Journal of the Neurological Sciences 341 (2014) 17–21
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Features of anti-aquaporin 4 antibody-seronegative Thai patients with neuromyelitis optica spectrum disorders: A comparison with seropositive cases S. Siritho a,b, M. Apiwattanakul c, I. Nakashima d, T. Takahashi d,e, K. Fujihara d,f, N. Prayoonwiwat a,⁎ a
Division of Neurology, Department of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand Bumrungrad International Hospital, Bangkok 10110, Thailand c Department of Neurology, Prasat Neurological Institute, Bangkok 10400, Thailand d Department of Neurology, Tohoku University School of Medicine, Sendai 980-8574, Japan e Department of Neurology, Yonezawa National Hospital, Yonezawa 992-1202, Japan f Department of Multiple Sclerosis Therapeutics, Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan b
a r t i c l e
i n f o
Article history: Received 22 January 2014 Received in revised form 7 March 2014 Accepted 17 March 2014 Available online 25 March 2014 Keywords: Neuromyelitis optica spectrum disorder AQP4-antibody-negative Thai
a b s t r a c t Objective: The aim of this study is to investigate the unique features of seronegative neuromyelitis optica spectrum disorders (NMOSD) in Thailand. Background: It remains unknown whether seronegative NMOSD patients possess clinical and paraclinical features that are distinct from those with seropositivity. Methods: In a Thai cohort of idiopathic inflammatory CNS disorders (n = 122), 52 patients fulfilled the Wingerchuk 2007 criteria for NMOSD. We determined anti-AQP4 antibody statuses using three different assays (an in-house cell-based assay [CBA], a commercially available CBA and a tissue-based indirect immunofluorescence [IIF] assay). Results: Among the NMOSD patients, the percentage of seropositive cases was 54.5% based on the in-house and commercial CBAs and 30.8% based on the IIF assay. Using the in-house CBA, seronegative NMOSD patients exhibited distinct features compared with seropositive patients, such as a lack of female preponderance (F/M = 1.2 vs. 6.0), frequent simultaneous bilateral optic involvement (33.3% vs. 0.04%), a lower annual relapse rate (0.4 ± 0.3 vs. 0.7 ± 0.6), fewer spinal cord lesions (1.0 ± 4.3 vs. 1.4 ± 0.6), and lower CSF cell counts (20 ± 72 vs. 80 ± 285). Use of the commercial CBA yielded essentially similar results, but some of these differences were not significant using IIF. Conclusions: Sensitive anti-AQP4 antibody assays reveal features of seronegative NMOSD patients that differ from those of seropositive patients from Thailand. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The diagnosis of neuromyelitis optica (NMO) mandates that a patient exhibits both optic neuritis and transverse myelitis, as well as 2 of the 3 supportive criteria: spinal cord involvement equal to or longer than 3 vertebral segments (VBs), a negative brain MRI at onset and the presence of either NMO-IgG or AQP4-antibody[1]. The term “neuromyelitis optica spectrum disorder” (NMOSD) has been proposed to refer to the spectrum of AQP4-antibody related diseases, including
⁎ Corresponding author at: Siriraj Hospital, Mahidol University, 2 Prannok Road, Bangkoknoi, Bangkok, Thailand 10700. Tel.: +66 2 4197101; fax: +66 2 4122400. E-mail addresses: [email protected] (S. Siritho), [email protected] (M. Apiwattanakul), [email protected] (I. Nakashima), [email protected] (T. Takahashi), [email protected] (K. Fujihara), [email protected] (N. Prayoonwiwat).
http://dx.doi.org/10.1016/j.jns.2014.03.033 0022-510X/© 2014 Elsevier B.V. All rights reserved.
definite NMO [2]. Although the anti-AQP4 antibody is an important pathogen of NMO, some NMO patients cannot be confirmed using this autoantibody, even via sensitive assays. It has been debated whether so-called seronegative NMO patients present with the same disease as seropositive NMO patients [3]. Previous reports indicate that NMOSD has some clinical manifestations that differ from those of seropositive NMOSD [4–6]. A French study of patients with definite NMO reported no male or female preponderance, more simultaneous development of optic neuritis (ON) and myelitis at onset and less frequent severe visual impairment at the most recent follow-up [4]. A German study found that seronegative NMOSD patients exhibited less female preponderance, more frequent bilateral ON at onset and more widespread simultaneous ON and myelitis [6]. However, these studies were conducted using Caucasian-dominant cohorts and there were some conflicting results. Several methods are available for AQP4-antibody detection including the original tissue-based indirect immunofluorescence assay, an enzyme-linked immunosorbent assay (ELISA), and an indirect
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immunofluorescence cell-based assay (CBA), each with differences in sensitivity and specificity [4,7–10]. We previously reported that Thai patients with idiopathic inflammatory demyelinating diseases exhibited a high frequency of seropositivity for the anti-AQP4 antibody. In our cohort, many of the anti-AQP4 antibody seropositive patients did not fulfill the criteria of NMO, and some of these patients had been misdiagnosed with multiple sclerosis (MS) because they lacked the characteristic longitudinal extensive spinal cord lesion [11]. This is a single-center cohort study using a consecutive case series including patients exhibiting clinical characteristics of seronegative NMOSD, identified using three different antibody assays, including an established sensitive and specific CBA assay. Our objective is to describe the different clinical manifestations between AQP4 positive and negative patients in Thai NMOSD.
2.2.3. Tissue-based indirect immunofluorescence assay (IIF-TBA) This assay was performed according to the method described elsewhere [10], which uses a composite substrate of mouse tissue (cerebellum, midbrain and kidney). AQP4-antibodies were detected using a fluorescence-conjugated goat antibody to human IgG. The serum was screened at a concentration of 1:120. Antibody reactivity was considered positive when the staining pattern of the Virchow–Robin spaces, the continuous pial vessels, the albuminal staining of the microvessels in the cerebellar molecular layer, the granular layer, the white matter and in the pial and subpial vessels of the midbrain were evident.
2. Methods
2.3. Statistical analysis
2.1. Patients and study design
Subject characteristics were described using descriptive statistics, including means, standard deviation, median, minimum and maximum, frequencies and percentages. PASW Statistics (SPSS) version 18 software was used to perform the statistical analysis. We applied Student's t-test and the Mann–Whitney U test for quantitative data and the chi-square test and the Fisher exact test for qualitative data. All P-values were two-tailed, and values b0.05 were considered statistically significant.
1) This study was a retrospective observational study that included 135 Thai patients suspected of idiopathic inflammatory demyelinating central nervous system diseases (IIDCD) who visited the MS clinic at Siriraj Hospital, Mahidol University, Thailand between May 1, 2009 and February 28, 2010. The previous study reported the prevalence of AQP4-antibody seropositivity in Thai IIDCD, using cellbased assay with the Sendai method [11]. The same sera, which were stored at − 80 °C, were later tested using the other two methods utilized in this study during November 2011 to May 2012. Only 122 patients who had serum tested with all three AQP4-antibody detection assays were included in this study. We identified 52 consecutive patients with clinical and paraclinical features associated with myelitis and optic neuritis in patients with NMOSD regardless of AQP4-Ab serostatus. Our definition of patient with opticospinal MS (OSMS) was a patient who had optic neuritis and acute myelitis with spinal cord MRI lesion not extending over N 3 VB and with normal brain MRI, same as in the previous study [11]. Since the definition of OSMS is still conflicting, we did not include OSMS in the analysis.
