding whether to treat for TBM due to the time required for a positive result and
35
suboptimal sensitivity.
SC
M AN U
36
RI PT
26
Efforts to identify new diagnostic markers for TBM in CSF have been ongoing, mostly in HIV-infected populations, and some of the markers identified may be used as aids in
38
diagnosing TBM.6-14 Currently, there is no single test with optimal sensitivity in patients with
39
suspected TBM. New molecular-based microbiological tests, as well as AFB smear
40
microscopy and mycobacterial culture, had lower sensitivity in HIV-uninfected patients
41
because the bacterial loads may have been lower than in HIV-infected patients.8,10 A recently
42
developed fully automated real-time polymerase chain reaction (PCR), Xpert MTB/RIF, for
43
the diagnosis of TBM was less sensitive among HIV-uninfected patients (0–48%) than among
44
HIV-infected patients (67–79%).8,10 Furthermore, its diagnostic utility was hampered by a
45
very poor sensitivity for probable or possible TBM, reported to be as low as 0–3%.8,10
AC C
EP
TE D
37
46
Where the bacterial burden is low, an immunodiagnostic tool may be more useful than
47
microbiological tools for detecting Mycobacterium tuberculosis at infection sites. Recently,
48
interferon-gamma release assays (IGRAs) using mononuclear cells that are
49
compartmentalized at the site of infection, such as pleural fluid,15,16 peritoneal fluid,17,18
50
pericardial effusion,19 and CSF,6 have yielded promising results in the diagnosis of active TB. 2
ACCEPTED MANUSCRIPT In our earlier preliminary studies undertaken among HIV-uninfected patients (with 50–82
52
evaluable CSF samples) we concluded that the enzyme-linked immunosorbent spot
53
(ELISPOT) assay using CSF-mononuclear cells (CSF-MC) appeared to be a promising
54
adjunct to current tests for diagnosing TBM.13,20 This study builds on those earlier
55
publications.13,20 In it we evaluated on a larger number of subjects, the clinical utility of
56
peripheral blood mononuclear cells (PBMC) and CSF-MC ELISPOT assays for diagnosing
57
TBM. The study was conducted in a country with an intermediate TB burden and a low
58
prevalence of HIV infection (5 days (P < 0.001) and a miliary pattern on chest radiograph (P
S0163-4453(16)00029-3
DOI:
10.1016/j.jinf.2016.01.012
Reference:
YJINF 3667
To appear in:
Journal of Infection
Received Date: 30 October 2015 Revised Date:
6 January 2016
Accepted Date: 7 January 2016
Please cite this article as: Park K-H, Lee MS, Kim S-M, Park S-J, Chong YP, Lee S-O, Choi S-H, Kim YS, Woo JH, Kang JK, Lee S-A, Kim S-H, Diagnostic usefulness of T-cell based assays for tuberculous meningitis in HIV-uninfected patients, Journal of Infection (2016), doi: 10.1016/j.jinf.2016.01.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
REVISED MANUSCRIPT#1: YJINF-D-15-00940
2
Diagnostic usefulness of T-cell based assays for tuberculous meningitis in HIV-
3
uninfected patients
4 Ki-Ho Parka, Mi Suk Leea, Sun-Mi Kimb, Su-Jin Parkb, Yong Pil Chongb, Sang-Oh Leeb,
6
Sang-Ho Choib, Yang Soo Kimb, Jun Hee Woob, Joong Koo Kangc, Sang-Ahm Leec, and
7
Sung-Han Kimb,*
RI PT
5
9
a
SC
8
Division of Infectious Diseases, Department of Internal Medicine, Kyung Hee University
Hospital, Kyung Hee University School of Medicine, Seoul, Republic of Korea
11
b
12
Medicine, Seoul, Republic of Korea
13
c
14
Seoul, Republic of Korea
M AN U
10
Department of Infectious Diseases, Asan Medical Center, University of Ulsan College of
TE D
Department of Neurology, Asan Medical Center, University of Ulsan College of Medicine,
15
* Correspondence. Sung-Han Kim, MD, Department of Infectious Diseases, Asan Medical
17
Center, University of Ulsan College of Medicine, 88, Olympic-ro 43-gil, Songpa-gu, Seoul
18
138-736, Republic of Korea. E-mail: [email protected]
20 21
AC C
19
EP
16
Running head: ELISPOT assay for tuberculous meningitis
22
Word count for abstract: 250
23
Word count for text: 3736
24
Number of tables: 4
25
Number of figures: 3
ACCEPTED MANUSCRIPT Number of supplementary tables: 2
27
Number of supplementary figures: 1
AC C
EP
TE D
M AN U
SC
RI PT
26
ACCEPTED MANUSCRIPT Summary
2
Objectives: Early diagnosis and treatment of tuberculous meningitis (TBM) is essential for a
3
positive outcome, but sensitive, specific, and rapid diagnostic tests for TBM are lacking. We
4
evaluated the diagnostic utility of enzyme-linked immunosorbent spot (ELISPOT) assays in
5
HIV-uninfected patients with suspected TBM.
