26 - bichuetti 2018_Treating neuromyelitis optica with azathioprine_ 20-year clinical practice

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DOI: 10.1177/ 
1352458518776584
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Introduction
Neuromyelitis optica spectrum disorder (NMOSD) is 
an autoimmune disease of the central nervous system 
associated primarily to anti-aquaporin 4 antibody that 
affects preferentially the optic nerve and spinal cord 
distinct from multiple sclerosis (MS).1,2 Its prevalence 
is estimated in 4–6/100,000 worldwide, with higher 
prevalence in South America and Asia, as it affects 
more persons with Asian and African ethnic back-
ground.3,4 Nevertheless, these numbers are possibly 
miscalculated since there are more studies from North 
America and Europe. Indeed, in two Brazilian studies, 
NMOSD comprised 15%–20% of the whole idio-
pathic demyelinating disease cohort.5,6 Preventive 
treatment is based on case series and revision papers 
evaluating the use of systemic immunosuppressants, 
mainly steroids, azathioprine, mycophenolate, metho-
trexate, and rituximab,7–9 and only one direct head-to-
head study comparing rituximab and azathioprine 
limited to 1-year follow-up is available.10
Brazil is a country with a mixed health-care system; 
while some have access to high-cost services and 
treatments, most depend on public health care, 
Sistema Único de Saúde (SUS) for treatment and out-
patient drug supplying.11 Azathioprine and prednisone 
are low-cost medication provided by SUS and have 
been the primary regimen for treating NMOSD in our 
center for the past two decades. The main objective of 
this study is to review the treatment of NMOSD at our 
center focusing on azathioprine and prednisone in a 
real-life scenario.
Methodology
This study is part of a cohort of patients with NMOSD 
from the Neuroimmunology Clinic of the Universidade 
Federal de São Paulo.12–14 The university’s Ethics 
Committees approved the study, and participants pro-
vided written informed consent.
Treating neuromyelitis optica with 
azathioprine: 20-year clinical practice
Denis Bernardi Bichuetti, Marília Mamprim de Moraes Perin, Nilton Amorim de Souza 
and Enedina Maria Lobato de Oliveira
Abstract
Background: Neuromyelitis optica leads to severe disability. Preventive treatment includes steroids and 
immunosuppressants, and indications are based on retrospective and observational studies.
Methods: We analyzed 158 patients with neuromyelitis optica regarding disease course, prognostic fac-
tors, and treatment response to azathioprine, a widely available low-cost drug. Disability accumulation 
was used as an endpoint to treatment response.
Results: Eight patients with monophasic and 150 with relapsing disease with a median 7 years of dis-
ease duration and 4.6 years of follow-up were evaluated. All relapsing patients received preventive treat-
ment, 100 with azathioprine. Only 30% reached Expanded Disability Status Scale (EDSS) 6, and 69% 
of patients presented no disability accumulation along follow-up. The time under azathioprine and pred-
nisone use were inversely correlated to the hazard of disability accumulation (hazard ratio (HR) = 0.981 
and 0.986, respectively; p < 0.01). Each month under azathioprine use reduced disability accumulation by 
2.6% (HR = 0.974, p < 0.01), corresponding to an 80% decrease in EDSS progression over 5 years.
Interpretation: We report less disability accumulation than previous series on patients with neuromyeli-
tis optica, two-thirds presenting no disability accumulation along follow-up. Continued azathioprine used 
from early disease onset was strongly associated to maintenance of neurological function and should be 
offered as a viable option for low-income scenarios.
Keywords: Neuromyelitis optica, prognosis, epidemiology, treatment, azathioprine
Date received: 9 February 2018; revised: 3 April 2018; accepted: 21 April 2018
Correspondence to: 
DB Bichuetti 
Neuroimmunology Clinic, 
Disciplina de Neurologia, 
Universidade Federal de 
São Paulo (UNIFESP), Rua 
Botucatu, 740, São Paulo, SP 
04023-900, Brazil. 
bichuetti@unifesp.br
Denis Bernardi Bichuetti 
Marília Mamprim de 
Moraes Perin 
Nilton Amorim de Souza 
Enedina Maria Lobato de 
Oliveira 
Neuroimmunology Clinic, 
Disciplina de Neurologia, 
Universidade Federal de 
São Paulo (UNIFESP), São 
Paulo, Brazil
776584MSJ0010.1177/1352458518776584Multiple Sclerosis JournalDB Bichuetti, MM de M Perin
research-article2018
Original Research Paper
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Case definition and inclusion criteria
The files of all patients followed-up for a demyelinat-
ing disease from 1994 to 31 December 2015 were 
reviewed. Inclusion criteria were clinical presentation 
compatible with NMOSD per the International Panel 
for NMO Diagnosis (IPND)2 and follow-up longer 
than 6 months. Patients evaluated before the publica-
tion of the IPND criteria had their files and exams 
revised for diagnostic ascertainment. All patients 
were evaluated for rheumatologic diseases, hepatitis 
B/C, syphilis, and HIV as part of our standard investi-
gation for demyelinating diseases. The presence of 
any infectious syndrome and uncomplete medical 
record were considered as exclusion criteria.
