Zika Virus infection and Guillain-Barré syndrome in Northeastern Mexico: A case-control study.

BACKGROUND: Beginning August 2017, we conducted a prospective case-control investigation in Monterrey, Mexico to assess the association between Zika virus (ZIKV) and Guillain-Barré syndrome (GBS).
METHODS: For each of 50 GBS case-patients, we enrolled 2-3 afebrile controls (141 controls in total) matched by sex, age group, and presentation to same hospital within 7 days.
RESULTS: PCR results for ZIKV in blood and/or urine were available on all subjects; serum ZIKV IgM antibody for 52% of case-patients and 80% of controls. Subjects were asked about antecedent illness in the two months prior to neurological onset (for case-patients) or interview (for controls). Laboratory evidence of ZIKV infection alone (PCR+ or IgM+) was not significantly different between case-patients and controls (OR: 1.26, 95% CI: 0.45-3.54) but antecedent symptomatic ZIKV infection [a typical ZIKV symptom (rash, joint pain, or conjunctivitis) plus laboratory evidence of ZIKV infection] was higher among case-patients (OR: 12.45, 95% CI: 1.45-106.64). GBS case-patients with laboratory evidence of ZIKV infection were significantly more likely to have had typical ZIKV symptoms than controls with laboratory evidence of ZIKV infection (OR: 17.5, 95% CI: 3.2-96.6). This association remained significant even when only GBS case-patients who were afebrile for 5 days before onset were included in the analysis, (OR 9.57 (95% CI: 1.07 to 85.35).
CONCLUSIONS: During ZIKV epidemics, this study indicates that increases in GBS will occur primarily among those with antecedent symptomatic ZIKV.

Introduction

Guillain-Barré syndrome (GBS) is an acute inflammatory immune-mediated polyradiculoneuropathy presenting classically with ascending progressive weakness, sensory changes, and hyporeflexia [1]. It is the most common cause of acute flaccid paralysis worldwide [1, 2]. GBS is usually precipitated by a preceding infection or other antigenic stimuli [3]. One of the pathophysiological mechanisms linked to GBS is molecular mimicry, as some pathogens may have antigens similar to peripheral nerve myelin and/or axonal epitopes [1, 2, 4]. The most commonly identified triggering agents are Campylobacter jejuni, cytomegalovirus, Epstein-Barr virus, and Mycoplasma pneumoniae [1, 2, 4].

Arthropod-borne viruses (arboviruses) such as Zika virus (ZIKV), dengue virus (DENV) and chikungunya virus (CHIKV) have become an increasingly important global health threat. DENV and ZIKV are arboviruses belonging to the Flavivirus genus of the Flaviviridae family, and are both transmitted by the Aedes species mosquito vectors [5, 6]. CHIKV is an arbovirus belonging to the alphavirus genus of the Togaviridae family [6]. Some arboviruses have been temporally associated with the occurrence of GBS [5].

A case-control study carried out during the French Polynesia ZIKV outbreak in 2013–2014 was the first to demonstrate an association between ZIKV and a large increase in the incidence of GBS [7]. Subsequently, ZIKV was introduced to South and Central America where excess reports of GBS were also reported in ZIKV-affected areas, and where DENV and CHIKV were already endemic [8, 9]. In Mexico, the first cases of ZIKV were reported in late 2015 [10, 11]. In this study, we assessed whether post-outbreak endemic circulation of ZIKV in Northeastern Mexico was associated with the development of GBS.

Methods

Ethics statement

The Institutional Review Board at the Universidad Autonoma de Nuevo Leon reviewed this protocol and approved it as research. The U.S. Centers for Disease Control and Prevention (CDC) relied on the IRB determination of UANL. All subjects provided written informed consent prior to investigation participation. The investigation was financially supported by CDC.

Study design and participants

We conducted a case-control study in the northeast of Mexico, including Coahuila, Nuevo León and Tamaulipas States. The study period was from August 1, 2017 to June 30, 2018. The study protocol was approved by the institutional review boards of Universidad de Nuevo León-Hospital Universitario (HU), the Instituto Mexicano del Seguro Social (IMSS), and the Centers for Disease Control and Prevention (CDC) before recruitment of GBS patients and controls. Informed consent was obtained from all GBS patients and control subjects before inclusion in the study. Patients were enrolled from three referral hospitals (emergency department visits or inpatient wards) of Monterrey City metropolitan area.

We identified suspected GBS case-patients based on onset of compatible neurologic symptoms (e.g., flaccid limb weakness, areflexia, cranial nerve palsies) reported by physicians and hospitals to a committee of investigators during the study period. To verify a GBS diagnosis, we performed medical record reviews to ascertain characteristics of the clinical illness and diagnostic testing, including cerebrospinal fluid, neuroimaging, and electro diagnostic test results, if available. Suspected GBS case-patients were classified according to diagnostic certainty of the Brighton Collaboration criteria case definitions for GBS [12]. Case-patients meeting levels 1–3 of diagnostic certainty were classified as confirmed GBS and eligible for enrollment in the investigation.

For each GBS case-patient, we enrolled three controls from the same hospitals seen in the emergency department or inpatient service within seven days of the GBS case with a non-febrile illness (no report or documentation of fever 48 hours before enrolment) that were matched to case-patients by sex and age ±10 years.

We interviewed all available case-patients and controls to obtain information about demographics, risk factors (age, male sex), and exposures in the two months prior to interview, for controls or to onset of neurological symptoms for the GBS case-patients. Functional outcomes in patients with GBS were assessed based on residual motor deficits using the Hughes GBS Disability Scale. Following the interviews, serum and urine samples were collected from case-patients and controls, to determine exposure to ZIKV, DENV and CHIKV.

Laboratory analysis

Viral RNA detection

Viral RNA was extracted from the serum and urine samples by using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). The Superscript III Platinum OneStep Real Time RT-PCR system (Invitrogen, Carlsbad, CA, USA) was used for RNA amplification to analyze gene expression. PCRs specific for DENV, CHIKV, and ZIKV were performed using the CDC Trioplex Real-time RT-PCR Assay (Trioplex Real-time RT-PCR Assay, method available at http://www.fda.gov/downloads/MedicalDevices/Safety/EmergencySituations/UCM491592.pdf).

IgM antibodies against ZIKV

IgM antibodies against ZIKV were determined by the use of the InBios ZIKV via an IgM antibody capture enzyme-linked immunosorbent assay (InBios MAC-ELISA; InBios International, Inc., Seattle, WA). InBios ELISA was performed and results interpreted as described by the manufacturer. An immune status ratio (ISR) was determined by dividing the OD of the patient sample with the ZIKV recombinant. ISR values of 1.7 were considered presumptive positive for IgM antibodies to ZIKV.

Statistical analysis

To determine a possible association between GBS and a preceding ZIKV infection, we estimated that 49 case-patients and 147 controls would provide a power of 80% to detect a difference of 20% in ZIKV prevalence, with an alpha level of 5%. Descriptive statistics was used to summarize clinical and demographic data. We conducted analyses assessing potential associations between GBS and demographic characteristics, known GBS risk factors, antecedent illness in the two months prior to hospitalization, and molecular and serological evidence of ZIKV infections. To assess possible differences between GBS case-patients and the matched controls, we calculated matched odds ratios and 95% confidence intervals by conditional maximum likelihood estimation, aside from comparisons for which these calculations did not converge, for which we calculated unconditional maximum likelihood estimates with confidence intervals produced using normal approximation. For comparisons between ZIKV positive and ZIKV negative GBS case-patients, we calculated unconditional maximum likelihood estimates with confidence intervals produced using normal approximation. For comparisons with zero values in any cells (such that odds ratio calculations were not calculable) we assessed differences using Fisher exact p-values (p ≤ 0.05 was considered statistically significant). We considered the presence of one or more of the following three antecedent symptoms–rash, joint pain and/or conjunctivitis–as having “typical” Zika symptoms.

IgM antibody testing was not available to be performed for all GBS case-patients and controls. Therefore, we used two different measures to assess ZIKV status by laboratory testing:

Positive PCR assay (all patient in cohort included in analysis)

Positive PCR assay or positive IgM assay (only patients with available IgM results used in analysis)

To assess antecedent symptomatic ZIKV infection, we used the following measures:

Positive PCR assay and at least one of the typical Zika symptoms (all patients in cohort included in analysis)

Positive PCR or IgM assay, and at least one of the typical Zika symptoms (only patients with available IgM results used in analysis)

All data were analyzed using R version 3.3.3 (The R Foundation for Statistical Computing, 2017).

Results

During the study period, 50 GBS case-patients, and 141 (24 outpatient, 117 hospitalized) controls were enrolled. Demographics of the case-patients and controls as well as prevalence of virus infection is shown in Table 1.

Table 1

Demographics, geographic origin and virus infection.