2.2. Anti-AQP4 antibody assays 2.2.1. In-house CBA at Tohoku University (Sendai CBA) AQP4-live transfected human embryonic kidney cells (HEK293) expressing the untagged AQP4-M23 isoform were incubated in diluted serum, washed and incubated in fluorescein-conjugated goat antihuman IgG. Fixed cells were photographed via confocal microscopy and scoring of antibody seropositivity was based on comparison with mock-transfected cells that did not express AQP4. Assays were performed as previously described [7]. 2.2.2. Commercially available CBA kit An alternative CBA was performed using the Neurology Mosaics kit (EUROIMMUN, AG, Luebeck, Germany). The CBA kit utilizes HEK 293 cells transfected with recombinant full-length human AQP4, M1 isoform, to detect AQP4 antibodies using wild-type HEK 293 cells as a control substrate. According to the manufacturer's instructions [8], 25 μL of 1:10 diluted sera was applied to slides on which mouse tissue or HEK cells were attached. After 30 min of incubation at room temperature, the slides were washed and fluorescein-labeled anti-human globulin was added. Fluorescence was observed under a microscope after 30 min of incubation. AQP4 antibody reactivity was considered positive if the cytoplasm of AQP4-transfected cells displayed a flat, smooth, fine, granular, fluorescent staining pattern.
2.2.4. Standard PROTOCOL APPROVAL, REGISTRATION AND PATIENT CONSENT The study received approval from an ethical standards committee. All participants gave written informed consent.
3. Results Of the 122 IIDCD patients who had AQP4-antibody testing with all the three different assays, we found that 24 patients were consistently AQP4antibody positive and 66 patients were consistently AQP4-antibody negative by all tests. The full report of the comparison tests will be separately published elsewhere. In brief commercial CBA-kit and in house CBASendai had 82.9% co-negativity and 74.1% co-positivity with a Kappa value of 0.758. While IIF-TBA vs commercial CBA-kit and IIF-TBA vs in house CBA-Sendai had 75.3% and 73.2% co-negativity and 49% and 49% co-positivity with Kappa values of 0.546 and 0.518, respectively. There were 18 definite NMO and 34 NMOSD, 4 OSMS, 51 CMS, and 12 CIS and 3 patients with other autoimmune diseases regardless of AQP4antibody serostatus. Eighty-three percent of the definite NMO and 41.2% of the NMOSD had seropositivity according to CBA-Sendai method. Of those 52 patients clinically diagnosed with NMOSD, there were 24 seronegative NMOSD cases based on the Sendai CBA. As compared with the 28 seropositive NMOSD, there were differences in gender ratio (p = 0.012), relapse frequency with respect to both the number of attacks (p b 0.001) and the annual relapse rate (p = 0.004), the frequency of simultaneous bilateral optic neuritis (p = 0.008), the initial brain MRI abnormality compatible with the Barkhof criteria (p = 0.029), the number of spinal cord lesions (p = 0.003), the CSF cell count (p = 0.015), and the predominance of polymorphonuclear cells (p = 0.027) (Tables 1 & 2). There were no differences between seronegative and seropositive NMOSD patients with respect to age at onset, duration of disease, or EDSS at the most recent follow-up. Although the same trends were identified in the 24 seronegative NMOSD patients, as determined by the commercial CBA kit, some features became less prominent compared with the results achieved using the Sendai CBA (Tables 1 & 2). In our study, IIF-TBA was not sensitive enough to properly differentiate seropositive NMOSD patients from seronegative NMOSD patients. 3.1. Clinical features of 24 seronegative NMOSD patients Of the 24 seronegative NMOSD patients determined using the Sendai CBA, 3 were diagnosed with NMO, 12 with idiopathic longitudinally extensive myelitis, 6 with recurrent or simultaneous bilateral optic
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Table 1 Demographic data and clinical manifestation of NMOSD stratified by different AQP4-Ab assay methods. Anti-AQP4-Ab assay
Sendai CBA
Parameter
Positive (n = 28)
Negative (n = 24)
P
CBA kit Positive (n = 28)
Negative (n = 24)
P
IIF-TBA Positive (n = 16)
Negative (n = 36)
P
Age at onset, yr Mean ± SD
40 ± 17
37 ± 13
0.464
39 ± 16
39 ± 15
0.936
40 ± 20
39 ± 13
0.845
Sex, n (%) Female Male Ratio, F/M
24 (86) 4 (14) 6.0
13 (54) 11 (46) 1.2
0.012*
23 (82) 5 (18) 4.6
14 (58) 10 (42) 1.4
0.059
13 (81) 3 (19) 4.3
24 (67) 12 (33) 2.0
0.340
Disease duration, yr Mean ± SD
7.2 ± 3.5
6.9 ± 4.8
0.219
7.7 ± 3.8
6.3 ± 4.5
0.038*
7.0 ± 3.8
7.1 ± 4.3
0.843
Number of attacks, n Mean ± SD
5.5 ± 6.5
2.1 ± 1.7
b0.001*
5.6 ± 6.5
2.0 ± 1.5
b0.001*
6.1 ± 7.8
3.0 ± 3.1
0.071
Annual relapse rate Mean ± SD
0.7 ± 0.6
0.4 ± 0.3
0.004*
0.7 ± 0.6
0.4 ± 0.3
0.015*
0.8 ± 0.6
0.5 ± 0.4
0.068
EDSS Median
3.5
3.5
0.211
3.5
3.5
0.697
5.0
3.5
0.112
Bilateral ON at first presentation, n (%) Yes 1(4) No 27 (96)
8 (33) 16 (67)
0.008*
2 (7) 26 (93)
7 (29) 17 (71)
0.064
1 (6) 15 (94)
8 (22) 28 (78)
0.245
ON + TM at first presentation, n (%) Yes 1 (4) No 27 (96)
1 (4) 23 (96)
1.000
1 (4) 27 (96)
1 (4) 23 (96)
1.000
0 (0) 16 (100)
2 (6) 34 (94)
1.000
Anti-AQP4-Ab: anti-aquaporin-antibody; NMOSD: neuromyelitis optica spectrum disorders; EDSS: expanded disability status scale; ON: optic neuritis; TM: transverse myelitis; Sendai CBA: cell-based indirect immunofluorescence AQP4 antibody assay using the Sendai method; CBA kit: cell-based indirect immunofluorescence AQP4 antibody assay using a commercial kit from Euroimmun; IIF-TBA: indirect immunofluorescence AQP4 antibody assay using tissue; *P b 0.05 for significance.
