NEWS & VIEWS NEUROIMMUNOLOGY
Towards more-accurate diagnosis in neuromyelitis optica Kazuo Fujihara and Jacqueline Palace
Neuromyelitis optica spectrum disorders (NMOSD) can mimic multiple sclerosis (MS). Avoiding misdiagnosis is crucial, because some diseasemodifying drugs for MS can aggravate NMOSD, causing blindness and paraplegia. A recent study reports that misdiagnosis of NMOSD as MS occasionally occurs, and that a two-step antibody assay could improve differential diagnosis. Fujihara, K. & Palace, J. Nat. Rev. Neurol. advance online publication 11 November 2014; doi:10.1038/nrneurol.2014.216
Neuromyelitis optica spectrum disorders (NMOSD) are associated with high mortality and morbidity if left untreated. Immunopathomechanisms and, therefore, immunomodulatory treatment of NMOSD differ from those in multiple sclerosis (MS). Early diagnostic distinction of NMOSD from MS is critically important, because some disease-modifying drugs for MS (IFN‑β, natalizumab, and fingolimod) exacerbate NMOSD.1 Thus, it is highly relevant to determine how often NMOSD is misdiagnosed as MS in clinical practice; however, such studies have not been done on a large scale. A recent study by Sean Pittock and colleagues, published in JAMA Neurology, has addressed this issue by quantifying the misdiagnosis rate in an MS cohort based in northern California, USA.2
‘‘
…some disease-modifying drugs for MS … exacerbate NMOSD
’’
NMOSD are clinically characterized by severe optic neuritis (which is commonly bilateral owing to chiasmal lesions), longitudinally extensive transverse myelitis (LETM; >3 vertebral segments), and/or involvement of the brain or the brain stem including area postrema syndrome manifesting as intract able hiccup, nausea and vomiting.3,4 NMOSD encompasses these individual core clinical characteristics as well as typical NMO (the diagnostic criteria for NMO require both optic neuritis and LETM).3 Because
both optic neuritis and acute myelitis are also common in MS, the relation of NMOSD to MS had long been debated until the discovery of IgG antibodies against the aquaporin 4 (AQP4) water channel, which is densely expressed in the end-feet of astrocytes. This antibody is highly specific for NMOSD.4,5 The discovery of AQP4-IgG gave a boost to the field of NMO research—according to PubMed, NMO-related publications have steadily increased from less than 30 per year before 2004 to over 350 in 2013—and revolutionized our understanding of the disease. NMOSD is now recognized to be an autoimmune astrocytopathic disease rather than a demyelinating disease;6 however, the similarity of the clinical presentation in NMOSD and MS poses a substantial risk of misdiagnosis. Pittock et al. reviewed the electronic medical records of adult patients with MS from a population of 3.2 million individuals participating in a local health care plan in northern California.2 The authors initially identified 3,293 patients with potential MS, and 98 patients with a diagnosis of NMO or NMOSD. The rough estimates of prevalence were 100/100,000 for MS and 3/100,000 for NMOSD, both of which are reasonable figures in Western populations. Following further scrutiny, 1,040 patients with confirmed MS enrolled in the study (91% non-Hispanic white; 68.4% with relapsing– remitting MS, 15% with secondary progressive MS, 7.2% with primary progressive MS). The participants’ blood samples were sent for AQP4-IgG testing with ELISA using
NATURE REVIEWS | NEUROLOGY
the full-length isoform (M1) of AQP4, and with AQP4‑M1-transfected cell-based assay (CBA) using pre-fixed cells. Samples that were AQP4-IgG seropositive in the initial assays were further tested with an inhouse validated, quantitative fluorescence- activated cell sorting (FACS) assay using live AQP4‑M1-transfected cells. Samples from seven patients were AQP4IgG-seropositive on the initial screening assays; four by ELISA alone and three by both ELISA and CBA. Five of these patients were confirmed to have MS on the basis of their clinical and MRI data, and although all five were positive on the ELISA, only one was positive on the CBA and none were seropositive by FACS. The remaining two patients were seropositive according to FACS, ELISA (with high AQP4-IgG titres) and CBA, and had optic neuritis and LETM, thereby fulfilling the Wingerchuk criteria of definite NMO. The authors concluded that NMOSD is rarely misdiagnosed as MS in clinical practice in the USA (0.2% of patients), and that the false positive rates were 0.5% for ELISA and 0.1% for CBA.