	Case-patients, n = 50	Controls, n = 141
Demographics
Median age (range)	40.5 (3–66)	40 (2–70)
Male n (%)	31 (62)	90 (63.8)
State of origin n (%)
Nuevo León	32 (64)	99 (70.2)
Coahuila	9 (18)	23 (16.3)
Tamaulipas	9 (18)	18 (12.8)
San Luis Potosi	0 (0)	1 (.7)
Virus infection (PCR+) n (%)
ZIKV+ n (%)	11 (22)	31 (22)
DENV+ n (%)	1 (2)	2 (1)
CHIKV+ n (%)	1 (2)	7 (5)
Antibody response (IgM+)
ZIKV+ n (%)	3 (12)*	9 (8)&

* out of 26 case-patients

& out of 113 controls

Current infection by arboviruses (supported by the detection of ZIKV, DENV and/or CHIKV qRT-PCR) was not significantly different between groups (Table 1).

ZIKV was the most commonly detected infection, in 22% each of case-patients and controls (Table 1). IgM antibody testing for ZIKV was performed for 26 of 50 GBS case-patients (52%) and 113 of 141 controls (80%) (Table 2).

Table 2

Number of GBS case-patients and matched controls by results of Zika PCR test, Zika IgM test, and presence of Zika symptoms.

GBS case-patients (n = 50)
		IgM assay performed (n = 26)		IgM assay not performed	Total
		IgM positive	IgM negative
PCR positive	≥1 Zika symptoms	0	4	2	11
	No Zika symptoms	0	3	2
PCR negative	≥1 Zika symptoms	3	2	5	39
	No Zika symptoms	0	14	15
Matched controls (n = 141)
		IgM assay performed (n = 113)		IgM assay not performed	Total
		IgM positive	IgM negative
PCR positive	≥1 Zika symptoms	0	3	0	31
	No Zika symptoms	3	22	3
PCR negative	≥1 Zika symptoms	1	5	0	110
	No Zika symptoms	5	74	25

The median times between symptom onset and sample collection did not significantly differ by ZIKV status (Table 3).

Table 3

Time (days) from neurological symptom onset to sample collection from 50 case-patients by Zika status.

			Days from neuro onset to sample collection
Category		N	Median	IQR	Mean	Min	Max
All case-patients		50	11	(7–17)	13.46	2	52
PCR only	Zika+	11	15	(9–23.5)	19.27	5	52
	Zika-	39	11	(7–16.5)	11.82	2	24
PCR and rash, joint pain, or conjunctivitis	Zika+	6	20.5	(11.25–35)	24.83	8	52
	Zika-	44	11	(7–17)	11.91	2	24
All case patients who received an IgM test		26
PCR or IgM	Zika+	10	17.5	(8.5–20.5)	19	2	52
	Zika-	16	14	(9–18)	14.19	5	24
PCR or IgM and rash, joint pain, or conjunctivitis	Zika+	7	17	(9–28)	20.71	2	52
	Zika-	19	15	(9–20)	14.32	5	24

In the two months before their admission, case-patients reported a variety of symptoms, including typical symptoms of ZIKV infection such as rash, joint pain and conjunctivitis (Table 4).

Table 4

Association between prior illness and GBS for 50 GBS case-patients and 141 matched controls–clinical symptoms reported prior to neurological onset/interview and virus infection.

Prior illness	Numbers (%) reported with antecedent symptoms		Matched odds ratio (cMLE)
	Case-patients, n = 50	Controls, n = 141	Estimate	LL	UL
Fever	16 (32)	13 (9.2)	5.93	2.27	15.50
Chills	2 (4)	11 (7.8)	0.50	0.10	2.37
Nausea	4 (8)	17 (12.1)	0.65	0.20	2.16
Diarrhea	22 (44)	5 (3.5)	12.17	4.60	32.19
Muscle Pain	8 (16)	12 (8.5)	2.84	0.87	9.25
Joint Pain	8 (16)	8 (5.7)	3.49	1.18	10.27
Skin Rash	6 (12)	1 (.7)	17.10	2.05	142.37
Conjunctivitis	6 (12)	1 (.7)	18.00	2.17	149.51
Headache	10 (20)	13 (9.2)	3.00	1.10	8.22
Retro Ocular Pain1	4 (8)	0 (0.0)	-	-	-
Nuchal Rigidity1	0 (0)	1 (0.7)	-	-	-
Confusion1	1 (2)	0 (0.0)	-	-	-
Abdominal Pain	8 (16)	11 (7.8)	2.16	0.85	5.52
Cough	9 (18)	11 (7.8)	3.08	1.11	8.53
Nasal Secretion	5 (10)	5 (3.5)	3.00	0.87	10.36
Odynophagia2	3 (6)	1 (0.7)	8.94	0.91	87.99
Periarticular Edema1	0 (0)	2 (1.4)	-	-	-
Lower Back Pain	1 (2)	3 (2.1)	1.00	0.08	11.93
Typical Zika symptoms3	16 (32)	9 (6.4)	9.58	3.16	29.09

1Undefined odds ratio/confidence limits.

2Odds ratio and confidence limits calculated by unconditional maximum likelihood estimation with normal approximation.

3Any of the following: rash, joint pain, conjunctivitis

When comparing the rates of previous illness, case-patients reported typical ZIKV symptoms more frequently than controls (OR: 9.58, 95% CI: 3.16–29.09) (Table 4). Of GBS case-patients, 38.5% had evidence of ZIKV by PCR or IgM, compared to 30.1% of controls (OR: 1.26, 95% CI: 0.45–3.54). Case-patients were more likely than controls to have laboratory evidence of ZIKV infection in conjunction with a history of typical ZIKV symptoms (OR: 12.45, 95% CI: 1.45–106.64) (“symptomatic ZIKV”; Table 5).

Table 5

Association between prior illness and GBS for 50 GBS case-patients and 141 matched controls–laboratory tests.

Prior illness	Number (%)		Matched odds ratio (cMLE)
	Case-patients	Controls	Estimate	LL	UL
All observations (50 case-patients, 141 controls)
Zika (PCR)	11 (22)	31 (22.0)	1.03	0.44	2.39
Zika (PCR) w/o typical symptoms1	5 (10)	28 (19.9)	0.42	0.14	1.26
Zika (PCR) with typical symptoms2	6 (12)	3 (2.1)	14.26	1.68	120.98
Patients receiving IgM tests (26 case-patients, 113 controls)
Zika (PCR or IgM)	10 (38.5)	34 (30.1)	1.26	0.45	3.54
Zika (PCR or IgM) w/o typical symptoms1	3 (11.5)	30 (26.5)	0.41	0.11	1.45
Zika (PCR or IgM) with typical symptoms2	7 (26.9)	4 (3.5)	12.45	1.45	106.64

1 Laboratory evidence of Zika but none of the following: rash, joint pain, conjunctivitis.

2 Laboratory evidence of Zika with any of the following: rash, joint pain, conjunctivitis.

Of the 16 GBS case-patients with an antecedent typical ZIKV symptom, six (38%) had a positive PCR test for ZIKV; none had a positive PCR test for DENV or CHIKV. For GBS case-patients, seven of 10 (70%) that had laboratory evidence for ZIKV infection by PCR or IgM also had typical ZIKV symptoms compared to two of 16 (13%) of those that tested negative for ZIKV (OR: 16.3, 95% CI: 2.2–121). In comparison, only four of 34 (12%) controls that had tested positive for ZIKV had typical ZIKV symptoms compared to five of 79 (6%) that tested negative for ZIKV (OR: 2.0, 95% CI 0.50–7.9). GBS case-patients with laboratory evidence of ZIKV infection were significantly more likely to have had typical ZIKV symptoms than controls with laboratory evidence of ZIKV infection (OR: 17.5, 95% CI: 3.2–96.6). This association remained statistically significant even in an analysis that included only case-patients with no febrile illnesses within 5 days prior to onset of GBS (OR 9.57 (95% CI: 1.07 to 85.35).

The majority of GBS case-patients-patients had paresis and areflexia, and 22% had facial diplegia (Table 6).

Table 6

Neurological signs and symptoms at onset or nadir of GBS case-patients: n (%).