neuritis, 2 with Asian optic-spinal multiple sclerosis, and 1 with myelitis with a brainstem lesion typical of NMO. There was no female predominance among the seronegative NMOSD patients. Bilateral optic neuritis was frequently detected as an initial manifestation. Although there was no difference between
seronegative and seropositive NMOSD with respect to disease duration or EDSS at the most recent follow-up, the relapse frequency and the number of spinal cord lesions were substantially decreased in the seronegative NMOSD patients. There was a trend of less spinal cord involvement over 3 vertebral segments (VBs) in the seronegative NMOSD
Table 2 Paraclinical data of NMOSD patients stratified by different AQP4-Ab assay methods. Anti-AQP4-Ab assay
Sendai CBA
CBA kit
Parameter
Positive (n = 28)
IIF
Negative (n = 24)
P
Positive (n = 28)
Negative (n = 24)
P
Positive (n = 16)
Negative (n = 36)
P
Initial brain abnormality, n (%) Yes 14/24 (58) No 10/24 (42)
10/18 (56) 8/18 (44)
0.857
13/23 (57) 10/23 (43)
11/19 (58) 8/19 (42)
0.929
8/13 (62) 5/13 (38)
16/29 (55) 13/29 (45)
0.700
Initial MRI meeting Barkhof criteria, n (%) Yes 6/24 (25) No 18/24 (75)
0/18 (0) 18/18 (100)
0.029*
5/23 (22) 18/23 (78)
1/19 (5) 18/19 (95)
0.197
4/13 (31) 9/13 (69)
2/29 (7) 27/29 (93)
0.063
Number of spinal cord lesions, n Mean ± SD 1.4 ± 0.6
1.0 ± 0.4
0.003*
1.4 ± 0.6
1.0 ± 0.4
0.002*
1.4 ± 0.6
1.1 ± 0.5
0.135
Number of spinal cord lesions (excluded patients with bilateral ON), n# Mean ± SD 1.4 ± 0.6 1.1 ± 0.3 0.010*
1.4 ± 0.6
1.1 ± 0.2
0.007*
1.4 ± 0.6
1.2 ± 0.4
0.318
Spinal cord lesion length N3 VBs, n (%) Yes 25/28 (89) No 3/28 (11)
13/20 (65) 7/20 (35)
0.070
25/27 (93) 2/27 (7)
13/21 (62) 8/21 (38)
0.013*
15/16 (94) 1/16 (6)
23/32 (72) 9/32 (28)
0.132
20 ± 72
0.015*
77 ± 285
23 ± 72
0.108
128 ± 377
19 ± 60
0.045*
CSF polymorphonuclear cells (cells/mm ) Mean ± SD 6.2 ± 17
3.5 ± 13
0.027*
4.3 ± 16
5.5 ± 15
0.719
7.5 ± 21
3.8 ± 13
0.042*
CSF oligoclonal IgG bands, n (%) Yes 3/21 (14) No 18/21 (86)
4/20 (20) 16/20 (80)
0.697
3/21 (14) 18/21 (86)
4/20 (20) 16/20 (80)
0.697
1/11 (9) 10/11 (91)
6/30 (20) 24/30 (80)
0.651
CSF cell count (cells/mm3) Mean ± SD 80 ± 285 3
Anti-AQP4-Ab: anti-aquaporin-antibody; NMOSD: Neuromyelitis optica spectrum disorders; Sendai CBA: cell-based indirect immunofluorescence AQP4 antibody assay using the Sendai method; CBA kit: cell-based indirect immunofluorescence AQP4 antibody assay using a commercial kit from Euroimmun; IIF-TBA: indirect immunofluorescence AQP4 antibody assay using tissue; ON: optic neuritis; CSF: cerebrospinal fluid; IgG: immunoglobulin *P b 0.05 for significance.# After excluding 9 patients with bilateral ON, there were 27, 26 and 15 patients who were seropositive by the Sendai CBA, CBA kit and IIF-TBA methods, respectively.
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S. Siritho et al. / Journal of the Neurological Sciences 341 (2014) 17–21
patients. We did not detect any significant differences between the two groups in other aspects based on either brain or spinal MRI. Further study using a larger sample size is needed to clarify this issue. 3.2. Discrepancy of the results between the Sendai CBA and the CBA kit (Table 3) Six patients received different results from the Sendai CBA and the commercial CBA kit. The first three were patients who were seropositive based on the Sendai CBA but seronegative based on the CBA kit. Patient 1 was a female who presented with recurrent transverse myelitis (TM) with poor recovery, which is not compatible with MRI McDonald criteria at onset and the presence of CSF-OCB. She was treated with mitoxantrone and then switched to azathioprine. Later, after the study period, she developed another attack of ON with poor recovery and was therefore diagnosed with definite NMO. Patient 2 was a female who presented with recurrent TM, brainstem attack and recurrent optic neuritis (ON). She displayed an abnormal brain MRI at onset. Both patients were CSF-OCB positive and exhibited spinal involvement longer than 3 VBs. Patient 3 was a 70-year-old female who had a single brainstem attack with extension to the cervicomedullary junction, CSF pleocytosis and negative OCB. All 3 patients, who were AQP4-antibody seropositive based on the Sendai CBA, exhibited the characteristic features of NMOSD. The other three patients were AQP4-antibody seropositive only using the CBA kit. Patient 4 was a male who presented with unilateral ON and then later developed brainstem and spinal cord involvement 4 VBs in length, but there was no available brain MRI at onset. He was also found to be positive for P-ANCA and ANA. Patient 5 presented with a single TM with 2 foci of spinal cord involvement, as well as CSF-positive and negative brain MRI at onset. She was treated with azathioprine and thus far has had no further attack. Patient 6 presented with recurrent ON with good recovery, a brain MRI not compatible with the McDonald criteria and an absence of CSF-OCB. 4. Discussion We identified the clinical features of seronegative NMOSD using consecutive patients in a large Thai cohort. Although the principal manifestation and prognosis did not differ between seronegative and seropositive NMOSD, substantial differences were found with respect to gender ratio and the frequency of relapse. Unlike previous reports, we did not detect a significant difference in favor of simultaneous development of optic neuritis (ON) and myelitis at onset in the seronegative group. Interestingly, similar to the findings of the German study, we observed that bilateral ON at the first episode appears to be a characteristic feature of these patients [6]. Although similar disease duration and EDSS suggest a similar prognosis between the two groups, long-term followup may identify some differences.