Patients were classified as monophasic neuromyelitis 
optica spectrum disorder (mNMOSD), if presented a 
single optic neuritis and longitudinal extensive trans-
verse myelitis (LETM) relapse, tested negative for the 
anti-aquaporin 4 antibody and maintained without 
immunosuppressive treatment; and relapsing neuro-
myelitis optica spectrum disorder (rNMOSD), if pre-
sented more than one relapse or positivity for 
anti-aquaporin 4 antibody.2,15,16 The time to “index 
event” was considered as the interval for occurrence 
of both optic neuritis and acute myelitis in a single 
patient, as defined by Wingerchuk et al.15 Considering 
that knowledge and treatment of NMOSD changed 
dramatically in the last 20 years,1 we divided the 
relapsing cohort into two: one including all patients 
ever seen in the clinic and other including only those 
with regular follow-up visits after 1 January 2013, 
including those that deceased. This division was cre-
ated to offer a general view of the history of NMOSD 
in our clinic and then evaluate a more uniform group, 
that is, those with recent follow-up information. The 
rNMOSD was divided into three groups according to 
their main clinical presentation: relapsing neuromy-
elitis optica (rNMO, when two of the three core symp-
toms were present, optic neuritis, LETM, or area 
postrema syndrome), relapsing longitudinal extensive 
transverse myelitis (rLETM, when only LETM was 
present) and relapsing optic neuritis (rON, when only 
optic neuritis was present).
Data collection
We collected demographic, clinical, laboratory, and 
magnetic resonance imaging (MRI) data on all 
patients who met inclusion criteria. To avoid bias due 
to disease duration, we normalized the Expanded 
Disability Status Scale (EDSS)17 on last follow-up 
visit and total number of relapses by the total time of 
disease (in years), thus using the progression index 
(PI, EDSS/years of disease) and annualized relapse 
rate (ARR, relapses/years of disease) to evaluate dis-
ease progression and severity.
Statistical analysis
Descriptive analysis
Statistical analysis was performed using GraphPad 
Prism version 7.00 (GraphPad Software Incorporated®) 
and Stata version 14 (StataCorp LLC®). D’Agostino–
Pearson test was used to evaluate the departure from 
normality and data are presented as mean ± standard 
deviation if met normal distributioncriteria and 
median and quartiles if not. Unpaired t-test or Mann–
Whitney test was used when comparing two groups; 
analysis of variance when comparing multiple groups; 
and chi-square or Fisher’s exact test, when appropri-
ate. Significance was set at p < 0.05.
Treatment analysis
Most patients received one or a combination of drugs, 
including corticosteroids, azathioprine, methotrexate, 
mycophenolate, cyclosporine, rituximab, cyclophos-
phamide, interferon beta, glatiramer acetate, and 
intravenous (IV) immunoglobulin. Since 2006, the 
mainstream recommendation in our clinic is azathio-
prine + prednisone,13,18,19 as they are reimbursed by 
SUS20 and are the focus of the treatment evaluation in 
this study. Immunosuppressive therapy was indicated 
for all patients positive for anti-aquaporin antibodies 
or with a relapsing course, regardless of anti-aqua-
porin status. Patients with a single event and negative 
for anti-aquaporin antibodies were not treated besides 
pulse IV steroids for their acute clinical event and 
excluded from this treatment analysis.
Retrieving complete relapse history for all therapies 
in every single patient was very difficult due to 
changes in our patient record system, from different 
paper charts to electronic medical record. Since 
patients with NMOSD do not develop a secondary 
progressive phase,21 we created a disability accumu-
lation index based on EDSS variation from the first to 
the last follow-up appointment, as performed on long-
term observation studies with MS:22 an increase of 
≥1.0 EDSS point-variation was considered for reach-
ing “outcome 1”; whereas only ≥0.5 EDSS point-
variation was already considered for reaching 
“outcome 2.” The two products were tested as ways of 
assessing disability accumulation with outcome 1 as a 
more lenient criterion and outcome 2 as more 
stringent and less tolerant regarding disability accumu-
lation. This way, we assumed that if minimal disabil-
ity progression happened along follow-up, either an 
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individual patient suffered no relapses or very mild 
relapses.
The variables chosen as predictors for each outcome 
were as follows: age of onset (age at first relapse), 
gender, type of NMOSD (rNMO, rLETM, and rON), 
EDSS on first appointment, time on azathioprine and 
prednisone (period that each patient ever used one of 
each drug, in months), time on each drug over disease 
duration (as percentage of the disease length that the 
patient received each treatment), and time to pred-
nisone or azathioprine (time lapsed from the first 
symptom to start of each drug).
The two different outcomes were analyzed as sur-
vival data with the event defined as each outcome 
described above. Thus, whenever the increased 
value in EDSS had not been reached, data were 
censored at the last evaluation. Median times were 
estimated using the Kaplan–Meier method, and 
survival curves were compared using the log-rank 
test. For continuous covariates (baseline EDSS, age 
of onset and time on/to each treatment), we used 
univariate Cox regression. To congregate the influ-
ence of the putative risk factors, we performed 
multivariate Cox models throughout forward selec-
tion procedures which resulted from the univariate 
analysis (i.e. whether for each covariate, it was 
associated with the outcome conditional with a 
p-value cutoff in a regression model of 0.10). The 
variable that was most strongly associated with 
outcome (based on having the smallest p-value 
below the cutoff) was then added to the collection 
of variables for which control was made.23 P-values 
of less than 0.05 on two-tailed tests were consid-
ered as statistically significant in the multivariate 
Cox regression.