‘‘
…the similarity of the clinical presentation in NMOSD and MS poses a substantial risk of misdiagnosis
’’
The low rate of NMOSD misdiagnosis suggests that the process for differentiation of NMOSD from MS is well conducted in clinical practice in the USA. However, it is noteworthy that this cohort had longstanding disease with a mean duration of 17 years from disease onset to enrollment. The risk of misdiagnosis could be higher in the early stage of disease. It is also worth a mention that this study was not able to assess whether patients with seronegative NMOSD were misdiagnosed as MS. An internet-based survey of MS specialists in the USA found that 95% of respondents reported having seen patients whose initial diagnosis of MS was incorrect. The most common alternative diagnosis was nonspecific white matter MRI abnormalities (reported by 81% of the survey respondents), but NMO ranked fifth (reported by 41%). ADVANCE ONLINE PUBLICATION | 1
© 2014 Macmillan Publishers Limited. All rights reserved
NEWS & VIEWS In Asia and some other regions where NMOSD:MS ratios are much higher than in the USA (from 1:1 to 1:10, mainly because of a low MS prevalence of about 5/100,000), the misdiagnosis rates might be high unless reliable AQP4-IgG assays are used. Indeed, a study conducted at an MS clinic in Bangkok showed that 24% (11/46) of patients in Thailand who had been clinically diagnosed with MS turned out to be AQP4IgG-seropositive, but in retrospect assessment, the majority of them had at least one feature characteristic of NMOSD, such as intractable hiccup or LETM.7 Moreover, in a Japanese trial on the efficacy of fingolimod in MS, 5.9% of patients tested positive for AQP4 antibodies despite an exclusion criterion for patients with LETM. Importantly, all these patients relapsed within a month of starting treatment—three with marked associated disability. In the study by Pittock et al., two patients whose AQP4-IgGseropositivity was confirmed by FACS were later found to have definite NMO after the review of medical records.2 These findings clearly demonstrate the importance of thorough history taking and evaluation of clinical, MRI and laboratory data as a whole in the initial diagnostic workup to differentiate NMOSD from MS. No single finding in NMOSD can confirm or rule out the diagnosis (for example, a spinal cord lesion extending >3 ver tebral segments does not always indicate NMOSD, whereas myelitis in some AQP4IgG-seropositive patients with NMOSD can be shorter than 3 vertebral segments8), and the new international consensus diagnostic criteria of NMOSD emphasizes the need to rigorously evaluate all available information and exclude other diseases.3 As Pittock et al. mention, giving disease-modifying drugs for MS to misdiagnosed patients with NMOSD could cause blindness, paraplegia and other permanent disabilities. Pittock and co-workers also showed that, in their MS cohort, the frequencies of false positive results were 0.5% for ELISA and 0.1% for CBA.2 Their two-step examination—s ending the samples that were seropositive in high-throughput tests like ELISA and CBA for confirmatory testing by FACS—could be a feasible way to avoid misdiagnosis. Because this study was set up to investigate the frequency of misdiagnosing NMOSD as MS in an MS
cohort using AQP4-IgG serostatus as the identifier, the relative sensitivities of AQP4IgG assays were not analyzed, but the findings by Pittock et al. seem to corroborate the previous work,9 which has established FACS as the most sensitive test. It is possible that FACS as a primary screening assay could have detected a few additional misdiagnosed patients. Using a sensitive and specific assay of AQP4-IgG is obviously important for reliable identification of patients with NMOSD. ELISA with linear AQP4 antigens is less sensitive than cell-based assays such as CBA and FACS, because pathogenic AQP4-IgG binds to extracellular loops of AQP4 that are conformationally intact in AQP4transfected cells, and irrelevant antibodies may occasionally be detected with ELISA, limiting its specificity. CBA is a ready-touse kit, but has the disadvantage that the pre-fixed cells can increase nons pecific binding of antibodies, potentially compromising assay sensitivity and specificity compared with FACS and other assays based on live