Although patients with NMOSD according to the Wingercuck criteria 2007 only included patients who were seropositive with the characteristic clinical features of NMO, there were continuous reports of the definite NMO who were seronegative. Our seronegative NMOSD patients in this study included the patients with clinical and paraclinical features associated with myelitis and optic neuritis in patients with NMOSD regardless of AQP4-Ab serostatus, similar to the German study [6]. In our study, some seronegative NMOSD patients exhibited clinical features similar to those reported for anti-MOG antibody seropositive NMO patients with severe optic neuritis and/or longitudinal extensive transverse myelitis with good recovery after treatment with steroids and plasma exchange [5]. Although we have not measured the antiMOG antibodies in our patients, it is likely that some seronegative NMOSD patients have a common pathomechanism that is possibly associated with other autoantibodies, such as the anti-MOG antibody. At our institute, we tend to perform close follow-up on the seronegative NMOSD patients rather than treat them with immunosuppressants to prevent future relapse unless they exhibit recurrent brainstem episodes or high cervical cord involvement with respiratory insufficiency or if they exhibit major neurologic deficits after the attack. Although the differences in treatment are not considered in this study, the responses to treatment should be analyzed more carefully in the future. Further studies of NMO/NMOSD and other demyelinating conditions are required to clarify the diagnostic and prognostic relevance of anti-MOG antibodies. Although the CBA kit could differentiate seronegative NMOSD as well as the Sendai CBA, there appears to be some discrepancy with respect to false positives and false negatives. The Sendai CBA used an untagged AQP4-M23 isoform from live cells, in which its conformation can form orthogonal arrays of particles (OAP) that are more readily detected by the AQP4-antibody than by the commercial CBA kit using the AQP4M1 isoform [4]. Additionally, incubation of patient serum in the prefixed CBA method increased nonspecific binding and generated a higher signal-to-noise ratio [3]. Therefore, this noise may possibly yield a falsepositive result. The other possibility is that the post-fixed CBA method used M23 whereas the pre-fixed CBA method used the M1 isoform, which might result in differences. One patient with a positive result based on the CBA kit but a negative result based on the Sendai CBA presented with various other autoantibodies. This included a high titer of an anti-nuclear antibody that may cause non-specific binding when using a pre-fixed assay. We could not identify whether other patients exhibited falsepositive or false-negative results. However, because there were fewer differences between seropositive NMOSD and seronegative NMOSD patients based on the CBA kit, this may indicate lower sensitivity and specificity compared with the Sendai CBA. Our study had a few limitations. Firstly, we did not test a patient serially in the disease course to firmly define seronegativity. All but one of the samples were obtained during an acute phase and before steroid treatment. Additionally, treatment with immunosuppressants or high doses of steroids can lower the titer of the AQP4-antibody such that it
Table 3 Patients with differing results between the Sendai CBA and the CBA kit. Patient
Age at onset (yr)
1
38.3
2
Disease duration (yr)
Sex
Number of relapses
Clinical manifestations
EDSS at last F/U
CSFOCB
CSFWBC
MRI compatible with McDonald 2010
NMO 2006 criteria
Current Rx
5.8
F
4
Recurrent TM(C2–6)
8.0
Yes
21
No
No
35.5
5.8
F
3
4.0
Yes
8
Yes
No
3 4
70.0 37.8
2.9 15.8
F M
1 7
3.0 6.0
No Yes
44 1
Yes N/A
No Yes
Aza Aza
5 6
38.6 31.5
7.4 4
F F
1 2
Recurrent TM(C4–6)-brainstem-unilateral ON Single brainstem with extension to C1–2 Recurrent unilateral ON-brainstem-TM; whole cord Single TM (C2–6, T6–7 with cord swelling) Recur bilateral ON
Mitoxantrone to Aza Aza
2.0 1.0
Yes No
13 9
No No
No No
Aza Aza
EDSS: expanded disability status scale; CSF: cerebrospinal fluid; OB: oligoclonal bands; NMO: neuromyelitis optica. N/A: not available.
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becomes undetectable. Repeated AQP4-antibody testing and long-term follow-up in cases that are highly suspicious for NMOSD are warranted. There is a possibility that we missed some cases with seropositive NMOSD despite the use of the most sensitive CBA because the AQP4antibody is detectable only in the CSF and not in the serum in a few cases. Secondly, the sample size was not large enough. Further study with a larger group of NMOSD patients is needed to validate the difference between the two groups. Thirdly, we did not perform an analysis of the response to the treatment because there is a limitation of access to the drug within the current health-care system.
5. Conclusions Seronegative NMOSD patients exhibited different clinical features from seropositive NMOSD patients. The differences became more apparent using a relatively sensitive and specific assay. NMOSD may include diseases that mimic NMO but involve distinct pathomechanisms due to the nearly equivalent number of patients who were seropositive and seronegative for the anti-AQP4 antibody. Treatment responses and the long-term prognosis of seronegative NMOSD should be analyzed separately from seropositive NMO in the future, and the search for novel pathogens such as anti-MOG antibodies should continue.
Conflict of interest We declared no conflict of interest regarding this study.
Funding This study was supported by Siriraj Research Grant number IOR015532049 and a research grant from the Ministry of Public Health, Thailand.
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Acknowledgments We are grateful to Wanichada Kosolthammakorn M.T., Natakorn Sangnumpong B.Sc., Leatchai Wachiruttamungkur, M.Sc, Nopwan Siwasiriyanont, M.Sc. and Laddawan Champa M.Sc. for their technical support. References [1] Wingerchuk DM, Lennon VA, Pittock SJ, Lucchinetti CF, Weinshenker BG. Revised diagnostic criteria for neuromyelitis optica. Neurology 2006;66(10):1485–9. [2] Wingerchuk DM, Lennon VA, Lucchinetti CF, Pittock SJ, Weinshenker BG. The spectrum of neuromyelitis optica. Lancet Neurol Sep 2007;6(9):805–15 [PubMed PMID: 17706564]. [3] Fujihara K, Leite MI. Seronegative NMO: a sensitive AQP4 antibody test clarifies clinical features and next challenges. Neurology Jun 11 2013;80(24):2176–7 [PubMed PMID: 23658387]. [4] Marignier R, Bernard-Valnet R, Giraudon P, Collongues N, Papeix C, Zephir H, et al. Aquaporin-4 antibody-negative neuromyelitis optica: distinct assay sensitivitydependent entity. Neurology Jun 11 2013;80(24):2194–200 [PubMed PMID: 23658379]. [5] Kitley J, Woodhall M, Waters P, Leite MI, Devenney E, Craig J, et al. Myelinoligodendrocyte glycoprotein antibodies in adults with a neuromyelitis optica phenotype. Neurology Sep 18 2012;79(12):1273–7 [PubMed PMID: 22914827]. [6] Jarius S, Ruprecht K, Wildemann B, Kuempfel T, Ringelstein M, Geis C, et al. Contrasting disease patterns in seropositive and seronegative neuromyelitis optica: a multicentre study of 175 patients. J Neuroinflammation 2012;9:14 [PubMed PMID: 22260418. Pubmed Central PMCID: 3283476]. [7] Takahashi T, Fujihara K, Nakashima I, Misu T, Miyazawa I, Nakamura M, et al. Antiaquaporin-4 antibody is involved in the pathogenesis of NMO: a study on antibody titre. Brain May 2007;130(Pt 5):1235–43. [8] Jarius S, Probst C, Borowski K, Franciotta D, Wildemann B, Stoecker W, et al. Standardized method for the detection of antibodies to aquaporin-4 based on a highly sensitive immunofluorescence assay employing recombinant target antigen. J Neurol Sci 2010;291(1–2):52–6. [9] Fazio R, Malosio ML, Lampasona V, De Feo D, Privitera D, Marnetto F. Antiacquaporin 4 antibodies detection by different techniques in neuromyelitis optica patients. Mult Scler Oct 2009;15(10):1153–63. [10] Lennon VA, Kryzer TJ, Pittock SJ, Verkman AS, Hinson SR. IgG marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel. J Exp Med 2005;202(4):473–7. [11] Siritho S, Nakashima I, Takahashi T, Fujihara K, Prayoonwiwat N. AQP4 antibodypositive Thai cases: clinical features and diagnostic problems. Neurology 2011;77(9):827–34.