Results
A total of 1748 files were reviewed and 216 selected 
for analysis: 58 were excluded (37 with uncomplete 
medical records and 21 without NMOSD) and 158 
selected, including 8 with mNMOSD and 150 with 
rNMOSD (from which 115 had regular follow-up 
after 1 January 2013; Table 1 and Figure 1). From the 
whole relapsing cohort, 112 presented rNMO, 16 
rLETM, and 22 rON (Table 2). The three presenta-
tions were similar in demographic and clinical 
aspects with the exception that patients with rLETM 
were older and had shorter time interval to second 
relapse than rNMO, and rNMO had a higher EDSS 
on last appointment than rON (Table 2). Only 12% of 
the patients with rNMOSD did not fulfill the 2015 
diagnostic criteria for NMOSD,2 mainly those with 
relapsing optic neuritis or relapsing LETM and nega-
tive anti-aquaporin 4 results. All eight patients with 
mNMOSD were anti-aquaporin 4 negative, did not 
receive chronic immunosuppressants, and presented 
acute myelitis and optic neuritis within less than 
30 days apart, similar to Wingerchuk et al.’s original 
cohort.15
Five of the eight patients with mNMOSD had infor-
mation on brain MRI, and two presented abnormali-
ties: one in the medulla and the other as unspecific 
cerebral white matter lesions. Around 140 of the 150 
patients with rNMOSD had information on brain 
MRI, and 73 (48.7%) presented abnormalities, none 
fulfilling MS criteria.24 The lesions sites were com-
patible to previously described,25,12 present in the fol-
lowing topographies: medulla (43.8%), pons (23.3%), 
midbrain (20.5%), optic chiasm (12.3%), diencepha-
lon (17.8%), internal capsule (8.2%), corpus callosum 
(15.1%), and unspecific cerebral white matter lesions 
(72.6%). Patients with brain abnormalities presented 
no statistical difference in age of onset, disease dura-
tion, EDSS, RR, or PI compared to patients without 
brain abnormalities.
Around 29 patients (19.3%) with rNMOSD had one 
or more comorbid autoimmune disease: systemic 
lupus erythematosus (7), Sjogren syndrome (5), 
Behcet syndrome (1), Hashimoto’s thyroiditis (7), 
myasthenia gravis (4), cyclic autoimmune neutrope-
nia (1), recurrent deep venous thrombosis (1), posi-
tive anticardiolipin antibody (1), dermatopolimiositis 
(1), type 1 diabetes mellitus (1), rheumatoid arthritis 
(1), and Graves thyroiditis (1). None of the patients 
with mNMOSD had an autoimmune disease. Six 
patients with rNMOSD deceased during follow-up; 
cause of death was either cervical spinal cord relapse 
leading to respiratory arrest or infectious complica-
tions of immunosuppression. Six patients developed 
neoplasms during follow-up: two skin cancer and 
the others breast, rectum, uterus, and thyroid, and 
none within less than 5 years from the start of 
immunosuppression.
All patients with rNMOSD received at least one pre-
ventive treatment, including disease-modifying thera-
pies for MS (16), prednisone (101), azathioprine 
(100), mycophenolate (4), cyclosporine (2), metho-
trexate (20), cyclophosphamide (16), rituximab, (2) 
and IV immunoglobulin (8). EDSS variation is pre-
sented in Table 1. Eleven patients under azathioprine 
suffered severe side effects demanding treatment 
interruption: gastrointestinal intolerance (4), severe 
infection (2), alopecia (1), liver toxicity (2), and 
allergy/skin reactions (2).
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Table 1. Clinical demographic and laboratory data of 158 patients with neuromyelitis optica spectrum disorder.
Feature (subjects) Monophasic 
NMOSD (N = 8)
rNMOSD* (N = 150) rNMOSD** 
(N = 115)
Age of onset, years 33.6 (±13.3) 33.0 (24.0–43.3) 33.8 (±12.3)
Sex (F:M) 1:1 4.4:1 4.2:1
Ethnicity, n (%)
 Caucasian 3 (38) 77 (51.3) 52 (45.2)
 Afro descendant (Mulatto + Afro) 4 (50) 71 (47.3) 61 (53.0)
 Asian descendant 1 (13) 2 (1.3) 2 (1.7)
 America Indian 0 0 0