Neurological signs and symptoms	All (n = 50)	Zika diagnosis by PCR or IgM (n = 26)*			Zika diagnosis by PCR or IgM and rash, joint pain, or conjunctivitis (n = 26)*
		Zika+ (n = 10)	Zika- (n = 16)	Odds Ratio	Zika+ (n = 7)	Zika- (n = 19)	Odds Ratio
Acute bilateral paresis
Upper extremities	46 (92.0)	8 (80.0)	16 (100.0)	-	6 (85.7)	18 (94.7)	0.33 (0.02–6.19)
Lower extremities	46 (92.0)	8 (80.0)	15 (93.8)	0.27 (0.02–3.41)	6 (85.7)	17 (89.5)	0.71 (0.05–9.27)
Areflexia
Upper extremities	47 (94.0)	9 (90.0)	14 (87.5)	1.29 (0.10–16.34)	6 (85.7)	17 (89.5)	0.71 (0.05–9.27)
Lower extremities	47 (94.0)	9 (90.0)	14 (87.5)	1.29 (0.10–16.34)	6 (85.7)	17 (89.5)	0.71 (0.05–9.27)
Paresthesia/Sensory changes
Upper extremities	19 (38.0)	1 (10.0)	8 (50.0)	0.11 (0.01–1.09)	1 (14.3)	8 (42.1)	0.23 (0.02–2.30)
Lower extremities	21 (42.0)	4 (40.0)	7 (43.8)	0.86 (0.17–4.27)	3 (42.9)	8 (42.1)	1.03 (0.18–5.95)
Dyspnea	11 (22.0)	3 (30.0)	1 (6.2)	6.43 (0.56–73.35)	3 (42.9)	1 (5.3)	13.50 (1.10–165.97)
Facial diplegia	11 (22.0)	3 (30.0)	4 (25.0)	1.29 (0.22–7.50)	3 (42.9)	4 (21.1)	2.81 (0.44–18.06)
Dysphagia	6 (12.0)	2 (20.0)	1 (6.2)	3.75 (0.29–47.99)	2 (28.6)	1 (5.3)	7.20 (0.54–96.64)
Ophthalmoparesis	12 (24.0)	2 (20.0)	5 (31.2)	0.55 (0.08–3.59)	1 (14.3)	6 (31.6)	0.36 (0.04–3.70)
Dysarthria	4 (8.0)	2 (20.0)	0 (0.0)	-	2 (28.6)	0 (0.0)	-
Ataxia	4 (8.0)	2 (20.0)	1 (6.2)	3.75 (0.29–47.99)	1 (14.3)	2 (10.5)	1.42 (0.11–18.59)
Dysautonomia	1 (2.0)	0 (0.0)	0 (0.0)	-	0 (0.0)	0 (0.0)	-

* Of the 50 case-patients, 26 had IgM testing done, and Zika diagnosis was determined for these 26 case-patients by either: 1) positive PCR or positive IgM test, or 2) positive PCR or positive IgM test, and one of the following symptoms: rash, conjunctivitis, joint pain.

Overall, there were few clinical differences between GBS patients with laboratory evidence of recent ZIKV infection and those without. Patients with both laboratory evidence of ZIKV infection and at least one antecedent typical ZIKV symptom (“symptomatic ZIKV”) reported dyspnea more frequently (43% vs 5%, OR: 13.50, 95% CI: 1.10–165.97 (Table 6). Hughes score at nadir was not significantly different between ZIKV+ and ZIKV- GBS case-patients (Table 7).

Table 7

Treatment and clinical results for GBS case-patients by Zika status: n (%).

Treatment and clinical results	All (n = 50)	Zika diagnosis by PCR or IgM (n = 26)*			Zika diagnosis by PCR or IgM and rash, joint pain, or conjunctivitis (n = 26)*
		Zika+ (n = 10)	Zika- (n = 16)	Odds Ratio	Zika+ (n = 7)	Zika- (n = 19)	Odds Ratio
Intravenous immunoglobulin	34 (68.0)	6 (60.0)	10 (62.5)	0.90 (0.18–4.55)	3 (42.9)	13 (68.4)	0.35 (0.06–2.06)
Plasma exchange	10 (20.0)	2 (20.0)	4 (25.0)	0.75 (0.11–5.11)	2 (28.6)	4 (21.1)	1.50 (0.21–10.82)
Mechanical ventilation	6 (12.0)	1 (10.0)	0 (0.0)	-	1 (14.3)	0 (0.0)	-
Hughes score at nadir: mean (SD)	3.5 (1.1)	3.2 (1.2)	3.4 (0.7)	-	3.3 (1.4)	3.4 (0.8)	-
Neurophysiological study
AMAN1	10 (20.0)	1 (10.0)	3 (18.8)	0.48 (0.04–5.40)	1 (14.3)	3 (15.8)	0.89 (0.08–10.30)
AIDP2	6 (12.0)	1 (10.0)	2 (12.5)	0.78 (0.06–9.88)	1 (14.3)	2 (10.5)	1.42 (0.11–18.59)
AMSAN3	4 (8.0)	0 (0.0)	2 (12.5)	-	0 (0.0)	2 (10.5)	-
Other	4 (8.0)	2 (20.0)	2 (12.5)	1.75 (0.21–14.93)	1 (14.3)	3 (15.8)	0.89 (0.08–10.30)

1AMAN = Acute motor axonal neuropathy.

2AIDP = Acute inflammatory demyelinating polyneuropathy.

3AMSAN = Acute motor and sensory axonal neuropathy.

* Of the 50 case-patients, 26 had IgM testing done, and Zika diagnosis was determined for these 26 case-patients by either: 1) positive PCR or positive IgM test, or 2) positive PCR or positive IgM test, and one of the following symptoms: rash, conjunctivitis, joint pain.

In total, 48% had complete neurophysiological studies for analysis. These revealed a predominance of AMAN compared to demyelinating subtype (Table 7). Treatment was initiated with intravenous immunoglobulin in 68% and plasmapheresis in 20% (Table 7). Mechanical ventilation was required in 12% of patients, and significantly more ZIKV-positive case-patients (diagnosed by PCR and at least one antecedent typical ZIKV symptom) required mechanical ventilation than ZIKV negative case-patients (OR: 13.67, 95% CI: 1.88–99.35). There was one in-hospital death after a long stay in the intensive care unit.

Discussion

Our study suggests that symptomatic ZIKV infection (laboratory evidence of ZIKV infection plus one or more typical symptoms) but not asymptomatic ZIKV infection was associated with GBS compared to controls. When we combined laboratory evidence of ZIKV infection and the presence of typical symptoms of ZIKV, there appeared to be more symptomatic ZIKV case-patients in the GBS group than in the control group. Furthermore, this subgroup also showed some subtle differences in their clinical presentation. The only other study in which symptoms and laboratory evidence consistent with a ZIKV illness were combined in order to assess an association of increased ZIKV with increased cases of GBS was one conducted in Brazil after ZIKV was first introduced into that country. The previous study reported no significant association between recent Flavivirus infection (a positive or equivocal IgM test result for ZIKV or DENV) and GBS. However, being a case-patient was significantly associated with evidence of recent Flavivirus infection when combined with clinical criteria for suspected ZIKV disease (rash with at least two other ZIKV-like symptoms). At the time of assessment in that study, unlike the current study, all living GBS case-patients were at least five months out from neurologic symptom onset and the laboratory criteria were based on a recent Flavivirus infection (a positive or equivocal IgM test result for ZIKV or dengue). [13].

The seminal French-Polynesian study found a strong association between ZIKV and GBS [7], although similar to other assessments, the authors did not compare the strength of the GBS association with symptomatic ZIKV infection compared to asymptomatic ZIKV infection. Unlike the French Polynesia, Puerto Rico, and New Caledonia studies [7, 14, 15], our study does not support an association between ZIKV and GBS in Northeastern Mexico when using laboratory evidence of infection alone. However, other studies from Latin American and Asia Pacific do not show a significant association between ZIKV and GBS [16-18]. Methods and designs of these studies are heterogeneous, with differences in inclusion criteria and laboratory assays.

Other observational studies have also suggested a close association between GBS and ZIKV. In one Dutch study of cases returning from Suriname with ZIKV infection, one out of 18 patients (5.5%) developed GBS [19]. Also, in a cross-sectional study of 42 GBS cases in a region of Colombia, 40% had positive PCR and 32% had a positive anti-ZIKV IgM [20]. And yet, other similar reports have yielded contrasting results. For example, a recent report from Thailand, a country endemic for ZIKV, reported 1,417 cases of ZIKV infection but only two (0.14%) cases with concomitant GBS [21], a rate considerably lower than those previously observed in Polynesia and the Americas. In one early study of a ZIKV-infection outbreak in Yap (Micronesia), it was estimated that 73% of the population over three years of age had been infected (in a population of around 10,000 people), and no cases of GBS were reported [22]. Lastly, in a recent study carried out in the Gulf Mexican state of Veracruz, Mexico, where 28 cases of GBS were described, only two (7.1%) had positive anti-ZIKV IgG and none had positive IgM or PCR in serum [23]. And, although there are numerous case control studies related to Zika virus infections and GBS, our study is unusual and particularly valuable because it highlights differences between symptomatic vs asymptomatic Zika virus infections as they relate to GBS, not just the relationship of Zika virus infection in general to GBS.

Observational studies on the association of ZIKV and GBS have many limitations, and selection bias due to non-random selection is a significant issue, as is the loss of follow up and the lack of adjustment for overall ZIKV prevalence in a given region [24]. In this study, we did find a higher prevalence of positive ZIKV PCR compared to studies done in Puerto Rico [14], and French Polynesia [7].