cells. Besides the assay technique, the isoform of AQP4 used as the antigen can influence assay accuracy: AQP4-IgG binds to the AQP4‑M23 isoform (which lacks the initial 22 amino acids from the N‑terminus of the AQP4 molecule) with higher affinity than the AQP4‑M1 isoform, which is likely to result from the M23 isoform forming orthogonal array of particles (OAP) This assumption has been supported by the observation that green fluorescence protein that tags at the N‑terminus of AQP4 to confirm successful insertion of transfected AQP4 inhibits the formation of OAP and lowers the assay sensitivity. These methodological factors that influence the sensitivity and specificity of AQP4-IgG assays should be taken into consideration in the diagnostic workup for new patients suspected of NMOSD or MS to further reduce the rate of misdiagnosis, which can have severe consequences for the patient.9,10 Department of Multiple Sclerosis Therapeutics, Tohoku University Graduate School of Medicine, Aobaku, Sendai 980‑8574, Japan (K.F.). Nuffield Department of Clinical Neurosciences, West Wing, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK (J.P.). Correspondence to: K.F. [email protected]
2 | ADVANCE ONLINE PUBLICATION
Acknowledgements K.F. has received funding from the Grants-in-Aid for Scientific Research form the Ministry of Education, Science and Techonology and the Ministry of Health, Labor and Welfare of Japan. J.P. acknowledges highly specialized funding from NHS England to run a national NMO service. Competing interests: K.F. serves on scientific advisory boards for Alexion Pharmaceuticals, Bayer Schering Pharma, Biogen Idec, Chugai Pharmaceutical, Merck Serono, Mitsubishi Tanabe Pharma Corporation, Nihon Pharmaceutical, Novartis Pharma and Ono Pharmaceutical. He has received travel funding and speaker honoraria from Asahi Kasei Medical Co., Astellas Pharma Inc., Bayer Schering Pharma, Biogen Idec, Cosmic Corporation (a distributer of ELISA to detect aquaporin AQP4-IgG in Japan), Daiichi Sankyo, Eisai Inc., Mitsubishi Tanabe Pharma Corporation, Novartis Pharma and Takeda Pharmaceutical Company, Ltd; and research funding from Asahi Kasei Medical Co., Bayer Schering Pharma, Biogen Idec Japan, Eisai Inc., Kowa Pharmaceuticals America, Inc., Mitsubishi Tanabe Pharma Corporation, Teva Pharmaceutical K.K., Teijin Pharma, and The Chemo-Sero-Therapeutic Research Institute. J.P. has received conference funding for scientific meetings and honoraria for advisory work from Bayer Schering, Biogen Idec, Chugai Pharma, Merck Serono, Novartis, Ono Pharmaceutical Co. Ltd and Teva. She has received unrestricted research grants from Bayer Schering, Biogen Idec, Merck Serono and Novartis. 1.
Kimbrough, D. J. et al. Treatment of neuromyelitis optica: review and recommendations. Mult. Scler. Relat. Disord. 1, 180–187 (2012). 2. Pittock, S. J. et al. Seroprevalence of aquaporin‑4‑IgG in a northern California population representative cohort of multiple sclerosis. JAMA Neurol. http://dx.doi.org/ 10.1001/jamaneurol.2014.1581. 3. Wingerchuk, D. et al. Revised diagnostic criteria of neuromyelitis optica spectrum disorders (S63.001). Neurology 82 (Suppl. 63), S63.001 (2014). 4. Lennon, V. A. et al. A serum autoantibody marker of neuromyelitis optica: distinction from multiple sclerosis. Lancet 364, 2106–2112 (2004). 5. Lennon, V. A., Kryzer, T. J., Pittock, S. J., Verkman, A. S. & Hinson, S. R. IgG marker of optic-spinal multiple sclerosis binds to the aquaporin‑4 water channel. J. Exp. Med. 202, 473–477(2005). 6. Fujihara, K. et al. Neuromyelitis optica should be classified as an astrocytopathic disease rather than a demyelinating disease. Clin. Exp. Neuroimmunol. 3, 58–73 (2012). 7. Siritho, S., Nakashima, I., Takahashi, T., Fujihara, K. & Prayoonwiwat, N. AQP4 antibodypositive Thai cases, clinical features and diagnostic problems. Neurology 77, 827–834 (2011). 8. Sato, D. K. et al. Aquaporin‑4 antibody-positive cases beyond current diagnostic criteria for NMO spectrum disorders. Neurology 80, 2210–2216 (2013). 9. Waters, P. et al. Serologic diagnosis of NMO: a multicenter comparison of aquaporin‑4‑IgG assays. Neurology 78, 665–671 (2012). 10. Marignier, R. et al. Aquaporin‑4 antibody– negative neuromyelitis optica: distinct assay sensitivity–dependent entity. Neurology 80, 2194–2200 (2013).