Years of follow-up 7.4 (1.5–11.2) 4.6 (2.1–8.7) 5.8 (2.7–9.8)
Years of disease 8.3 (2.8–21.2) 7.0 (3.7–12.6) 8.0 (4.2–12.8)
First relapse, n (%)
 Myelitis 60 (40.0) 49 (42.6)
 Optic neuritis 56 (37.3) 41 (35.7)
 Brainstem 9 (6.0) 7.0 (6.1)
 Encephalitic 2 (1.3) 2 (1.7)
 Optic neuritis+ myelitis 8 (100) 13 (8.7) 9 (7.8)
 Optic neuritis + brainstem 2 (1.3) 1 (0.9)
 Myelitis + brainstem 5 (3.3) 4 (3.5)
 ON + brainstem + myelitis 1 (0.7) 1 (0.9)
 Encephalitic + brainstem + myelitis 1 (0.7) 1 (0.9)
 Unregistered 1 (0.7) 0
Months to index event*** NA 11.6 (2.0–36.1) 12.7 (2.1–36.1)
Months to second relapse NA 11.3 (3.1–25.9) 10.4 (3.0–25.4)
Months from first relapse to evaluation 17.5 (1.9–51.8) 15.9 (5.1–41.3) 15.2 (4.6–38.2)
EDSS on first appointment 3.0 (2.0–3.0) 3.0 (3.0–4.0) 3.0 (3.0–4.0)
EDSS on last appointment 3.0 (2.0–3.0) 4.0 (3.0–6.5) 4.0 (3.0–6.0)
Relapse rate NA 0.6 (0.4–1.0) 0.6 (0.4–0.8)
Progression Index NA 0.5 (0.3–1.1) 0.4 (0.3–0.9)
Abnormal brain MRI not meeting criteria 
for MS, n (%)
2 (25.0) 73 (48.7) 57 (49.6)
Unknown/unavailable, n (%) 3 (37.5) 10 (6.7) 7 (6.1)
Patients reaching EDSS, n (%)
 3.0 5 (62.5) 126 (84.0) 106 (92.2)
 4.0 1 (12.5) 88 (58.7) 72 (62.6)
 6.0 1 (12.5) 50 (33.3) 38 (33.0)
 6.5 1 (12.5) 44 (29.3) 32 (27.8)
 7.0 1 (12.5) 39 (26.0) 29 (25.2)
 10 0 (0) 6 (4.0) 6 (5.2)
EDSS variation from first to last 
appointment, n (%)
NA 
 0–0.5 103 (69) 78 (67)
 1.0–1.5 19 (13) 16 (14)
 2.0–2.5 9 (6) 6 (5)
 3.0–3.5 7 (5) 7 (6)
 ≥4.0 10 (7) 8 (7)
Patients reaching Outcomes, n (%) NA 
 Outcome 1 34 (22.7) 26 (22.6)
 Outcome 2 50 (33.3) 38 (33.0)
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Feature (subjects) Monophasic 
NMOSD (N = 8)
rNMOSD* (N = 150) rNMOSD** 
(N = 115)
CSF analysis (number of patients available) 5 105 81
 WBC/mm3 5.0 (3.5–21.5) 7.0 (2.0–19.5) 5.0 (2.0–18.0)
 >50 WBC/mm3, n (%) 0 (100) 15/105 (14.3) 13/81 (16.0)
OCB (analyzed/positive (+)) 1/0 32/5 26/4
Anti aquaporin 4. tested/positive (%) 6/0 (0) 126/68 (54.0) 107/56 (52.3)
Patients fulfilling 2015 NMOSD criteria, n (%)
 Yes NA 124 (82.7) 97 (84.3)
 No 18 (12.0) 16 (13.9)
 Incomplete information 8 (5.3) 2 (1.7)
NMOSD type, n (%)
 rNMO NA 112 (74.7) 87 (75.7)
 rLETM 16 (10.7) 13 (11.3)
 rON 22 (14.7) 15 (13.0)
Comorbid autoimmunity, n (%) 0 (0) 29 (19.3) 25 (21.7)
EDSS: Expanded Disability Status Scale; MS: multiple sclerosis; NA: not applicable; NMOSD: neuromyelitis optica spectrum 
disorder; rNMOSD: relapsing neuromyelitis optica spectrum disorder; rNMO: relapsing neuromyelitis optica; rLETM: relapsing 
longitudinal extensive transverse myelitis; ON: optic neuritis; rON: relapsing optic neuritis; CSF: cerebrospinal fluid; WBC: white 
blood cell; MRI: magnetic resonance imaging: ANA: anti-nuclear antibodies. Index event: the interval for occurrence of any optic 
neuritis and acute myelitis in a single patient; relapse rate: total number of relapses/disease duration; progression index: EDSS on last 
appointment/disease duration.
*rNMO 150: comprise all patients ever seen by our staff; *rNMO 115: comprise all patients that had regular follow-up visits up to 
December 2013; ***For those that had optic neuritis. myelitis or area postrema syndrome as a first event.
Normality test = D’Agostino–Pearson test.
Table 1. (Continued)
Figure 1. Sample description according to inclusion and exclusion criteria.
NMOSD: neuromyelitis optica spectrum disorder; mNMOSD: monophasic neuromyelitis optica spectrum disorder; rNMOSD: relapsing 
neuromyelitis optica spectrum disorder.
Table 3 presents the univariate Cox regression for the 
categorical and continuous predictors for treatment 
analysis. Time on prednisone and on azathioprine 
were statistically significant for both samples with a 
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Table 2. Clinical data of 150 patients with relapsing NMOSD according to type of NMOSD.