Systematic reviews and meta-analyses of studies of ZIKV and GBS have been published. In a recent meta-analysis, from a total pooled number of 164,651 ZIKV-infected individuals, 1,513 developed ZIKV-associated GBS, 1.23% (95% CI = 1.17–1.29%) [25]. Another mathematical inference framework study utilizing data from 11 locations that had reported suspect ZIKV and GBS cases (including nine in the Americas), estimated that 2 (95% CI = 0.5–4.5) of reported GBS cases may occur per 10,000 ZIKV-infections [26].

ZIKV may be associated with particular phenotypic presentations of GBS. ZIKV-associated GBS has been associated with more dysautonomia, facial nerve palsy, and a more rapid onset of clinical GBS signs [27, 28]. Although our numbers are small, we found that dyspnea was more common in symptomatic ZIKV GBS case-patients, and symptomatic ZIKV infection was associated with more frequent need for mechanical ventilation.

This study is subject to several limitations. Using reports of antecedent illness may lead to several sources of bias, such as the non-specific nature of the symptoms, possible underreporting of acute illnesses by controls, and recall bias in reporting of symptoms by case-patients. Although selection of controls with non-febrile illness risks bias toward over-estimation of the significance of the predictive value of Zika-associated symptoms, analyses including only GBS case-patients who had no febrile illness within 5 days prior to onset of GBS were statistically significantly different from the controls. The limited number of subjects who had ZIKV-specific IgM antibodies tested for is another limitation, as is the inability to do serologic testing for DENV and CHIKV. The finding of up to 22% of GBS case-patients and controls having PCR-positivity for ZIKV was admittedly surprising; ordinarily, it would be expected that persons developing GBS would be outside of the time window for continuing to have ZIKV viremia. In the absence of confirmatory ZIKV-specific neutralization assay testing, we cannot say for certain that a certain amount of false-positivity may not have been present in our PCR results. However, the PCR positivity seemed specific for ZIKV; one might expect that if the problem was general false-positivity, one would observe unusually high percentages of DENV and CHIKV positivity as well, which was not the case. In addition, one might expect that false positivity would have been present in both GBS case-patients and controls; rather, the PCR results seemed preferentially present in the GBS case-patients rather than both case-patients and controls. Controls were obtained to account for geographic location, sex, and age, but other factors such as socioeconomic condition were not controlled for and may have affected some of the findings. Finally, given a finding of 44% of case-patients reporting a diarrheal illness, we were unable to test for enteric pathogens, such as Campylobacter jejuni, which may have contributed to the overall burden of GBS in this group. The lack of a commercially available and standardized ELISA test for detecting anti-Campylobacter antibodies made pursuing this diagnosis logistically challenging.

Conclusions

The accumulated evidence suggests a link between ZIKV infection and/or illness and GBS. Our study found a statistically significant association with symptomatic ZIKV but not with asymptomatic ZIKV infection alone. This finding supports a conclusion that the ZIKV association with GBS is stronger with ZIKV illness. Although the incidence of asymptomatic ZIKV is known to be several-fold higher than symptomatic ZIKV, our study suggests that during ZIKV epidemics, increases in GBS will occur primarily among those with antecedent symptomatic ZIKV.

16 Dec 2019

PONE-D-19-30389

Zika Virus infection and Guillain-Barré syndrome in Northeastern Mexico: a case-control study

PLOS ONE

Dear Dr Sejvar,

Thank you very much for submitting your manuscript "Zika Virus infection and Guillain-Barré syndrome in Northeastern Mexico: a case-control study" (#PONE-D-19-30389) for review by PLOS ONE. As with all papers submitted to the journal, your manuscript was fully evaluated by academic editor (myself) and by independent peer reviewers. The reviewers appreciated the attention to an important health topic, but they raised substantial concerns about the paper that must be addressed before this manuscript can be accurately assessed for meeting the PLOS ONE criteria. Therefore, if you feel these issues can be adequately addressed, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. We can’t, of course, promise publication at that time.

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PLOS ONE

Additional Editor Comments:

I invited and received three reviews of your manuscript. All reviews raised substantial concerns about your manuscript as it currently stands. I read the manuscript myself and i must say that i completely agree that the reviews are detailed and solid. So, my decision is "major revision". Please address all these concerns before submitting a revised version of your manuscript. I would kindly ask the authors to confirm that they follow the Journal guidelines available via the link https://journals.plos.org/plosone/s/submission-guidelines. I would love to see the revised version of your manuscript as soon (i.e. within the next two weeks), so, we will save much time in the review process. Thanks too much for choosing PLOS ONE for your submission!

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**********

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**********

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Reviewer #1: In this article, the authors included patients from three states of northeastern Mexico between August 2017 and June 2018, in order to establish whether the endemic circulation of Zika virus (ZIKV) was associated with the development of Guillaín-Barre syndrome (GBS). It is a case-control study, the cases corresponded to hospitalized or outpatient with GBS, with diagnostic certainty of grades 1 to 3 according to the classification of Brighton Collaboration Criteria. For each patient they included 3 controls (2.8) seen in emergency visits or hospitalized, with non-febrile illness for 48 hours, paired with cases by sex and age + minus 10 years, and seen in the same hospital within seven days of each case. After the interview, serum and urine were taken in both groups to determine exposure to ZIKV, dengue virus and Chikungunya virus by viral RNA amplification (PCR) and detection of IgM antibodies to ZIKV.

They studied 50 cases and 141 controls with similar demography. They found 22% of ZIKV positive PCR samples, both in cases and controls. The detection of IgM to ZIKV was performed in 26/50 (52%) of the cases and in 113/141 (80%) of the controls, with no difference in times between the onset of symptoms and the collection of samples.

The analysis prior to the neurological picture showed that the cases had with more frequency: Fever, diarrhea, myalgia, arthralgia, rash, conjunctivitis, headache, odynophagia and a composite outcome of typical ZIKV symptom (rash, joint pain, or conjunctivitis). The comparisons were carried out with conditional maximum likelihood estimation.

The first problem we encountered with the study is that the characteristics of the control group are not well described, at least in general terms. Such as the proportion of outpatients and hospitalized patients. It would be important to describe if in the control group there were patients with neurological disease, especially with GBS and, if among the emergency included patients, there were patients with minor trauma or trivial conditions and otherwise previously healthy, who would be representative of the “open” population. With some of these data, the general characteristics of the control group would be clearer.

Finally, with ZIKV, something similar to other viral diseases seems to happen in their post-epidemic transition to endemic status, and it is the fact that a high number of infections are asymptomatic as the clear example of the study of the island of Yap, where it was estimated that the 73% of the population was infected with ZIKV but only 18% of those infected (95% CI, 10 to 27), had a clinical illness that was probably attributable to ZIKV infection or 1 symptomatic person in 4.4 ZIKV infected persons (1).

A surprising finding in this study is that the controls had exactly the same percentage of positive PCR tests (22%) as the cases, which leads us to conclude on the importance of studying GBS patients as thoroughly as available resources allow it, to investigate coinfections, particularly gastrointestinal and thus be able to establish with greater solidity the association of ZIKV with GBS and the prevalence of other potential pathogens in endemic areas of ZIKV.

We recommend that a specific header must be opened prior to the conclusions, to clearly indicate the limitations of the study:

That electrophysiological studies were carried out in 48% of patients with GBS, that 20% of controls did not have the IgM antibody tests against ZIKV. That in the cases group, diarrhea occurred in 44% of the patients and it is not mentioned if the presence of intestinal pathogenic microorganisms was investigated, especially Campylobacter jejuni, which seems to be associated with Guillain Barré syndrome in the state of Veracruz, Mexico according to a publication by del Carpio-Orantes (2), this is a very important matter, which opens the possibility that the GBS was caused by concomitant enteric pathogens as an epiphenomenon to asymptomatic ZIKA virus infection as it would seem to occur in the controls.

Finally, there are some inaccuracies in the tables that merit a careful review:

1. In Table 3, in the line 9, conjunctivitis, the number of patients is 6, but the percentage in brackets is indicated 1 and should correspond to 12.

2. In Table 5. Neurological signs and symptoms at onset or nadir of GBS case-patients: n (%)

The first line reads neurological signs and symptoms all n = 50, the thrid column indicates Zika diagnosis by PCR or IgM (n = 26), and the fourth column shows Zika diagnosis by PCR or IgM and rash, joint pain, or conjunctivitis (n = 26), the sum is 52, no 50. In addition, if the individual data of the next line 10 + 16 +7 + 19 are added, it is equal to 52, so it should be reviewed why the total number of patients is exceeded by 2.

References

1. Duffy MR, Chen TH, Hancock WT, Powers AM, Kool JL, Lanciotti RS, Pretrick M,

Marfel M, Holzbauer S, Dubray C, Guillaumot L, Griggs A, Bel M, Lambert AJ, Laven

J, Kosoy O, Panella A, Biggerstaff BJ, Fischer M, Hayes EB. Zika virus outbreak

on Yap Island, Federated States of Micronesia. N Engl J Med. 2009 Jun

11;360(24):2536-43. doi: 10.1056/NEJMoa0805715. PubMed PMID: 19516034.