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Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Features of anti-aquaporin 4 antibody-seronegative Thai patients with neuromyelitis optica spectrum disorders: A comparison with seropositive cases S. Siritho a,b, M. Apiwattanakul c, I. Nakashima d, T. Takahashi d,e, K. Fujihara d,f, N. Prayoonwiwat a,⁎ a
Division of Neurology, Department of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand Bumrungrad International Hospital, Bangkok 10110, Thailand c Department of Neurology, Prasat Neurological Institute, Bangkok 10400, Thailand d Department of Neurology, Tohoku University School of Medicine, Sendai 980-8574, Japan e Department of Neurology, Yonezawa National Hospital, Yonezawa 992-1202, Japan f Department of Multiple Sclerosis Therapeutics, Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan b
a r t i c l e
i n f o
Article history: Received 22 January 2014 Received in revised form 7 March 2014 Accepted 17 March 2014 Available online 25 March 2014 Keywords: Neuromyelitis optica spectrum disorder AQP4-antibody-negative Thai
a b s t r a c t Objective: The aim of this study is to investigate the unique features of seronegative neuromyelitis optica spectrum disorders (NMOSD) in Thailand. Background: It remains unknown whether seronegative NMOSD patients possess clinical and paraclinical features that are distinct from those with seropositivity. Methods: In a Thai cohort of idiopathic inflammatory CNS disorders (n = 122), 52 patients fulfilled the Wingerchuk 2007 criteria for NMOSD. We determined anti-AQP4 antibody statuses using three different assays (an in-house cell-based assay [CBA], a commercially available CBA and a tissue-based indirect immunofluorescence [IIF] assay). Results: Among the NMOSD patients, the percentage of seropositive cases was 54.5% based on the in-house and commercial CBAs and 30.8% based on the IIF assay. Using the in-house CBA, seronegative NMOSD patients exhibited distinct features compared with seropositive patients, such as a lack of female preponderance (F/M = 1.2 vs. 6.0), frequent simultaneous bilateral optic involvement (33.3% vs. 0.04%), a lower annual relapse rate (0.4 ± 0.3 vs. 0.7 ± 0.6), fewer spinal cord lesions (1.0 ± 4.3 vs. 1.4 ± 0.6), and lower CSF cell counts (20 ± 72 vs. 80 ± 285). Use of the commercial CBA yielded essentially similar results, but some of these differences were not significant using IIF. Conclusions: Sensitive anti-AQP4 antibody assays reveal features of seronegative NMOSD patients that differ from those of seropositive patients from Thailand. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The diagnosis of neuromyelitis optica (NMO) mandates that a patient exhibits both optic neuritis and transverse myelitis, as well as 2 of the 3 supportive criteria: spinal cord involvement equal to or longer than 3 vertebral segments (VBs), a negative brain MRI at onset and the presence of either NMO-IgG or AQP4-antibody[1]. The term “neuromyelitis optica spectrum disorder” (NMOSD) has been proposed to refer to the spectrum of AQP4-antibody related diseases, including
⁎ Corresponding author at: Siriraj Hospital, Mahidol University, 2 Prannok Road, Bangkoknoi, Bangkok, Thailand 10700. Tel.: +66 2 4197101; fax: +66 2 4122400. E-mail addresses: [email protected] (S. Siritho), [email protected] (M. Apiwattanakul), [email protected] (I. Nakashima), [email protected] (T. Takahashi), [email protected] (K. Fujihara), [email protected] (N. Prayoonwiwat).
http://dx.doi.org/10.1016/j.jns.2014.03.033 0022-510X/© 2014 Elsevier B.V. All rights reserved.
definite NMO [2]. Although the anti-AQP4 antibody is an important pathogen of NMO, some NMO patients cannot be confirmed using this autoantibody, even via sensitive assays. It has been debated whether so-called seronegative NMO patients present with the same disease as seropositive NMO patients [3]. Previous reports indicate that NMOSD has some clinical manifestations that differ from those of seropositive NMOSD [4–6]. A French study of patients with definite NMO reported no male or female preponderance, more simultaneous development of optic neuritis (ON) and myelitis at onset and less frequent severe visual impairment at the most recent follow-up [4]. A German study found that seronegative NMOSD patients exhibited less female preponderance, more frequent bilateral ON at onset and more widespread simultaneous ON and myelitis [6]. However, these studies were conducted using Caucasian-dominant cohorts and there were some conflicting results. Several methods are available for AQP4-antibody detection including the original tissue-based indirect immunofluorescence assay, an enzyme-linked immunosorbent assay (ELISA), and an indirect
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immunofluorescence cell-based assay (CBA), each with differences in sensitivity and specificity [4,7–10]. We previously reported that Thai patients with idiopathic inflammatory demyelinating diseases exhibited a high frequency of seropositivity for the anti-AQP4 antibody. In our cohort, many of the anti-AQP4 antibody seropositive patients did not fulfill the criteria of NMO, and some of these patients had been misdiagnosed with multiple sclerosis (MS) because they lacked the characteristic longitudinal extensive spinal cord lesion [11]. This is a single-center cohort study using a consecutive case series including patients exhibiting clinical characteristics of seronegative NMOSD, identified using three different antibody assays, including an established sensitive and specific CBA assay. Our objective is to describe the different clinical manifestations between AQP4 positive and negative patients in Thai NMOSD.
2.2.3. Tissue-based indirect immunofluorescence assay (IIF-TBA) This assay was performed according to the method described elsewhere [10], which uses a composite substrate of mouse tissue (cerebellum, midbrain and kidney). AQP4-antibodies were detected using a fluorescence-conjugated goat antibody to human IgG. The serum was screened at a concentration of 1:120. Antibody reactivity was considered positive when the staining pattern of the Virchow–Robin spaces, the continuous pial vessels, the albuminal staining of the microvessels in the cerebellar molecular layer, the granular layer, the white matter and in the pial and subpial vessels of the midbrain were evident.
2. Methods
2.3. Statistical analysis
2.1. Patients and study design
Subject characteristics were described using descriptive statistics, including means, standard deviation, median, minimum and maximum, frequencies and percentages. PASW Statistics (SPSS) version 18 software was used to perform the statistical analysis. We applied Student's t-test and the Mann–Whitney U test for quantitative data and the chi-square test and the Fisher exact test for qualitative data. All P-values were two-tailed, and values b0.05 were considered statistically significant.
1) This study was a retrospective observational study that included 135 Thai patients suspected of idiopathic inflammatory demyelinating central nervous system diseases (IIDCD) who visited the MS clinic at Siriraj Hospital, Mahidol University, Thailand between May 1, 2009 and February 28, 2010. The previous study reported the prevalence of AQP4-antibody seropositivity in Thai IIDCD, using cellbased assay with the Sendai method [11]. The same sera, which were stored at − 80 °C, were later tested using the other two methods utilized in this study during November 2011 to May 2012. Only 122 patients who had serum tested with all three AQP4-antibody detection assays were included in this study. We identified 52 consecutive patients with clinical and paraclinical features associated with myelitis and optic neuritis in patients with NMOSD regardless of AQP4-Ab serostatus. Our definition of patient with opticospinal MS (OSMS) was a patient who had optic neuritis and acute myelitis with spinal cord MRI lesion not extending over N 3 VB and with normal brain MRI, same as in the previous study [11]. Since the definition of OSMS is still conflicting, we did not include OSMS in the analysis.