Feature (subjects, n) 150 150 150 p-value 115 115 115 p-value
NMOSD 1 rNMO (112) 2 rLETM (16) 3 rON (22) 1 rNMO (87) 2 rLETM (13) 3 rON (15) 
Age of onset, years 32.5 (±12.9) 41.4 (±8.7) 35.2 (±10.7) 0.0226 32.8 (±13.0) 40.1 (±6.1) 33.4 (±10.4) 0.1040
Sex (F:M) 3.7:1 4.3:1 21.0:1 0.1780 3.8:1 3.3:1 14.0:1 0.4119
Years of follow-up 4.7 (2.1–9.1) 6.5 (±4.2) 2.4 (0.9–5.5) 0.0802 5.8 (2.7–9.8) 7.6 (±3.8) 4.9 (±5.2) 0.1017
Years of disease 7.7 (3.9–13.4) 7.9 (±4.3) 4.2 (2.0–7.9) 0.0783 8.7 (4.5–14.0) 8.8 (±4.2) 5.7 (2.5–10.9) 0.1798
First relapse, n (%)
 Myelitis 44 (39.3) 16 (100) 35 (40.2) 13 (100) 
 Optic neuritis 34 (30.4) 22 (100) 27 (31.0) 15 (100) 
 Brainstem 9 (8.0) NA 7 (8.0) 
 Encephalitic 2 (1.8) 2 (2.3) 
 Optic neuritis + myelitis 13 (11.6) 9 (10.3) NA
 Optic neuritis + brainstem 2 (1.8) 1 (1.1) 
 Myelitis + brainstem 5 (4.5) 4 (4.6) 
 ON + brainstem + myelitis 1 (0.9) 1 (1.1) 
 Encephalitic + brainstem + myelitis 1 (0.9) 1 (1.1) 
 Unregistered 1 (0.9) 0 
Months to index event 11.6 (2.0–36.1) NA NA NA 12.6 (2.1–36.1) NA NA 
Months to second relapse 11.7 (3.1–30.5) 3.8 (±2.3) 7.1 (3.0–19.7) 0.0047 10.1 (3.0–27.4) 12.3 (±9.4) 6.1 (2.1–12.2) 0.6291
Months from first relapse to 
evaluation
19.3 (5.6–48.7) 16.5 (±15.3) 12.2 (6.7–43.4) 0.2798 15.2 (4.6–48.6) 14.5 (±13.5) 16.6 (5.4–37.5) 0.3568
EDSS on first appointment 3.5 (3.0–5.0) 3.8 (±2.3) 2.9 (±1.1) 0.1588 3.5 (3.0–4.0) 4.0 (±2.2) 3.1 (±1.0) 0.5701
EDSS on last appointment 4.0 (3.0–7.0) 4.0 (±2.4) 2.9 (±1.2) 0.0059 4.0 (3.0–6.5) 4.3 (±2.4) 3.1 (±1.1) 0.0850
Relapse rate 0.6 (0.4–1.0) 0.6 (±0.3) 0.7 (0.3–1.2) 0.6767 0.6 (0.4–0.8) 0.6 (±0.3) 0.7 (0.3–1.2) 0.7482
Progression Index 0.5 (0.3–1.1) 0.4 (0.3–1.1) 0.7 (0.3–1.0) 0.7814 0.4 (0.3–0.9) 0.4 (0.3–0.9) 0.7 (0.3–1.0) 0.8323
Abnormal brain MRI not meeting 
criteria for MS, n (%)
56 (50.0) 8 (50.0) 9 (40.9) 0.4836 46 (52.9) 5 (38.5) 6 (35.3) 0.2506
 Unknown/unavailable, n (%) 10 (8.9) 0 (0) 0 (0) 7 (8.0) 0 (0) 0 (0) 
Patients reaching EDSS, n (%) NA NA
 3.0 96 (85.7) 14 (87.5) 16 (72.7) 81 (93.1) 13 (100.0) 12 (80.0) 
 4.0 71 (63.4) 7 (43.8) 10 (45.5) 57 (65.5) 6 (46.2) 9 (60.0) 
 6.0 45 (40.2) 5 (31.3) 0 34 (39.1) 4 (30.8) 0 
 6.5 39 (34.8) 5 (31.3) 0 28 (32.2) 4 (30.8) 0 
 7.0 35 (31.3) 4 (25.0) 0 25 (28.7) 4 (30.8) 0 
 10 6 (5.4) 0 (0) 0 6 (6.9) 0 0 
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Feature (subjects, n) 150 150 150 p-value 115 115 115 p-value
CSF analysis (number of patients 
available)
72 16 16 0.0046 57 13 11 0.0709
 WBC/mm3 9.0 (3.0–21.8) 22.6 (±32.5) 2.9 (±1.2) 7.0 (3.0–19.5) 26.9 (±35.3) 2.0 (1.0–5.0) 
 >50 WBC/mm3, n (%) 10 (13.9) 4 (25.0) 0 (0.0) 9 (15.8) 4 (30.8) 0 (0.0) 
OCB (analyzed/positive(+)) 22/4 3/1 7/0 NP 19/3 1/1 6/0 
Anti aquaporin 4 tested/positive (%) 95 (57.9) 16 (56.3) 15 (26.7) NA 81 (56.8) 13 (61.5) 13 (15.4) NA
Patients fulfilling 2015 NMOSD 
criteria, n (%)
NA NA
 Yes 111 (99.1) 9 (56.3) 4 (18.2) 87 (100) 8 (61.5) 2 (13.3) 
 No 0 7 (43.8) 11 (50.0) 0 5 (38.5) 11 (73.3) 
Incomplete information 1 (0.9) 0 7 (31.8) 0 0 2 (13.3) 
Comorbid autoimmunity, n (%) 24 (21.4) 3 (18.8) 2 (9.1) NP 21 (24.1) 13 (23.1) 1 (6.7) 
EDSS: expanded disability status scale; MS: multiple sclerosis; NA: not applicable; NMOSD: neuromyelitis optica spectrum disorder; rNMOSD: relapsing neuromyelitis optica spectrum disorder; CSF: 
cerebrospinal fluid; WBC: white blood cell; rNMO: relapsing neuromyelitis optica; rLETM: relapsing longitudinal extensive transverse myelitis; ON: optic neuritis; rON: relapsing optic neuritis; MRI: 
magnetic resonance imaging: ANA: anti-nuclear antibodies. Index event: the interval for occurrence of any optic neuritis and acute myelitis in a single patient; relapse rate: total number of relapses/
disease duration; progression index: EDSS on last appointment/disease duration; NP: not performed due to low number of subjects.