2. Del Carpio-Orantes L, Da Silva IRF, Moguel KGP, Díaz JSS, Del Pilar Mata

Miranda M, García-Méndez S, Perfecto-Arroyo MA, Solís-Sánchez I, Del Rosario

Pola-Ramírez M. Guillain Barré syndrome in arbovirus outbreak, Campylobacter claims

his throne. J Neurol Sci. 2019 Jan 15;396:254-255. doi: 10.1016/j.jns.2018.10.029.

Reviewer #2: Soares et al. describe a case-control study in Mexico assessing the association between Zika infection and GBS. They enroll 50 cases and match 1:3 141 controls to these. They assess Zika infection using RT-PCR in all and IgM in some patients. They find a similar and high proportion of Zika infection in both groups based on RT-PCR. Lab results in combination with at least one symptom does provide a signal that favors the hypothesis that there is an association between Zika infection and GBS.

From surveillance reports (PAHO, https://www.paho.org/hq/dmdocuments/2017/2017-phe-zika-situation-report-mex.pdf), it seems that the ZIKV outbreak took place in 2016, 2017. Could the authors in the introduction give some indication of the level of circulation, especially during the study period (August 2017, June 2018). Is this the tail of the epidemic, especially into 2018 was there any ZIKV circulation? How were the cases distributed over time? If the authors could provide an epidemic curve combined with when cases were sampled?

Regarding the diagnostic methods: The authors present a surprising proportion of RT-PCR positive patients both in the cases as control group (22%). We tend to believe that RT-PCR in general is highly specific, and that a positive result could be considered as Zika. False negatives are much more common, since, in settings as these, we are often too late. We know from GBS cases that were preceded by symptomatic ZIKV infection, that these would often occur 5-10 days after symptom onset. If we allow time before sampling these patients (here:5-52 days, we expect few to still have viral RNA in their blood or urine. Even in a peak of an epidemic, sampling symptomatic patients would likely not yield such high counts. Cross-validation of the results with a different method, IgM/neutralization would be of great value here to interpret the results. Can the authors clarify the diagnostics by at least providing crosstabulation with the IgM. It would be crucial to get more clarity on this issue, since this is one of the most relevant exposure assessments in the manuscript.

More specific: Should we indeed trust these results, or can the authors provide some additional verification of the results? (confirmation of the analyses, re-analyses?)

Much of the ‘significance’ relies on the combination of Lab results and one symptom (Rash, joint pain, conjunctivitis). Could the authors be more precise in explaining what these symptoms mean and how they were obtained: from clinical examination or the survey? E.g. does ‘joint pain’ mean that the subject had one day of joint pain 55 days before GBS onset (since the interview period mentions 2 months). A survey example could be provided in the supplementary material. Are these self-reported? What would classify as ‘conjunctivitis’ and what as ‘rash’? Do the authors agree that this information is crucial to interpret the likelihood of these (often aspecific) symptoms to be truly indicators for Zika virus infection.

How sensitive are the results to the selection of symptoms? Do combinations of symptoms, “at least two symptoms” still yield the same results?

Table 1: Could the number of IgM tested individuals and positive samples be added here? Crosstabulation of IgM and PCR would be of value as discussed above.

Table 2: the number of patients with IgM OR PCR (10) is lower than PCR only (11), that seems strange, since the first group would include at least the second.

Table 5: The sample size seems to be reduced to 26, although all patients have been tested using at least PCR? It seems that these are patients that have been assessed using IgM AND PCR instead IgM or PCR? What is the rationale to only take this subset here, where the other tables use the full sample as denominator? The conclusions seem to be thin, and based on multiple testing and wide confidence intervals. The text reports a ‘significant’ difference in dyspnea and a ‘trend’ in facial diplegia, dysphagia and dysarthria. What is a ‘trend’ and does the confidence interval take into account multiple testing? Would the authors consider phrasing these findings a bit more careful keeping in mind that these could as well be chance findings? E.g., we might as well say that there is a trend that PCR or IgM positivity (regardless of symptoms) is protective of GBS based on Table 4.

Specific comments:

Line 242-243: prevalence of RT-PCR positivity similar to Cao-Lormeau? They found 0/42 cases, this is not similar?

Line 248: that should be 2, not 2%?

Line 253: the ‘trend’ is here described as ‘slighlty more common’, what does this mean?

Line 262: The discussion of the RT-PCR results comes at a peculiar place in the discussion. This seems vital as this warrants care for the interpretation. It would be great to be clear and avoid the double negatives in sentence 266. You seem to state here that: “We are unsure about the validity of out RT-PCR results”.

It would be elegant to report according to a checklist like STROBE https://www.strobe-statement.org/index.php?id=available-checklists and provide the checklist as supplementary material.

Reviewer #3: The paper “Zika Virus infection and Guillain Barré syndrome in Northeastern Mexico: a case-control study”, authored by Gongora-Rivera F. et al., is an interesting and well written manuscript that assesses the relationship between the occurrence of Guillain Barré syndrome (GBS) and ZIKA virus infection. The authors did not find evidence of a link between laboratory evidence of ZIKA virus infection and the occurrence of GBS, but they did find a significant association when considering the antecedent of ZIKA’s typical signs and symptoms. I found the conclusion for this work supported previous evidence on this topic, assessing the impact on neurological complications related to acute infection with ZIKA virus in a specific population, in Northeast Mexico.

Nevertheless, I suggest some revisions to consider it for publication, as follows:

1-LINE 55: where it said, “We identified suspected GBS case-patients based on onset of compatible neurologic symptoms…” I suggest listing signs and symptoms considered to the case definition (can be in parentheses).

2-All sections of the manuscript where authors refer to the evaluation of exposure or serology for Dengue and Chikungunya (LINE 72: “…to determine exposure to ZIKV, DENV and CHIKV.”; LINE 94: “serological evidence of DENV, CHIKV and ZIKV infections.” need to be clarified and adjusted to the methodology applied to this work. Serological evidence of Dengue and Chikungunya virus was not evaluated. The authors tested for current infection with Zika, Dengue, and Chikungunya virus by RT-PCR, as well as to exposure to Zika with IgM antibodies against ZIKV.

3-LINE 93: List GBS risk factors (can be in parentheses).

4-LINE 107: Measures used to assess ZIKV status seem duplicative and create some confusion with respect to how the results are interpreted. For example, PCR-positive cases will be present also in the second group (Positive PCR assay or positive IgM assay) so that it is not clear how many patients were PCR- and IgM-positive, how many were only PCR-positive; and how many were only IgM-positive. Please clarify. This is very important since PCR and IgM positivity are not synonymous from a pathophysiological perspective, but rather reflect two different processes that can occur during the infection. It is my understanding that these criteria were chosen because not all cases and controls have a serology for ZIKV performed. This is unfortunate since GBS is considered a post-infectious complication such that antibodies rather than viremia seem more likely a measure of a post-infectious state. Still, this test was obtained in only half of the cases and 80% of controls. It will be very interesting to analyze the subgroup of patients with a serology test and its relation with GBS over the total of cases and controls with this test available, and also those patients with coexistence of PCR and IgM (if any). Therefore, authors can considerate these measures to assess ZIKV status: positive PCR; positive IgM; positive PCR and IgM.

5-Prolonged viremia is increasingly reported in ZIKV infection and had been related to pathogenesis [The Journal of Experimental Medicine (2019) 216 (10): 2302–2315]. Even if there is no way to know the duration of viremia in the cases with PCR positive presented in this work, do authors assume that the antecedent symptoms in the previous two months suggests prolonged viremia in cases with positive PCR?

RESULTS

6-Table 1. Results from the serology are missing. Please, add results from IgM assay against ZIKA. In a footnote it can be clarified for how many cases and how many controls serological testing was available. It also would be interesting express how many patients (if any?) were both PCR- and IgM- positive. Even when flavivirus infection is traditionally related to short viremias, followed by a rise of antibodies, some ZIKV infections have shown unusually prolonged viremias (more studies in pregnant woman). And even some recent studies link this prolonged viremia overlapping with peaking of specific antibodies, with the pathogenesis of congenital disease.

7-LINE 125 where it said “Recent infection by arbovirus…” must said “Current infection by arbovirus”. Since PCR refer to current infection, and IgM would refer to recent infection.

8-Table 2. I found this table very confusing to read, and I believe that confusion merge from the measures used to assess ZIKV status. I suggest follow directions previous suggest for METHODS.

9-Table 3. Fever, diarrhea and cough also had a correlation with cases. What do the authors think about that? Where these symptoms in relation with typical Zika symptoms, or they were observed in different patients? (and diarrhea is misspelled in the table)

10-Table 4. It is not clear. Again, this dual measure of ZIKV status causes confusion. Same comment to table 2.

11-Table 5. The same comment to previous tables. I suggest one block: Zika diagnosis by PCR and or IgM (n50) with two sub columns: Zika positive and Zika negative. Each one of these with two sub columns: with and without typical zika symptoms. Include (N and %) at the headline.