2.2. Anti-AQP4 antibody assays 2.2.1. In-house CBA at Tohoku University (Sendai CBA) AQP4-live transfected human embryonic kidney cells (HEK293) expressing the untagged AQP4-M23 isoform were incubated in diluted serum, washed and incubated in fluorescein-conjugated goat antihuman IgG. Fixed cells were photographed via confocal microscopy and scoring of antibody seropositivity was based on comparison with mock-transfected cells that did not express AQP4. Assays were performed as previously described [7]. 2.2.2. Commercially available CBA kit An alternative CBA was performed using the Neurology Mosaics kit (EUROIMMUN, AG, Luebeck, Germany). The CBA kit utilizes HEK 293 cells transfected with recombinant full-length human AQP4, M1 isoform, to detect AQP4 antibodies using wild-type HEK 293 cells as a control substrate. According to the manufacturer's instructions [8], 25 μL of 1:10 diluted sera was applied to slides on which mouse tissue or HEK cells were attached. After 30 min of incubation at room temperature, the slides were washed and fluorescein-labeled anti-human globulin was added. Fluorescence was observed under a microscope after 30 min of incubation. AQP4 antibody reactivity was considered positive if the cytoplasm of AQP4-transfected cells displayed a flat, smooth, fine, granular, fluorescent staining pattern.
2.2.4. Standard PROTOCOL APPROVAL, REGISTRATION AND PATIENT CONSENT The study received approval from an ethical standards committee. All participants gave written informed consent.
3. Results Of the 122 IIDCD patients who had AQP4-antibody testing with all the three different assays, we found that 24 patients were consistently AQP4antibody positive and 66 patients were consistently AQP4-antibody negative by all tests. The full report of the comparison tests will be separately published elsewhere. In brief commercial CBA-kit and in house CBASendai had 82.9% co-negativity and 74.1% co-positivity with a Kappa value of 0.758. While IIF-TBA vs commercial CBA-kit and IIF-TBA vs in house CBA-Sendai had 75.3% and 73.2% co-negativity and 49% and 49% co-positivity with Kappa values of 0.546 and 0.518, respectively. There were 18 definite NMO and 34 NMOSD, 4 OSMS, 51 CMS, and 12 CIS and 3 patients with other autoimmune diseases regardless of AQP4antibody serostatus. Eighty-three percent of the definite NMO and 41.2% of the NMOSD had seropositivity according to CBA-Sendai method. Of those 52 patients clinically diagnosed with NMOSD, there were 24 seronegative NMOSD cases based on the Sendai CBA. As compared with the 28 seropositive NMOSD, there were differences in gender ratio (p = 0.012), relapse frequency with respect to both the number of attacks (p b 0.001) and the annual relapse rate (p = 0.004), the frequency of simultaneous bilateral optic neuritis (p = 0.008), the initial brain MRI abnormality compatible with the Barkhof criteria (p = 0.029), the number of spinal cord lesions (p = 0.003), the CSF cell count (p = 0.015), and the predominance of polymorphonuclear cells (p = 0.027) (Tables 1 & 2). There were no differences between seronegative and seropositive NMOSD patients with respect to age at onset, duration of disease, or EDSS at the most recent follow-up. Although the same trends were identified in the 24 seronegative NMOSD patients, as determined by the commercial CBA kit, some features became less prominent compared with the results achieved using the Sendai CBA (Tables 1 & 2). In our study, IIF-TBA was not sensitive enough to properly differentiate seropositive NMOSD patients from seronegative NMOSD patients. 3.1. Clinical features of 24 seronegative NMOSD patients Of the 24 seronegative NMOSD patients determined using the Sendai CBA, 3 were diagnosed with NMO, 12 with idiopathic longitudinally extensive myelitis, 6 with recurrent or simultaneous bilateral optic
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Table 1 Demographic data and clinical manifestation of NMOSD stratified by different AQP4-Ab assay methods. Anti-AQP4-Ab assay
Sendai CBA
Parameter
Positive (n = 28)
Negative (n = 24)
P
CBA kit Positive (n = 28)
Negative (n = 24)
P
IIF-TBA Positive (n = 16)
Negative (n = 36)
P
Age at onset, yr Mean ± SD
40 ± 17
37 ± 13
0.464
39 ± 16
39 ± 15
0.936
40 ± 20
39 ± 13
0.845
Sex, n (%) Female Male Ratio, F/M
24 (86) 4 (14) 6.0
13 (54) 11 (46) 1.2
0.012*
23 (82) 5 (18) 4.6
14 (58) 10 (42) 1.4
0.059
13 (81) 3 (19) 4.3
24 (67) 12 (33) 2.0
0.340
Disease duration, yr Mean ± SD
7.2 ± 3.5
6.9 ± 4.8
0.219
7.7 ± 3.8
6.3 ± 4.5
0.038*
7.0 ± 3.8
7.1 ± 4.3
0.843
Number of attacks, n Mean ± SD
5.5 ± 6.5
2.1 ± 1.7
b0.001*
5.6 ± 6.5
2.0 ± 1.5
b0.001*
6.1 ± 7.8
3.0 ± 3.1
0.071
Annual relapse rate Mean ± SD
0.7 ± 0.6
0.4 ± 0.3
0.004*
0.7 ± 0.6
0.4 ± 0.3
0.015*
0.8 ± 0.6
0.5 ± 0.4
0.068
EDSS Median
3.5
3.5
0.211
3.5
3.5
0.697
5.0
3.5
0.112
Bilateral ON at first presentation, n (%) Yes 1(4) No 27 (96)
8 (33) 16 (67)
0.008*
2 (7) 26 (93)
7 (29) 17 (71)
0.064
1 (6) 15 (94)
8 (22) 28 (78)
0.245
ON + TM at first presentation, n (%) Yes 1 (4) No 27 (96)
1 (4) 23 (96)
1.000
1 (4) 27 (96)
1 (4) 23 (96)
1.000
0 (0) 16 (100)
2 (6) 34 (94)
1.000
Anti-AQP4-Ab: anti-aquaporin-antibody; NMOSD: neuromyelitis optica spectrum disorders; EDSS: expanded disability status scale; ON: optic neuritis; TM: transverse myelitis; Sendai CBA: cell-based indirect immunofluorescence AQP4 antibody assay using the Sendai method; CBA kit: cell-based indirect immunofluorescence AQP4 antibody assay using a commercial kit from Euroimmun; IIF-TBA: indirect immunofluorescence AQP4 antibody assay using tissue; *P b 0.05 for significance.