*For those that had optic neuritis, Myelitis, or area postrema syndrome as a first event.
rNMO 150: comprise all patients ever seen by our staff.
rNMO 115: comprise all patients that had regular follow-up visits up to December 2013.
Normality test = D’Agostino–Pearson test.
Table 2. (Continued)https://journals.sagepub.com/home/msj
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hazard ratio (HR) lower than 1, indicating that, as the 
value of the covariate increases, the event hazard 
decreases and thus the length of time to the outcome 
increases. Baseline EDSS, time on prednisone and on 
azathioprine were included in the multivariate Cox 
regression, and all predictors were statistically signifi-
cant with exception of time on prednisone (Table 4). 
For all other covariates, regardless of the evaluated 
sampling (150 or 115), all HR indicated that, as the 
value of covariate increases (time on azathioprine and 
baseline EDSS), the event hazard decreases.
This meant that each month under azathioprine use 
reduced the hazard for outcome 1 and outcome 2 by 
Table 3. Treatment analysis: univariate Cox regression for the categorical and continuous predictors.
rNMO (n = 150)
 Outcome 1 Outcome 2
 Estimate 
(HR)
95% CI p-value Missing Estimate 
(HR)
95% CI p-value Missing
Age of onset 1.008 0.976 1.041 0.633 10 1.005 0.978 1.033 0.717 10
Male 1.36 0.631 2.932 0.433 10 1.26 0.653 2.432 0.49 10
NMOSD * * * * * 0.156** 
Baseline EDSS 0.812 0.642 1.027 0.083 10 0.976 0.819 1.163 0.788 10
Azathioprine (ever 
used)
0.927 0.417 2.061 0.852 10 0.999 0.507 1.97 0.998 10
Time on prednisone 0.986 0.976 0.996 0.006 51 0.986 0.978 0.993 <0.001 51
Time on azathioprine 0.981 0.972 0.99 <0.001 52 0.983 0.976 0.991 <0.001 52
Time on prednisone/
disease duration
0.816 0.218 2.044 0.816 51 0.817 0.284 2.35 0.707 51
Time on azathioprine/
disease duration
0.461 0.109 1.946 0.292 52 0.743 0.229 2.414 0.622 52
Time to prednisone 0.997 0.989 1.005 0.451 57 0.995 0.988 1.002 0.129 57
Time to azathioprine 0.996 0.989 1.004 0.355 49 0.994 0.987 1.001 0.078 49
rNMO (n = 115)
 Outcome 1 Outcome 2
 Estimate 
(HR)
95% CI p-value Missing Estimate 
(HR)
95% CI p-value Missing
Age onset 1.033 0.994 1.074 0.097 2 1.026 0.993 1.06 0.122 2
Male 1.134 0.452 2.848 0.788 2 1.068 0.484 2.356 0.87 2
NMOSD * * * * * 0.47** 
Baseline EDSS 0.861 0.648 1.144 0.302 2 0.814 0.633 1.045 0.106 2
Azathioprine (ever 
used)
0.915 0.264 2.3 0.851 2 1.194 0.518 2.751 0.677 2
Time on prednisone 0.986 0.975 0.997 0.012 29 0.985 0.976 0.994 0.001 29
Time on azathioprine 0.982 0.972 0.992 0.001 29 0.983 0.975 0.992 <0.001 29
Time on prednisone/
disease duration
1.551 0.337 7.127 0.573 29 1.056 0.311 3.589 0.93 29
Time on azathioprine/
disease duration
0.779 0.159 3.813 0.758 29 0.817 0.219 3.051 0.764 29
Time to prednisone 0.997 0.988 1.006 0.516 35 0.996 0.989 1.003 0.279 35
Time to azathioprine 0.993 0.984 1.003 0.166 27 0.992 0.984 1 0.058 27
EDSS: expanded disability status scale; NMOSD: neuromyelitis optica spectrum disorder; rNMO: relapsing neuromyelitis optica; HR: hazard ratio; CI: 
confidence interval.
*Not possible to compute converge problems because all subject classified as optic neuritis were censored; **Overall significance (Wald test).
Outcome 1: ≥1 point of EDSS variation from the first to the last appointment.
Outcome 2: ≥0.5 point of EDSS variation from the first to the last appointment.
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DB Bichuetti, MM de M Perin et al.
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2.6% (HR = 0.974) and 2.1% (HR = 0.979), respec-
tively for the sample of 150 subjects. Considering the 
prednisone use, there was a reduction in the hazard 
for outcome 1 by 1.2% (HR = 0.988) and for outcome 
2 by 1% (HR = 0.990). Regarding long-term risk for 
outcome 1, comparing two patients with a difference 
of azathioprine use of 60 months yielded a decrease 
by 80% (HR = 0.2) of presenting EDSS progression 
when keeping prednisone and baseline EDSS values 
as constant, whereas for prednisone use there was a 
reduction by 50% (HR = 0.50) when keeping azathio-
prine and EDSS values constant, for the sample of 
150. For outcome 2, azathioprine continuously for 
60 months yielded a decreasing by 71.9% (HR = 0.281) 
and prednisone by 46% (HR = 0.54).