12-Table 6. Same comment table 5.

CONCLUSIONS

13-The authors develop a complete and correct analysis of appropriate bibliography. I consider the main limitation of this work, the lack of serology test for ZIKV for almost half of the cases (and actually, it is hard to find in the manuscript, how many of cases and controls had a positive result). This limitation is briefly mention in the discussion (LINE 261-262). Because GBS is a post-infectious event, is expected to find this clinical manifestation in synchrony with the presence of antibodies. It would be interesting to evaluate if exist an association with the presence of IgM against ZIKV and GBS, considering only that subgroup of cases and controls.

14-Another important limitation is the lack of Dengue and Chikungunya serology. The co-circulation of these Aedes borne diseases in the region, and the almost impossibility to clinical differentiate the clinical features make so relevant these tests. This limitation needs to be mention, and clarity it in methods as it was mentioned before.

15-I found very interesting the evaluation of previous typical ZIKV symptoms mainly for its moderate Positive Predictive Value for GBS diagnosis. The PPV could be calculated.

**********

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From: em.pone.0.67f9a8.15841351@editorialmanager.com on behalf of PLOS ONE

Sent: Monday, December 16, 2019 12:10:37 PM

To: Sejvar, James (CDC/DDID/NCEZID/DHCPP)

Subject: PLOS ONE Decision: Revision required [PONE-D-19-30389] - [EMID:e47842d417be5818]

PONE-D-19-30389

Zika Virus infection and Guillain-Barré syndrome in Northeastern Mexico: a case-control study

PLOS ONE

Dear Dr Sejvar,

Thank you very much for submitting your manuscript "Zika Virus infection and Guillain-Barré syndrome in Northeastern Mexico: a case-control study" (#PONE-D-19-30389) for review by PLOS ONE. As with all papers submitted to the journal, your manuscript was fully evaluated by academic editor (myself) and by independent peer reviewers. The reviewers appreciated the attention to an important health topic, but they raised substantial concerns about the paper that must be addressed before this manuscript can be accurately assessed for meeting the PLOS ONE criteria. Therefore, if you feel these issues can be adequately addressed, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. We can’t, of course, promise publication at that time.

We would appreciate receiving your revised manuscript by Dec 28 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

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We look forward to receiving your revised manuscript.

Kind regards,

Abdallah M. Samy, PhD

Academic Editor

PLOS ONE

Additional Editor Comments:

I invited and received three reviews of your manuscript. All reviews raised substantial concerns about your manuscript as it currently stands. I read the manuscript myself and i must say that i completely agree that the reviews are detailed and solid. So, my decision is "major revision". Please address all these concerns before submitting a revised version of your manuscript. I would kindly ask the authors to confirm that they follow the Journal guidelines available via the link https://journals.plos.org/plosone/s/submission-guidelines. I would love to see the revised version of your manuscript as soon (i.e. within the next two weeks), so, we will save much time in the review process. Thanks too much for choosing PLOS ONE for your submission!

Journal Requirements:

When submitting your revision, we need you to address these additional requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf

JJS: We have reviewed the style requirements, and believe that our manuscript does meet these requirements.

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JJS: We are happy to have our study data available; we have indicated the point of contact to access the complete, de-identified data.

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JJS: The corresponding author’s ORCID id is valid. 0000-0002-2536-3276.

4. One of the noted authors is a group or consortium: Guillain-Barré syndrome -University Hospital -Mexican Institute of Social Security Working Group (GBS HU-IMSS WORKING GROUP).

In addition to naming the author group, please list the individual authors and affiliations within this group in the acknowledgments section of your manuscript. Please also indicate clearly a lead author for this group along with a contact email address.

JJS: We have named a lead author for the GBS HU-IMSS Working Group. It is the following, along with email address:

GBS HU IMSS Working Group

Hector Jorge Villarreal, MD

Chief of Servicio de Neurología del Hospital Universitario, Monterrey. Mexico

Email: neurologia01@yahoo.com.mx

5. Your ethics statement must appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please also ensure that your ethics statement is included in your manuscript, as the ethics section of your online submission will not be published alongside your manuscript.

JJS: This has been done

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Partly

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

________________________________________

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

________________________________________

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

________________________________________

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this article, the authors included patients from three states of northeastern Mexico between August 2017 and June 2018, in order to establish whether the endemic circulation of Zika virus (ZIKV) was associated with the development of Guillaín-Barre syndrome (GBS). It is a case-control study, the cases corresponded to hospitalized or outpatient with GBS, with diagnostic certainty of grades 1 to 3 according to the classification of Brighton Collaboration Criteria. For each patient they included 3 controls (2.8) seen in emergency visits or hospitalized, with non-febrile illness for 48 hours, paired with cases by sex and age + minus 10 years, and seen in the same hospital within seven days of each case. After the interview, serum and urine were taken in both groups to determine exposure to ZIKV, dengue virus and Chikungunya virus by viral RNA amplification (PCR) and detection of IgM antibodies to ZIKV.

They studied 50 cases and 141 controls with similar demography. They found 22% of ZIKV positive PCR samples, both in cases and controls. The detection of IgM to ZIKV was performed in 26/50 (52%) of the cases and in 113/141 (80%) of the controls, with no difference in times between the onset of symptoms and the collection of samples.

The analysis prior to the neurological picture showed that the cases had with more frequency: Fever, diarrhea, myalgia, arthralgia, rash, conjunctivitis, headache, odynophagia and a composite outcome of typical ZIKV symptom (rash, joint pain, or conjunctivitis). The comparisons were carried out with conditional maximum likelihood estimation.

The first problem we encountered with the study is that the characteristics of the control group are not well described, at least in general terms. Such as the proportion of outpatients and hospitalized patients. It would be important to describe if in the control group there were patients with neurological disease, especially with GBS and, if among the emergency included patients, there were patients with minor trauma or trivial conditions and otherwise previously healthy, who would be representative of the “open” population. With some of these data, the general characteristics of the control group would be clearer.

JJS: We appreciate the comment by the reviewer. Of the 141 controls, 24 were outpatients, and 117 were hospitalized. This would suggest that the control population would not be an accurate reflection of the nonhospitalized population. We have included this information in the results.

Finally, with ZIKV, something similar to other viral diseases seems to happen in their post-epidemic transition to endemic status, and it is the fact that a high number of infections are asymptomatic as the clear example of the study of the island of Yap, where it was estimated that the 73% of the population was infected with ZIKV but only 18% of those infected (95% CI, 10 to 27), had a clinical illness that was probably attributable to ZIKV infection or 1 symptomatic person in 4.4 ZIKV infected persons (1).

JJS: We concur with the reviewer on this.

A surprising finding in this study is that the controls had exactly the same percentage of positive PCR tests (22%) as the cases, which leads us to conclude on the importance of studying GBS patients as thoroughly as available resources allow it, to investigate coinfections, particularly gastrointestinal and thus be able to establish with greater solidity the association of ZIKV with GBS and the prevalence of other potential pathogens in endemic areas of ZIKV.

JJS: The authors concur with the reviewer that other, alternative etiologies of GBS with a temporal association, such as Campylobacter jejuni, ideally would ideally have been tested for an excluded. However, the lack of a widely available and standardized ELISA serum assay for anti-Campylobacter antibodies made such a diagnosis challenging in this situation.

We recommend that a specific header must be opened prior to the conclusions, to clearly indicate the limitations of the study:

That electrophysiological studies were carried out in 48% of patients with GBS, that 20% of controls did not have the IgM antibody tests against ZIKV. That in the cases group, diarrhea occurred in 44% of the patients and it is not mentioned if the presence of intestinal pathogenic microorganisms was investigated, especially Campylobacter jejuni, which seems to be associated with Guillain Barré syndrome in the state of Veracruz, Mexico according to a publication by del Carpio-Orantes (2), this is a very important matter, which opens the possibility that the GBS was caused by concomitant enteric pathogens as an epiphenomenon to asymptomatic ZIKA virus infection as it would seem to occur in the controls.

JJS: We have included a section on limitations and will ensure that language to this effect will be placed in the limitations: ‘Diarrhea was present in approximately 44% of cases. Diarrhea is a common clinical manifestation of the gastrointestinal bacterium Campylobacter jejuni, which also has a strong association with the axonal form of GBS, acute motor axonal neuropathy (AMAN). However, the lack of a commercially available and standardized ELISA test for detecting anti-Campylobacter antibodies made pursuing this diagnosis logistically challenging. ‘

Finally, there are some inaccuracies in the tables that merit a careful review:

1. In Table 3, in the line 9, conjunctivitis, the number of patients is 6, but the percentage in brackets is indicated 1 and should correspond to 12.

JJS: This has been fixed in the manuscript.