neuritis, 2 with Asian optic-spinal multiple sclerosis, and 1 with myelitis with a brainstem lesion typical of NMO. There was no female predominance among the seronegative NMOSD patients. Bilateral optic neuritis was frequently detected as an initial manifestation. Although there was no difference between
seronegative and seropositive NMOSD with respect to disease duration or EDSS at the most recent follow-up, the relapse frequency and the number of spinal cord lesions were substantially decreased in the seronegative NMOSD patients. There was a trend of less spinal cord involvement over 3 vertebral segments (VBs) in the seronegative NMOSD
Table 2 Paraclinical data of NMOSD patients stratified by different AQP4-Ab assay methods. Anti-AQP4-Ab assay
Sendai CBA
CBA kit
Parameter
Positive (n = 28)
IIF
Negative (n = 24)
P
Positive (n = 28)
Negative (n = 24)
P
Positive (n = 16)
Negative (n = 36)
P
Initial brain abnormality, n (%) Yes 14/24 (58) No 10/24 (42)
10/18 (56) 8/18 (44)
0.857
13/23 (57) 10/23 (43)
11/19 (58) 8/19 (42)
0.929
8/13 (62) 5/13 (38)
16/29 (55) 13/29 (45)
0.700
Initial MRI meeting Barkhof criteria, n (%) Yes 6/24 (25) No 18/24 (75)
0/18 (0) 18/18 (100)
0.029*
5/23 (22) 18/23 (78)
1/19 (5) 18/19 (95)
0.197
4/13 (31) 9/13 (69)
2/29 (7) 27/29 (93)
0.063
Number of spinal cord lesions, n Mean ± SD 1.4 ± 0.6
1.0 ± 0.4
0.003*
1.4 ± 0.6
1.0 ± 0.4
0.002*
1.4 ± 0.6
1.1 ± 0.5
0.135
Number of spinal cord lesions (excluded patients with bilateral ON), n# Mean ± SD 1.4 ± 0.6 1.1 ± 0.3 0.010*
1.4 ± 0.6
1.1 ± 0.2
0.007*
1.4 ± 0.6
1.2 ± 0.4
0.318
Spinal cord lesion length N3 VBs, n (%) Yes 25/28 (89) No 3/28 (11)
13/20 (65) 7/20 (35)
0.070
25/27 (93) 2/27 (7)
13/21 (62) 8/21 (38)
0.013*
15/16 (94) 1/16 (6)
23/32 (72) 9/32 (28)
0.132
20 ± 72
0.015*
77 ± 285
23 ± 72
0.108
128 ± 377
19 ± 60
0.045*
CSF polymorphonuclear cells (cells/mm ) Mean ± SD 6.2 ± 17
3.5 ± 13
0.027*
4.3 ± 16
5.5 ± 15
0.719
7.5 ± 21
3.8 ± 13
0.042*
CSF oligoclonal IgG bands, n (%) Yes 3/21 (14) No 18/21 (86)
4/20 (20) 16/20 (80)
0.697
3/21 (14) 18/21 (86)
4/20 (20) 16/20 (80)
0.697
1/11 (9) 10/11 (91)
6/30 (20) 24/30 (80)
0.651
CSF cell count (cells/mm3) Mean ± SD 80 ± 285 3
Anti-AQP4-Ab: anti-aquaporin-antibody; NMOSD: Neuromyelitis optica spectrum disorders; Sendai CBA: cell-based indirect immunofluorescence AQP4 antibody assay using the Sendai method; CBA kit: cell-based indirect immunofluorescence AQP4 antibody assay using a commercial kit from Euroimmun; IIF-TBA: indirect immunofluorescence AQP4 antibody assay using tissue; ON: optic neuritis; CSF: cerebrospinal fluid; IgG: immunoglobulin *P b 0.05 for significance.# After excluding 9 patients with bilateral ON, there were 27, 26 and 15 patients who were seropositive by the Sendai CBA, CBA kit and IIF-TBA methods, respectively.
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patients. We did not detect any significant differences between the two groups in other aspects based on either brain or spinal MRI. Further study using a larger sample size is needed to clarify this issue. 3.2. Discrepancy of the results between the Sendai CBA and the CBA kit (Table 3) Six patients received different results from the Sendai CBA and the commercial CBA kit. The first three were patients who were seropositive based on the Sendai CBA but seronegative based on the CBA kit. Patient 1 was a female who presented with recurrent transverse myelitis (TM) with poor recovery, which is not compatible with MRI McDonald criteria at onset and the presence of CSF-OCB. She was treated with mitoxantrone and then switched to azathioprine. Later, after the study period, she developed another attack of ON with poor recovery and was therefore diagnosed with definite NMO. Patient 2 was a female who presented with recurrent TM, brainstem attack and recurrent optic neuritis (ON). She displayed an abnormal brain MRI at onset. Both patients were CSF-OCB positive and exhibited spinal involvement longer than 3 VBs. Patient 3 was a 70-year-old female who had a single brainstem attack with extension to the cervicomedullary junction, CSF pleocytosis and negative OCB. All 3 patients, who were AQP4-antibody seropositive based on the Sendai CBA, exhibited the characteristic features of NMOSD. The other three patients were AQP4-antibody seropositive only using the CBA kit. Patient 4 was a male who presented with unilateral ON and then later developed brainstem and spinal cord involvement 4 VBs in length, but there was no available brain MRI at onset. He was also found to be positive for P-ANCA and ANA. Patient 5 presented with a single TM with 2 foci of spinal cord involvement, as well as CSF-positive and negative brain MRI at onset. She was treated with azathioprine and thus far has had no further attack. Patient 6 presented with recurrent ON with good recovery, a brain MRI not compatible with the McDonald criteria and an absence of CSF-OCB. 4. Discussion We identified the clinical features of seronegative NMOSD using consecutive patients in a large Thai cohort. Although the principal manifestation and prognosis did not differ between seronegative and seropositive NMOSD, substantial differences were found with respect to gender ratio and the frequency of relapse. Unlike previous reports, we did not detect a significant difference in favor of simultaneous development of optic neuritis (ON) and myelitis at onset in the seronegative group. Interestingly, similar to the findings of the German study, we observed that bilateral ON at the first episode appears to be a characteristic feature of these patients [6]. Although similar disease duration and EDSS suggest a similar prognosis between the two groups, long-term followup may identify some differences.