www.nature.com/nrneurol © 2014 Macmillan Publishers Limited. All rights reserved
Towards more-accurate diagnosis in neuromyelitis optica Kazuo Fujihara and Jacqueline Palace
Neuromyelitis optica spectrum disorders (NMOSD) can mimic multiple sclerosis (MS). Avoiding misdiagnosis is crucial, because some diseasemodifying drugs for MS can aggravate NMOSD, causing blindness and paraplegia. A recent study reports that misdiagnosis of NMOSD as MS occasionally occurs, and that a two-step antibody assay could improve differential diagnosis. Fujihara, K. & Palace, J. Nat. Rev. Neurol. advance online publication 11 November 2014; doi:10.1038/nrneurol.2014.216
Neuromyelitis optica spectrum disorders (NMOSD) are associated with high mortality and morbidity if left untreated. Immunopathomechanisms and, therefore, immunomodulatory treatment of NMOSD differ from those in multiple sclerosis (MS). Early diagnostic distinction of NMOSD from MS is critically important, because some disease-modifying drugs for MS (IFN‑β, natalizumab, and fingolimod) exacerbate NMOSD.1 Thus, it is highly relevant to determine how often NMOSD is misdiagnosed as MS in clinical practice; however, such studies have not been done on a large scale. A recent study by Sean Pittock and colleagues, published in JAMA Neurology, has addressed this issue by quantifying the misdiagnosis rate in an MS cohort based in northern California, USA.2
‘‘
…some disease-modifying drugs for MS … exacerbate NMOSD
’’
NMOSD are clinically characterized by severe optic neuritis (which is commonly bilateral owing to chiasmal lesions), longitudinally extensive transverse myelitis (LETM; >3 vertebral segments), and/or involvement of the brain or the brain stem including area postrema syndrome manifesting as intract able hiccup, nausea and vomiting.3,4 NMOSD encompasses these individual core clinical characteristics as well as typical NMO (the diagnostic criteria for NMO require both optic neuritis and LETM).3 Because
both optic neuritis and acute myelitis are also common in MS, the relation of NMOSD to MS had long been debated until the discovery of IgG antibodies against the aquaporin 4 (AQP4) water channel, which is densely expressed in the end-feet of astrocytes. This antibody is highly specific for NMOSD.4,5 The discovery of AQP4-IgG gave a boost to the field of NMO research—according to PubMed, NMO-related publications have steadily increased from less than 30 per year before 2004 to over 350 in 2013—and revolutionized our understanding of the disease. NMOSD is now recognized to be an autoimmune astrocytopathic disease rather than a demyelinating disease;6 however, the similarity of the clinical presentation in NMOSD and MS poses a substantial risk of misdiagnosis. Pittock et al. reviewed the electronic medical records of adult patients with MS from a population of 3.2 million individuals participating in a local health care plan in northern California.2 The authors initially identified 3,293 patients with potential MS, and 98 patients with a diagnosis of NMO or NMOSD. The rough estimates of prevalence were 100/100,000 for MS and 3/100,000 for NMOSD, both of which are reasonable figures in Western populations. Following further scrutiny, 1,040 patients with confirmed MS enrolled in the study (91% non-Hispanic white; 68.4% with relapsing– remitting MS, 15% with secondary progressive MS, 7.2% with primary progressive MS). The participants’ blood samples were sent for AQP4-IgG testing with ELISA using
NATURE REVIEWS | NEUROLOGY
the full-length isoform (M1) of AQP4, and with AQP4‑M1-transfected cell-based assay (CBA) using pre-fixed cells. Samples that were AQP4-IgG seropositive in the initial assays were further tested with an inhouse validated, quantitative fluorescence- activated cell sorting (FACS) assay using live AQP4‑M1-transfected cells. Samples from seven patients were AQP4IgG-seropositive on the initial screening assays; four by ELISA alone and three by both ELISA and CBA. Five of these patients were confirmed to have MS on the basis of their clinical and MRI data, and although all five were positive on the ELISA, only one was positive on the CBA and none were seropositive by FACS. The remaining two patients were seropositive according to FACS, ELISA (with high AQP4-IgG titres) and CBA, and had optic neuritis and LETM, thereby fulfilling the Wingerchuk criteria of definite NMO. The authors concluded that NMOSD is rarely misdiagnosed as MS in clinical practice in the USA (0.2% of patients), and that the false positive rates were 0.5% for ELISA and 0.1% for CBA.