Discussion
The fast-changing scenario on knowledge about 
NMOSD demands a constant re-evaluation of former 
case series for a better understanding of the disease’s 
clinical presentation and treatment response. This 
revision of our former case series with now 150 
rNMOSD subjects disclosed that only 30% of them 
reached EDSS 6.0 during a median 7 years of disease 
(Table 1), a smaller percentage than our previous 
analysis.12,13 Furthermore, two-thirds of patients 
treated with immunosuppressants, mainly azathio-
prine ± prednisone, remained stable or with minimal 
EDSS variation during follow-up.
This encompasses the largest single-center cohort of 
patients with rNMOSD cared continuously by the 
same team up to submission date, with 150 patients 
evaluated and nearly 115 with recent follow-up evalu-
ations. This is important for data gathering unification 
and development of internal protocols, since the care 
for patients with any neurological disease can differ 
between countries, and drug availability is not ubiqui-
tous. Our patients present similar clinical and radio-
logical characteristics to our previous series and other 
cohorts,3,12,26 except for less disability accumulation 
over time, possibly influenced by earlier interventions 
and uniformity in the treatment protocol. Since 2006, 
we have offered continued immunosuppression 
immediately at the second relapse, regardless of anti-
aquaporin 4 status or at first relapse if anti-aquaporin 
status was positive. We start with prednisone 1 mg/kg 
for at least 3–6 months and then taper slowly 10 mg/
month until 10 or 5 mg/day; only in rare cases with 
side effect do we discontinue steroids. Concomitantly, 
we offer azathioprine aiming at 3–4 mg/kg/day in the 
first month and keep the highest tolerable dose among 
this range indefinitely unless severe side effects are 
observed. The rates of azathioprine discontinuation in 
our series is smaller than others,8,27 and we attribute 
this to the restricted access to mycophenolate and 
rituximab; therefore, we insist on its use and instruct 
patients on strategies for side effects reduction, such as 
consuming with meals, dividing the daily dose, use of 
proton pump inhibitors when indicated and educate 
them on the drug’s benefit for better adherence. 
Neoplasm occurence was not distinct to age-related 
expected, suggesting that azathioprine for up to 8 years 
is safe, but cancer screening is still mandatory for eve-
ryone in permanent immunosuppression.
Although there is consensus that patients with 
rNMOSD need long-term immunosuppression, the 
best treatment choice for each individual remains 
uncertain, and the comparison between these drugs 
have not yielded a specific superiority of one over 
another.8,9 Considering that the annual cost of azathio-
prine 150 mg/day is US$2311.44, mycophenolate 
Table 4. Treatment analysis: multivariate regression.
Sampling Covariates tested Outcome 1 Outcome 2
HR 95% CI p-value HR 95% CI p-value
rNMOSD, 150 
subjects (whole 
cohort)
Baseline EDSS 0.593 0.426 0.825 0.002 - - - -
Time on prednisone 0.988 0.976 1.001 0.068 0.990 0.981 0.999 0.032
Time on azathioprine 0.974 0.961 0.987 <0.001 0.979 0.970 0.989 <0.001
rNMOSD, 115 
subjects (patients 
with the last 
appointment after 
1 January 2013)
Time on prednisone 0.986 0.972 1.000 0.049 0.985 0.975 0.996 0.006
Time on azathioprine 0.975 0.961 0.989 <0.001 0.976 0.965 0.987 <0.001
EDSS: expanded disability status scale; rNMOSD: relapsing neuromyelitis optica spectrum disorder; HR: hazard ratio; CI: confidence interval.
Outcome 1: ≥1 point of EDSS variation from the first to the last appointment.
Outcome 2: ≥0.5 point of EDSS variation from the first to the last appointment.
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2500 mg/day is Us$14,464.77 and rituximab 4 g/year 
is US$41,694.24 (information retrieved from the 
UpToDate® database, www.uptodate.com, on22 
November 2017), knowing that patients on azathio-
prine have nearly 80% of chance reduction on the 
hazard of disability progression after 5 years is of 
uttermost importance for those practicing in countries 
with restricted access to monoclonal antibodies, espe-
cially in times when the very high prices of some 
drugs are being discussed.28
Recently, Nikoo et al.10 reported an open-label 
trial comparing azathioprine and rituximab for 68 
patients with neuromyelitis optica (NMO) for 1 year. 
Both groups presented statistically significant 
reduction in ARR and EDSS comparing to baseline. 
Although patients receiving rituximab presented 
higher reduction, they also presented higher base-
line ARR and EDSS, implying some selection bias. 
These results disclose that both drugs have positive 
effects and that even though patients under azathio-
prine still present relapses, there is disability reduc-
tion along follow-up, reinforcing this measure as a 
useful endpoint.