6
Methods: All HIV-uninfected patients with suspected TBM were prospectively enrolled at a
7
tertiary care hospital in an intermediate TB-burden country, during a 6-year period. ELISPOT
8
assays were performed on peripheral blood mononuclear cells (PBMC) and cerebrospinal
9
fluid-mononuclear cells (CSF-MC).
SC
RI PT
1
Results: Of the 276 evaluable patients, 90 (33%) were classified as having TBM (30 definite
11
cases, 19 probable, and 41 possible), and 186 (67%) as having non-TBM. When comparing
12
definite TBM versus non-TBM, the sensitivity and specificity of the PBMC ELISPOT assay
13
(≥6 spots; manufacturer’s recommended cut-off) for diagnosing TBM were 96% (95% CI,
14
82–100) and 58% (95% CI, 50–66), respectively. The CSF-MC ELISPOT assay (≥38 spots;
15
receiver operating characteristic [ROC]-derived cut-off) was a useful rule-in test with
16
specificity of 95% (96% CI, 90–98). Its sensitivity was 68% (95% CI, 45–86), which was
17
superior those of AFB smear microscopy (14%; P < 0.001) and CSF M. tuberculosis PCR
18
(41%; P = 0.07). Combining this assay with M. tuberculosis PCR, clinical score, and both
19
together increased sensitivity to 86%, 91%, and 95%, respectively, while retaining about 95%
20
specificity.
21
Conclusions: The CSF-MC ELISPOT assay appears to be a rapid and accurate rule-in test for
22
the diagnosis of TBM and a useful adjunct for diagnosing TBM in HIV-uninfected patients.
AC C
EP
TE D
M AN U
10
23 24
Keywords: Tuberculosis; Meningitis; Cerebrospinal fluid; Diagnosis.
25 1
ACCEPTED MANUSCRIPT Introduction
27
Tuberculous meningitis (TBM) is the most severe form of tuberculosis, causing substantial
28
morbidity and mortality. Early diagnosis and treatment is essential to save lives and reduce
29
long-term morbidity.1,2 However, TBM is sometimes difficult to diagnose with certainty,
30
especially in its early phase, because the initial clinical features are non-specific and the
31
laboratory tests are insensitive. Cerebrospinal fluid (CSF) acid-fast bacilli (AFB) smear
32
microscopy, although rapid and inexpensive, has very low sensitivity in routine clinical
33
practice (less than 20%).3-5 Culture is the gold standard for diagnosing TBM, but it is not
34
useful for deciding whether to treat for TBM due to the time required for a positive result and
35
suboptimal sensitivity.
SC
M AN U
36
RI PT
26
Efforts to identify new diagnostic markers for TBM in CSF have been ongoing, mostly in HIV-infected populations, and some of the markers identified may be used as aids in
38
diagnosing TBM.6-14 Currently, there is no single test with optimal sensitivity in patients with
39
suspected TBM. New molecular-based microbiological tests, as well as AFB smear
40
microscopy and mycobacterial culture, had lower sensitivity in HIV-uninfected patients
41
because the bacterial loads may have been lower than in HIV-infected patients.8,10 A recently
42
developed fully automated real-time polymerase chain reaction (PCR), Xpert MTB/RIF, for
43
the diagnosis of TBM was less sensitive among HIV-uninfected patients (0–48%) than among
44
HIV-infected patients (67–79%).8,10 Furthermore, its diagnostic utility was hampered by a
45
very poor sensitivity for probable or possible TBM, reported to be as low as 0–3%.8,10
AC C
EP
TE D
37
46
Where the bacterial burden is low, an immunodiagnostic tool may be more useful than
47
microbiological tools for detecting Mycobacterium tuberculosis at infection sites. Recently,
48
interferon-gamma release assays (IGRAs) using mononuclear cells that are
49
compartmentalized at the site of infection, such as pleural fluid,15,16 peritoneal fluid,17,18
50
pericardial effusion,19 and CSF,6 have yielded promising results in the diagnosis of active TB. 2
ACCEPTED MANUSCRIPT In our earlier preliminary studies undertaken among HIV-uninfected patients (with 50–82
52
evaluable CSF samples) we concluded that the enzyme-linked immunosorbent spot
53
(ELISPOT) assay using CSF-mononuclear cells (CSF-MC) appeared to be a promising
54
adjunct to current tests for diagnosing TBM.13,20 This study builds on those earlier
55
publications.13,20 In it we evaluated on a larger number of subjects, the clinical utility of
56
peripheral blood mononuclear cells (PBMC) and CSF-MC ELISPOT assays for diagnosing
57
TBM. The study was conducted in a country with an intermediate TB burden and a low
58
prevalence of HIV infection (5 days (P < 0.001) and a miliary pattern on chest radiograph (P