2. In Table 5. Neurological signs and symptoms at onset or nadir of GBS case-patients: n (%)

The first line reads neurological signs and symptoms all n = 50, the thrid column indicates Zika diagnosis by PCR or IgM (n = 26), and the fourth column shows Zika diagnosis by PCR or IgM and rash, joint pain, or conjunctivitis (n = 26), the sum is 52, no 50. In addition, if the individual data of the next line 10 + 16 +7 + 19 are added, it is equal to 52, so it should be reviewed why the total number of patients is exceeded by 2.

JJS: We added a footnote to clarify this. Specifically, this represents 26 people of the 50 who had IgM testing done; the two columns represent separate groups of 26. Thus, the sum of the two columns should not add up to 52.

References

1. Duffy MR, Chen TH, Hancock WT, Powers AM, Kool JL, Lanciotti RS, Pretrick M,

Marfel M, Holzbauer S, Dubray C, Guillaumot L, Griggs A, Bel M, Lambert AJ, Laven

J, Kosoy O, Panella A, Biggerstaff BJ, Fischer M, Hayes EB. Zika virus outbreak

on Yap Island, Federated States of Micronesia. N Engl J Med. 2009 Jun

11;360(24):2536-43. doi: 10.1056/NEJMoa0805715. PubMed PMID: 19516034.

2. Del Carpio-Orantes L, Da Silva IRF, Moguel KGP, Díaz JSS, Del Pilar Mata

Miranda M, García-Méndez S, Perfecto-Arroyo MA, Solís-Sánchez I, Del Rosario

Pola-Ramírez M. Guillain Barré syndrome in arbovirus outbreak, Campylobacter claims

his throne. J Neurol Sci. 2019 Jan 15;396:254-255. doi: 10.1016/j.jns.2018.10.029.

Reviewer #2: Soares et al. describe a case-control study in Mexico assessing the association between Zika infection and GBS. They enroll 50 cases and match 1:3 141 controls to these. They assess Zika infection using RT-PCR in all and IgM in some patients. They find a similar and high proportion of Zika infection in both groups based on RT-PCR. Lab results in combination with at least one symptom does provide a signal that favors the hypothesis that there is an association between Zika infection and GBS.

From surveillance reports (PAHO, https://www.paho.org/hq/dmdocuments/2017/2017-phe-zika-situation-report-mex.pdf), it seems that the ZIKV outbreak took place in 2016, 2017. Could the authors in the introduction give some indication of the level of circulation, especially during the study period (August 2017, June 2018). Is this the tail of the epidemic, especially into 2018 was there any ZIKV circulation? How were the cases distributed over time? If the authors could provide an epidemic curve combined with when cases were sampled?

JJS: As the reviewer rightly points out, part of the investigation was conducted during periods of relatively low, and even absent, Zika transmission. While we would like to give the readers a sense of the burden of Zika virus infection over the study period, unfortunately accurate, complete, and consistent data do not exist for these time periods in the Monterrey region, or for Mexico in general. Based upon the data that we were able to glean, Zika was still circulating during 2016 -2018, and based on what we know from 2016-2018, it seems like Zika incidence was highest during the fall through early winter, which seems to correspond roughly to an increase in GBS cases (and subsequent controls). We appreciate the reviewer’s request for an epidemiologic curve, but when we formatted one, it did not appear to very well address the question posed by the reviewer, and we chose to omit it.

Regarding the diagnostic methods: The authors present a surprising proportion of RT-PCR positive patients both in the cases as control group (22%). We tend to believe that RT-PCR in general is highly specific, and that a positive result could be considered as Zika. False negatives are much more common, since, in settings as these, we are often too late. We know from GBS cases that were preceded by symptomatic ZIKV infection, that these would often occur 5-10 days after symptom onset. If we allow time before sampling these patients (here:5-52 days, we expect few to still have viral RNA in their blood or urine. Even in a peak of an epidemic, sampling symptomatic patients would likely not yield such high counts. Cross-validation of the results with a different method, IgM/neutralization would be of great value here to interpret the results. Can the authors clarify the diagnostics by at least providing crosstabulation with the IgM. It would be crucial to get more clarity on this issue, since this is one of the most relevant exposure assessments in the manuscript.

JJS: We concur with the reviewer that the proportion / percentage of cases and controls having positive results by PCR testing was much higher than expected. We also agree that it is more likely to have false negatives by PCR due to the timing issue. Unfortunately, during this investigation, there was so little sample left after routine bloodwork and PCR testing that we were, in the vast majority of cases, unable to ‘confirm’ the PCR results with concomitant serology. However, the definitive testing to substantiate this is not feasible.

More specific: Should we indeed trust these results, or can the authors provide some additional verification of the results? (confirmation of the analyses, re-analyses?)

JJS: Again, unfortunately we are unable to provide additional verification of results, due to lack of sample. The reason that the controls were just as likely to have a positive PCR result is unclear.

Much of the ‘significance’ relies on the combination of Lab results and one symptom (Rash, joint pain, conjunctivitis). Could the authors be more precise in explaining what these symptoms mean and how they were obtained: from clinical examination or the survey? E.g. does ‘joint pain’ mean that the subject had one day of joint pain 55 days before GBS onset (since the interview period mentions 2 months). A survey example could be provided in the supplementary material. Are these self-reported? What would classify as ‘conjunctivitis’ and what as ‘rash’? Do the authors agree that this information is crucial to interpret the likelihood of these (often aspecific) symptoms to be truly indicators for Zika virus infection.

How sensitive are the results to the selection of symptoms? Do combinations of symptoms, “at least two symptoms” still yield the same results?

JJS: We thank the reviewer for this observation. Indeed, these were self-reported signs and symptoms reported among subjects. Per request, a copy of the case report form has been added to the supplementary material. ‘Conjunctivitis’ and ‘Rash’ were whatever was interpreted by the subject, but it is generally thought that most lay persons recognize a rash and conjunctivitis (red eyes).

When combining symptoms, we unfortunately found that there was an insufficient number of patients in each cell with 2 or more symptoms to evaluate this. Essentially, there were 1 of 26 cases, and 0 of 113 controls that are Zika positive and have 2 or more symptoms. This gives an uninterpretable odds ratio with an interval of 0 to infinity.

Table 1: Could the number of IgM tested individuals and positive samples be added here? Crosstabulation of IgM and PCR would be of value as discussed above.

JJS: Please see added New Table 2

Table 2: the number of patients with IgM OR PCR (10) is lower than PCR only (11), that seems strange, since the first group would include at least the second.

JJS: This is due to the fact that only 26 of the 50 cases received an IgM test. We restricted the assessment of Zika positivity by PCR or IgM to those 26 patients (this was to remove the possibility of bias from including all 50 patients: those who received an IgM test would be inherently more likely to be reported as Zika+, and if they were different in any way from the 24 who did not receive an IgM test, this could result in unforeseen biases).

Table 5: The sample size seems to be reduced to 26, although all patients have been tested using at least PCR? It seems that these are patients that have been assessed using IgM AND PCR instead IgM or PCR? What is the rationale to only take this subset here, where the other tables use the full sample as denominator? The conclusions seem to be thin, and based on multiple testing and wide confidence intervals. The text reports a ‘significant’ difference in dyspnea and a ‘trend’ in facial diplegia, dysphagia and dysarthria. What is a ‘trend’ and does the confidence interval take into account multiple testing? Would the authors consider phrasing these findings a bit more careful keeping in mind that these could as well be chance findings? E.g., we might as well say that there is a trend that PCR or IgM positivity (regardless of symptoms) is protective of GBS based on Table 4.

JJS: We added a footnote to clarify this. We have removed the term ‘trend’ and excluded facial diplegia, dysphagia, and dysarthria, mentioning only dyspnea that is ‘significant’ statistically, though we acknowledge the wide confidence intervals.

Specific comments:

Line 242-243: prevalence of RT-PCR positivity similar to Cao-Lormeau? They found 0/42 cases, this is not similar?

JJS: This was clearly an error on our part, and this has been corrected in the text.

Line 248: that should be 2, not 2%?

JJS: The reviewer is correct; this number should be 2, not 2%. This has been corrected.

Line 253: the ‘trend’ is here described as ‘slighlty more common’, what does this mean?

JJS: This has been corrected to indicate that only dyspnea was associated with a statistically significant measure of being more common among cases than controls (page 15 line 243, clean version).

Line 262: The discussion of the RT-PCR results comes at a peculiar place in the discussion. This seems vital as this warrants care for the interpretation. It would be great to be clear and avoid the double negatives in sentence 266. You seem to state here that: “We are unsure about the validity of out RT-PCR results”.

JJS: We apologize to the reviewer, but we cannot locate the sentence in which there is a double negative; we are simply saying that we cannot determine with absolute certainty that there were not some subjects with false-positive results. But then we lay out why we feel that the results are sound.

It would be elegant to report according to a checklist like STROBE https://www.strobe-statement.org/index.php?id=available-checklists and provide the checklist as supplementary material.