Although relapses with encephalitic symptoms with 
brain lesions tend to be more severe, there was no 
association between brain MRI abnormalities and 
disability accumulation measured by the EDSS, nor 
were the presence of brain lesion associated to longer 
disease duration. Patients with rNMO had higher 
EDSS than with rON, but this does not directly imply 
a worse prognosis, as moderate to severe visual com-
promise (final EDSS 3.0 or 4.0 for the visual func-
tional system17) can cause profound quality of life 
loss.29
Pivotal studies identified a monophasic course in 
15% of patients with NMOSD,15 whereas further 
recent studies corroborate our findings of a much 
smaller proportion of monophasic over relapsing 
NMOSD.3,4,7,30,31 Although aggressive immunosup-
pression is advocated for all patients with NMOSD, 
it is important to recall that some patients, especially 
if seronegative and that present a second clinical 
relapse within less than 30 days apart, might have a 
single relapse in their life and not need long-term 
immunosuppression.
Preserving neurologic function is a major goal for 
patients with NMOSD and this outcome could be 
applied to other series as well, but might be consid-
ered a limitation since current recommendations for 
prospective trials in NMOSD is event based (i.e. 
relapse).32 Although we did not compare azathioprine 
to other drugs, reinforcing the benefits of a widely 
available and inexpensive medication is comforting. 
We have tested nearly all patients for the anti-aqua-
porin 4 antibodies, but our positivity rates are lower 
than other series (52.3%), justified by the fact that 
many patients were tested with non-cell-based assays 
and after treatment implementation, which is known 
to reduce positivity rates33. Since testing for anti-mye-
lin oligodendrocyte glycoprotein antibodies is not 
available in Brazil outside specific research facilities, 
it was not performed, but that would not undermine 
this analysis as 83% of patients fulfill NMOSD diag-
nostic criteria, and we focused on disability mainte-
nance regardless of antibody status. We acknowledge 
that anti-aquaporin 4–positive patients with rLETM 
and rON may have a distinct pathophysiology mecha-
nism than seronegative ones, but so far anti-aqua-
porin-positive or anti-MOG-positive patients do not 
have distinct therapeutic recommendations,7,34 so we 
have grouped our patients according to their main 
clinical presentation (rNMO, rLETM, and rON) 
reflecting a real-life 20-year clinical practice.
In conclusion, patients described in this observa-
tional study have less neurological disability than 
previously reported. Immediate and long-term immu-
nosuppression with azathioprine ± prednisone can be 
an excellent option for nearly two-thirds of patients, 
especially if introduced early and used at adequate 
doses, as actual recommendations mandate pred-
nisone maintenance in low doses and the combina-
tion of these two drugs has not yielded severe side 
effects for the median 8 years’ observation period. 
Nevertheless, NMOSD remains a severe and still 
challenging syndrome outside referral centers and 
warrants continuous efforts for early diagnosis and 
adequate treatment.
Acknowledgements
This article has been written following the STROBE 
guidelines for adequate reporting observational data: 
The Strengthening the Reporting of Observational 
Studies in Epidemiology (STROBE) statement: 
guidelines for reporting observational studies. Lancet 
2007; 370: 1453–57. Approval by the Internal Review 
Board of the Federal University of São Paulo was 
obtained for this study as part of an observational 
study on demyelinating diseases, and all patients 
signed a written informed consent. Study registration 
number 14058213.9.0000.5505. This is indicated in 
the “Methodology” section. D.B.B. had full access to 
all the data in the study and takes responsibility for 
the integrity of the data and the accuracy of the data 
analysis; declares that all authors and contributors 
have agreed to conditions noted on the Authorship 
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DB Bichuetti, MM de M Perin et al.
journals.sagepub.com/home/msj 11
Agreement Form; and contributed to study concep-
tion and design, acquisition of data, analysis, and 
interpretation and critical revision of the manuscript 
for important intellectual content. M.M.d.M.P. con-
tributed to study conception and design, acquisition 
of data, and critical revision of the manuscript for 
important intellectual content. N.A.d.S. and 
E.M.L.d.O. contributed to study conception and 
design and critical revision of the manuscript for 
important intellectual content. Statistical analysis was 
performed by D.B.B. and Hugo Cogo-Moreira, pro-
fessor of Biostatistics, Universidade Federal de São 
Paulo. Hugo Cogo-Moreira received financial com-
pensation for statistical support.
Declaration of Conflicting Interests
The author(s) declared the following potential con-
flicts of interest with respect to the research, author-
ship, and/or publication of this article: D.B.B. has 
received speaking/consulting honoraria from Bayer 
HealthCare, Biogen Idec, Merck, Sanofi-Genzyme, 
TEVA, and Roche and had travel expenses to scien-
tific meetings sponsored by Bayer Health Care, Merck 
Serono, TEVA, and Roche. M.M.d.M.P has been 
awarded an educational grant by Bayer HealthCare to 
study for an online Masters in Neuroimmunology at 
Universitat Autònoma de Barcelona (UAB), has 
received speaking honoraria from Novartis, and 
financial assistance to attend neurology congress paid 
by Bayer HealthCare, Merck Serono, and TEVA. 
N.A.d.S. has received travel expenses to scientific 
meetings sponsored by Sanofi-Genzyme, Biogen, 
TEVA, Merck Serono, Novartis, and Bayer 
HealthCare. E.M.L.d.O. has received speaker fee 
from Teva, Biogen Idec, Sanofi-Genzyme, and 
Novartis; travel grant from Merck; and consulting 
honoraria from Merck and Sanofi-Genzyme.
Funding
The author(s) received no financial support for the 
research, authorship, and/or publication of this article.
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