Although patients with NMOSD according to the Wingercuck criteria 2007 only included patients who were seropositive with the characteristic clinical features of NMO, there were continuous reports of the definite NMO who were seronegative. Our seronegative NMOSD patients in this study included the patients with clinical and paraclinical features associated with myelitis and optic neuritis in patients with NMOSD regardless of AQP4-Ab serostatus, similar to the German study [6]. In our study, some seronegative NMOSD patients exhibited clinical features similar to those reported for anti-MOG antibody seropositive NMO patients with severe optic neuritis and/or longitudinal extensive transverse myelitis with good recovery after treatment with steroids and plasma exchange [5]. Although we have not measured the antiMOG antibodies in our patients, it is likely that some seronegative NMOSD patients have a common pathomechanism that is possibly associated with other autoantibodies, such as the anti-MOG antibody. At our institute, we tend to perform close follow-up on the seronegative NMOSD patients rather than treat them with immunosuppressants to prevent future relapse unless they exhibit recurrent brainstem episodes or high cervical cord involvement with respiratory insufficiency or if they exhibit major neurologic deficits after the attack. Although the differences in treatment are not considered in this study, the responses to treatment should be analyzed more carefully in the future. Further studies of NMO/NMOSD and other demyelinating conditions are required to clarify the diagnostic and prognostic relevance of anti-MOG antibodies. Although the CBA kit could differentiate seronegative NMOSD as well as the Sendai CBA, there appears to be some discrepancy with respect to false positives and false negatives. The Sendai CBA used an untagged AQP4-M23 isoform from live cells, in which its conformation can form orthogonal arrays of particles (OAP) that are more readily detected by the AQP4-antibody than by the commercial CBA kit using the AQP4M1 isoform [4]. Additionally, incubation of patient serum in the prefixed CBA method increased nonspecific binding and generated a higher signal-to-noise ratio [3]. Therefore, this noise may possibly yield a falsepositive result. The other possibility is that the post-fixed CBA method used M23 whereas the pre-fixed CBA method used the M1 isoform, which might result in differences. One patient with a positive result based on the CBA kit but a negative result based on the Sendai CBA presented with various other autoantibodies. This included a high titer of an anti-nuclear antibody that may cause non-specific binding when using a pre-fixed assay. We could not identify whether other patients exhibited falsepositive or false-negative results. However, because there were fewer differences between seropositive NMOSD and seronegative NMOSD patients based on the CBA kit, this may indicate lower sensitivity and specificity compared with the Sendai CBA. Our study had a few limitations. Firstly, we did not test a patient serially in the disease course to firmly define seronegativity. All but one of the samples were obtained during an acute phase and before steroid treatment. Additionally, treatment with immunosuppressants or high doses of steroids can lower the titer of the AQP4-antibody such that it
Table 3 Patients with differing results between the Sendai CBA and the CBA kit. Patient
Age at onset (yr)
1
38.3
2
Disease duration (yr)
Sex
Number of relapses
Clinical manifestations
EDSS at last F/U
CSFOCB
CSFWBC
MRI compatible with McDonald 2010
NMO 2006 criteria
Current Rx
5.8
F
4
Recurrent TM(C2–6)
8.0
Yes
21
No
No
35.5
5.8
F
3
4.0
Yes
8
Yes
No
3 4
70.0 37.8
2.9 15.8
F M
1 7
3.0 6.0
No Yes
44 1
Yes N/A
No Yes
Aza Aza
5 6
38.6 31.5
7.4 4
F F
1 2
Recurrent TM(C4–6)-brainstem-unilateral ON Single brainstem with extension to C1–2 Recurrent unilateral ON-brainstem-TM; whole cord Single TM (C2–6, T6–7 with cord swelling) Recur bilateral ON
Mitoxantrone to Aza Aza
2.0 1.0
Yes No
13 9
No No
No No
Aza Aza
EDSS: expanded disability status scale; CSF: cerebrospinal fluid; OB: oligoclonal bands; NMO: neuromyelitis optica. N/A: not available.
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becomes undetectable. Repeated AQP4-antibody testing and long-term follow-up in cases that are highly suspicious for NMOSD are warranted. There is a possibility that we missed some cases with seropositive NMOSD despite the use of the most sensitive CBA because the AQP4antibody is detectable only in the CSF and not in the serum in a few cases. Secondly, the sample size was not large enough. Further study with a larger group of NMOSD patients is needed to validate the difference between the two groups. Thirdly, we did not perform an analysis of the response to the treatment because there is a limitation of access to the drug within the current health-care system.
5. Conclusions Seronegative NMOSD patients exhibited different clinical features from seropositive NMOSD patients. The differences became more apparent using a relatively sensitive and specific assay. NMOSD may include diseases that mimic NMO but involve distinct pathomechanisms due to the nearly equivalent number of patients who were seropositive and seronegative for the anti-AQP4 antibody. Treatment responses and the long-term prognosis of seronegative NMOSD should be analyzed separately from seropositive NMO in the future, and the search for novel pathogens such as anti-MOG antibodies should continue.
Conflict of interest We declared no conflict of interest regarding this study.
Funding This study was supported by Siriraj Research Grant number IOR015532049 and a research grant from the Ministry of Public Health, Thailand.
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Acknowledgments We are grateful to Wanichada Kosolthammakorn M.T., Natakorn Sangnumpong B.Sc., Leatchai Wachiruttamungkur, M.Sc, Nopwan Siwasiriyanont, M.Sc. and Laddawan Champa M.Sc. for their technical support. References [1] Wingerchuk DM, Lennon VA, Pittock SJ, Lucchinetti CF, Weinshenker BG. Revised diagnostic criteria for neuromyelitis optica. Neurology 2006;66(10):1485–9. [2] Wingerchuk DM, Lennon VA, Lucchinetti CF, Pittock SJ, Weinshenker BG. The spectrum of neuromyelitis optica. Lancet Neurol Sep 2007;6(9):805–15 [PubMed PMID: 17706564]. [3] Fujihara K, Leite MI. Seronegative NMO: a sensitive AQP4 antibody test clarifies clinical features and next challenges. Neurology Jun 11 2013;80(24):2176–7 [PubMed PMID: 23658387]. [4] Marignier R, Bernard-Valnet R, Giraudon P, Collongues N, Papeix C, Zephir H, et al. Aquaporin-4 antibody-negative neuromyelitis optica: distinct assay sensitivitydependent entity. Neurology Jun 11 2013;80(24):2194–200 [PubMed PMID: 23658379]. [5] Kitley J, Woodhall M, Waters P, Leite MI, Devenney E, Craig J, et al. Myelinoligodendrocyte glycoprotein antibodies in adults with a neuromyelitis optica phenotype. Neurology Sep 18 2012;79(12):1273–7 [PubMed PMID: 22914827]. [6] Jarius S, Ruprecht K, Wildemann B, Kuempfel T, Ringelstein M, Geis C, et al. Contrasting disease patterns in seropositive and seronegative neuromyelitis optica: a multicentre study of 175 patients. J Neuroinflammation 2012;9:14 [PubMed PMID: 22260418. Pubmed Central PMCID: 3283476]. [7] Takahashi T, Fujihara K, Nakashima I, Misu T, Miyazawa I, Nakamura M, et al. Antiaquaporin-4 antibody is involved in the pathogenesis of NMO: a study on antibody titre. Brain May 2007;130(Pt 5):1235–43. [8] Jarius S, Probst C, Borowski K, Franciotta D, Wildemann B, Stoecker W, et al. Standardized method for the detection of antibodies to aquaporin-4 based on a highly sensitive immunofluorescence assay employing recombinant target antigen. J Neurol Sci 2010;291(1–2):52–6. [9] Fazio R, Malosio ML, Lampasona V, De Feo D, Privitera D, Marnetto F. Antiacquaporin 4 antibodies detection by different techniques in neuromyelitis optica patients. Mult Scler Oct 2009;15(10):1153–63. [10] Lennon VA, Kryzer TJ, Pittock SJ, Verkman AS, Hinson SR. IgG marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel. J Exp Med 2005;202(4):473–7. [11] Siritho S, Nakashima I, Takahashi T, Fujihara K, Prayoonwiwat N. AQP4 antibodypositive Thai cases: clinical features and diagnostic problems. Neurology 2011;77(9):827–34.