‘‘
…the similarity of the clinical presentation in NMOSD and MS poses a substantial risk of misdiagnosis
’’
The low rate of NMOSD misdiagnosis suggests that the process for differentiation of NMOSD from MS is well conducted in clinical practice in the USA. However, it is noteworthy that this cohort had longstanding disease with a mean duration of 17 years from disease onset to enrollment. The risk of misdiagnosis could be higher in the early stage of disease. It is also worth a mention that this study was not able to assess whether patients with seronegative NMOSD were misdiagnosed as MS. An internet-based survey of MS specialists in the USA found that 95% of respondents reported having seen patients whose initial diagnosis of MS was incorrect. The most common alternative diagnosis was nonspecific white matter MRI abnormalities (reported by 81% of the survey respondents), but NMO ranked fifth (reported by 41%). ADVANCE ONLINE PUBLICATION | 1
© 2014 Macmillan Publishers Limited. All rights reserved
NEWS & VIEWS In Asia and some other regions where NMOSD:MS ratios are much higher than in the USA (from 1:1 to 1:10, mainly because of a low MS prevalence of about 5/100,000), the misdiagnosis rates might be high unless reliable AQP4-IgG assays are used. Indeed, a study conducted at an MS clinic in Bangkok showed that 24% (11/46) of patients in Thailand who had been clinically diagnosed with MS turned out to be AQP4IgG-seropositive, but in retrospect assessment, the majority of them had at least one feature characteristic of NMOSD, such as intractable hiccup or LETM.7 Moreover, in a Japanese trial on the efficacy of fingolimod in MS, 5.9% of patients tested positive for AQP4 antibodies despite an exclusion criterion for patients with LETM. Importantly, all these patients relapsed within a month of starting treatment—three with marked associated disability. In the study by Pittock et al., two patients whose AQP4-IgGseropositivity was confirmed by FACS were later found to have definite NMO after the review of medical records.2 These findings clearly demonstrate the importance of thorough history taking and evaluation of clinical, MRI and laboratory data as a whole in the initial diagnostic workup to differentiate NMOSD from MS. No single finding in NMOSD can confirm or rule out the diagnosis (for example, a spinal cord lesion extending >3 ver tebral segments does not always indicate NMOSD, whereas myelitis in some AQP4IgG-seropositive patients with NMOSD can be shorter than 3 vertebral segments8), and the new international consensus diagnostic criteria of NMOSD emphasizes the need to rigorously evaluate all available information and exclude other diseases.3 As Pittock et al. mention, giving disease-modifying drugs for MS to misdiagnosed patients with NMOSD could cause blindness, paraplegia and other permanent disabilities. Pittock and co-workers also showed that, in their MS cohort, the frequencies of false positive results were 0.5% for ELISA and 0.1% for CBA.2 Their two-step examination—s ending the samples that were seropositive in high-throughput tests like ELISA and CBA for confirmatory testing by FACS—could be a feasible way to avoid misdiagnosis. Because this study was set up to investigate the frequency of misdiagnosing NMOSD as MS in an MS
cohort using AQP4-IgG serostatus as the identifier, the relative sensitivities of AQP4IgG assays were not analyzed, but the findings by Pittock et al. seem to corroborate the previous work,9 which has established FACS as the most sensitive test. It is possible that FACS as a primary screening assay could have detected a few additional misdiagnosed patients. Using a sensitive and specific assay of AQP4-IgG is obviously important for reliable identification of patients with NMOSD. ELISA with linear AQP4 antigens is less sensitive than cell-based assays such as CBA and FACS, because pathogenic AQP4-IgG binds to extracellular loops of AQP4 that are conformationally intact in AQP4transfected cells, and irrelevant antibodies may occasionally be detected with ELISA, limiting its specificity. CBA is a ready-touse kit, but has the disadvantage that the pre-fixed cells can increase nons pecific binding of antibodies, potentially compromising assay sensitivity and specificity compared with FACS and other assays based on live cells. Besides the assay technique, the isoform of AQP4 used as the antigen can influence assay accuracy: AQP4-IgG binds to the AQP4‑M23 isoform (which lacks the initial 22 amino acids from the N‑terminus of the AQP4 molecule) with higher affinity than the AQP4‑M1 isoform, which is likely to result from the M23 isoform forming orthogonal array of particles (OAP) This assumption has been supported by the observation that green fluorescence protein that tags at the N‑terminus of AQP4 to confirm successful insertion of transfected AQP4 inhibits the formation of OAP and lowers the assay sensitivity. These methodological factors that influence the sensitivity and specificity of AQP4-IgG assays should be taken into consideration in the diagnostic workup for new patients suspected of NMOSD or MS to further reduce the rate of misdiagnosis, which can have severe consequences for the patient.9,10 Department of Multiple Sclerosis Therapeutics, Tohoku University Graduate School of Medicine, Aobaku, Sendai 980‑8574, Japan (K.F.). Nuffield Department of Clinical Neurosciences, West Wing, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK (J.P.). Correspondence to: K.F. [email protected]
2 | ADVANCE ONLINE PUBLICATION
Acknowledgements K.F. has received funding from the Grants-in-Aid for Scientific Research form the Ministry of Education, Science and Techonology and the Ministry of Health, Labor and Welfare of Japan. J.P. acknowledges highly specialized funding from NHS England to run a national NMO service. Competing interests: K.F. serves on scientific advisory boards for Alexion Pharmaceuticals, Bayer Schering Pharma, Biogen Idec, Chugai Pharmaceutical, Merck Serono, Mitsubishi Tanabe Pharma Corporation, Nihon Pharmaceutical, Novartis Pharma and Ono Pharmaceutical. He has received travel funding and speaker honoraria from Asahi Kasei Medical Co., Astellas Pharma Inc., Bayer Schering Pharma, Biogen Idec, Cosmic Corporation (a distributer of ELISA to detect aquaporin AQP4-IgG in Japan), Daiichi Sankyo, Eisai Inc., Mitsubishi Tanabe Pharma Corporation, Novartis Pharma and Takeda Pharmaceutical Company, Ltd; and research funding from Asahi Kasei Medical Co., Bayer Schering Pharma, Biogen Idec Japan, Eisai Inc., Kowa Pharmaceuticals America, Inc., Mitsubishi Tanabe Pharma Corporation, Teva Pharmaceutical K.K., Teijin Pharma, and The Chemo-Sero-Therapeutic Research Institute. J.P. has received conference funding for scientific meetings and honoraria for advisory work from Bayer Schering, Biogen Idec, Chugai Pharma, Merck Serono, Novartis, Ono Pharmaceutical Co. Ltd and Teva. She has received unrestricted research grants from Bayer Schering, Biogen Idec, Merck Serono and Novartis. 1.
Kimbrough, D. J. et al. Treatment of neuromyelitis optica: review and recommendations. Mult. Scler. Relat. Disord. 1, 180–187 (2012). 2. Pittock, S. J. et al. Seroprevalence of aquaporin‑4‑IgG in a northern California population representative cohort of multiple sclerosis. JAMA Neurol. http://dx.doi.org/ 10.1001/jamaneurol.2014.1581. 3. Wingerchuk, D. et al. Revised diagnostic criteria of neuromyelitis optica spectrum disorders (S63.001). Neurology 82 (Suppl. 63), S63.001 (2014). 4. Lennon, V. A. et al. A serum autoantibody marker of neuromyelitis optica: distinction from multiple sclerosis. Lancet 364, 2106–2112 (2004). 5. Lennon, V. A., Kryzer, T. J., Pittock, S. J., Verkman, A. S. & Hinson, S. R. IgG marker of optic-spinal multiple sclerosis binds to the aquaporin‑4 water channel. J. Exp. Med. 202, 473–477(2005). 6. Fujihara, K. et al. Neuromyelitis optica should be classified as an astrocytopathic disease rather than a demyelinating disease. Clin. Exp. Neuroimmunol. 3, 58–73 (2012). 7. Siritho, S., Nakashima, I., Takahashi, T., Fujihara, K. & Prayoonwiwat, N. AQP4 antibodypositive Thai cases, clinical features and diagnostic problems. Neurology 77, 827–834 (2011). 8. Sato, D. K. et al. Aquaporin‑4 antibody-positive cases beyond current diagnostic criteria for NMO spectrum disorders. Neurology 80, 2210–2216 (2013). 9. Waters, P. et al. Serologic diagnosis of NMO: a multicenter comparison of aquaporin‑4‑IgG assays. Neurology 78, 665–671 (2012). 10. Marignier, R. et al. Aquaporin‑4 antibody– negative neuromyelitis optica: distinct assay sensitivity–dependent entity. Neurology 80, 2194–2200 (2013).
www.nature.com/nrneurol © 2014 Macmillan Publishers Limited. All rights reserved