JJS: Per the reviewer’s suggestion, we have utilized the STROBE checklist for this paper. We feel that criteria 1 – 7, 9-12, and 14-22 of the STROBE checklist for case-control studies was adequately addressed by our paper.

Reviewer #3: The paper “Zika Virus infection and Guillain Barré syndrome in Northeastern Mexico: a case-control study”, authored by Gongora-Rivera F. et al., is an interesting and well written manuscript that assesses the relationship between the occurrence of Guillain Barré syndrome (GBS) and ZIKA virus infection. The authors did not find evidence of a link between laboratory evidence of ZIKA virus infection and the occurrence of GBS, but they did find a significant association when considering the antecedent of ZIKA’s typical signs and symptoms. I found the conclusion for this work supported previous evidence on this topic, assessing the impact on neurological complications related to acute infection with ZIKA virus in a specific population, in Northeast Mexico.

Nevertheless, I suggest some revisions to consider it for publication, as follows:

1-LINE 55: where it said, “We identified suspected GBS case-patients based on onset of compatible neurologic symptoms…” I suggest listing signs and symptoms considered to the case definition (can be in parentheses).

JJS: Per the reviewer’s request, we have added some signs and symptoms compatible with GBS (page 5, line 104).

2-All sections of the manuscript where authors refer to the evaluation of exposure or serology for Dengue and Chikungunya (LINE 72: “…to determine exposure to ZIKV, DENV and CHIKV.”; LINE 94: “serological evidence of DENV, CHIKV and ZIKV infections.” need to be clarified and adjusted to the methodology applied to this work. Serological evidence of Dengue and Chikungunya virus was not evaluated. The authors tested for current infection with Zika, Dengue, and Chikungunya virus by RT-PCR, as well as to exposure to Zika with IgM antibodies against ZIKV.

JJS: We concur with the reviewer that this investigation did not assess for serologic evidence of DENV and CHIKV, and that this was misstated in the manuscript. This has been corrected.

3-LINE 93: List GBS risk factors (can be in parentheses).

JJS: This has been added (page 6, line 117)

4-LINE 107: Measures used to assess ZIKV status seem duplicative and create some confusion with respect to how the results are interpreted. For example, PCR-positive cases will be present also in the second group (Positive PCR assay or positive IgM assay) so that it is not clear how many patients were PCR- and IgM-positive, how many were only PCR-positive; and how many were only IgM-positive. Please clarify.

JJS: We concur with the reviewer that this information is important to the interpretation of the results here. We have defined, in the table, the number of subjects that were PCR-positive only; IgM-positive only; and those that were PCR- and IgM-positive. (See New Table 2)

This is very important since PCR and IgM positivity are not synonymous from a pathophysiological perspective, but rather reflect two different processes that can occur during the infection. It is my understanding that these criteria were chosen because not all cases and controls have a serology for ZIKV performed. This is unfortunate since GBS is considered a post-infectious complication such that antibodies rather than viremia seem more likely a measure of a post-infectious state. Still, this test was obtained in only half of the cases and 80% of controls. It will be very interesting to analyze the subgroup of patients with a serology test and its relation with GBS over the total of cases and controls with this test available, and also those patients with coexistence of PCR and IgM (if any). Therefore, authors can considerate these measures to assess ZIKV status: positive PCR; positive IgM; positive PCR and IgM.

JJS: We concur with the reviewer that PCR and IgM represent very different pathophysiological underpinnings of flavivirus infection, and that, as a post-infectious phenomenon, GBS would more likely be expected to be positive by IgM, rather than PCR, testing. So, per above, we are including the breakdown of PCR+, IgM+, and PCR / IgM+. This can be found in New Table 2.

5-Prolonged viremia is increasingly reported in ZIKV infection and had been related to pathogenesis [The Journal of Experimental Medicine (2019) 216 (10): 2302–2315]. Even if there is no way to know the duration of viremia in the cases with PCR positive presented in this work, do authors assume that the antecedent symptoms in the previous two months suggests prolonged viremia in cases with positive PCR?

JJS: We are aware of the reports of prolonged viremia, although the reports in the literature are relatively few. The reviewer is correct that we are unable to say with any degree of certainty the duration of viremia in our cases. We cannot, however, a priori assume that this is reflective of a prolonged viremia.

RESULTS

6-Table 1. Results from the serology are missing. Please, add results from IgM assay against ZIKA. In a footnote it can be clarified for how many cases and how many controls serological testing was available. It also would be interesting express how many patients (if any?) were both PCR- and IgM- positive. Even when flavivirus infection is traditionally related to short viremias, followed by a rise of antibodies, some ZIKV infections have shown unusually prolonged viremias (more studies in pregnant woman). And even some recent studies link this prolonged viremia overlapping with peaking of specific antibodies, with the pathogenesis of congenital disease.

JJS: The IgM results have been added to table 1, along with footnotes that specify the number of cases (26) and controls (113) that received IgM tests

7-LINE 125 where it said “Recent infection by arbovirus…” must said “Current infection by arbovirus”. Since PCR refer to current infection, and IgM would refer to recent infection.

JJS: This has been corrected.

8-Table 2. I found this table very confusing to read, and I believe that confusion merge from the measures used to assess ZIKV status. I suggest follow directions previous suggest for METHODS.

JJS: We edited Table 2 (now Table 3) so that the number of cases assessed for each group is easier to understand

9-Table 3. Fever, diarrhea and cough also had a correlation with cases. What do the authors think about that? Where these symptoms in relation with typical Zika symptoms, or they were observed in different patients? (and diarrhea is misspelled in the table)

JJS: Fever, diarrhea, and cough are very nonspecific findings of acute viral infection. Although it could be argued that rash, joint pain, and conjunctivitis are similarly nonspecific, during our several investigations into the clinical characteristics of Zika virus infection, it seemed that these three symptoms were strikingly more common in Zika virus infection as opposed to other general viral infections. Thus, we placed more emphasis on these three symptoms of ZIKV.

‘Diarrhea’ has been corrected.

10-Table 4. It is not clear. Again, this dual measure of ZIKV status causes confusion. Same comment to table 2.

JJS: We edited Table 4 (now Table 5) so that the number of cases assessed for each group is easier to understand; specifically, we added headings of “All Observations” and “Patients Undergoing IgM Test”.

11-Table 5. The same comment to previous tables. I suggest one block: Zika diagnosis by PCR and or IgM (n50) with two sub columns: Zika positive and Zika negative. Each one of these with two sub columns: with and without typical zika symptoms. Include (N and %) at the headline.

JJS: The main purpose of Table 5 (now Table 6) is to determine if patients with Zika-associated GBS were clinically different from those with non-Zika-associated GBS. This was done using two slightly different definitions for Zika diagnosis (Zika diagnosis by PCR or IgM, or Zika diagnosis by PCR or IgM and rash, joint pain, or conjunctivitis). The suggested changes would alter the comparisons: we would be seeing if, out of patients testing positive for Zika by PCR or IgM, whether those who had previous Zika symptoms differed clinically from those who did not have previous Zika symptoms (this also asks of those who tested negative for Zika by PCR or IgM, whether those who had previous Zika symptoms differed clinically from those who did not have previous Zika symptoms). These were not the research questions we were interested in. For the sake of clarity, we did add a footnote that specified which patients were included in these analyses.

12-Table 6. Same comment table 5.

JJS: Similar to Table 5, changing Table 6 (now Table 7) in the suggested manner would have changed the research question. We did add the same footnote from Table 5 in Table 6 for clarification.

CONCLUSIONS

13-The authors develop a complete and correct analysis of appropriate bibliography. I consider the main limitation of this work, the lack of serology test for ZIKV for almost half of the cases (and actually, it is hard to find in the manuscript, how many of cases and controls had a positive result). This limitation is briefly mention in the discussion (LINE 261-262). Because GBS is a post-infectious event, is expected to find this clinical manifestation in synchrony with the presence of antibodies. It would be interesting to evaluate if exist an association with the presence of IgM against ZIKV and GBS, considering only that subgroup of cases and controls.

JJS: Unfortunately, there are very few patients who are Zika+ by IgM. Of the cases, 3 of 26 (11.5%) had a positive IgM test; for controls, it was 9 of 113 (8.0%), for an odds ratio of 1.50 (0.37 – 5.95). Thus, we were unable to assess this question.

14-Another important limitation is the lack of Dengue and Chikungunya serology. The co-circulation of these Aedes borne diseases in the region, and the almost impossibility to clinical differentiate the clinical features make so relevant these tests. This limitation needs to be mention, and clarity it in methods as it was mentioned before.

JJS: We concur with the reviewer that the clinical signs and symptoms of dengue, Chikungunya, and Zika can be clinically indistinguishable. And, had we the necessary aliquots of serum to conduct these investigations, we would most certainly have done so. We were, however, limited by the availability of the necessary aliquots of serum. Nonetheless, there was no clear evidence of a great deal of circulating dengue or Chikungunya virus in Monterrey at the time, as evidenced by lack of PCR findings for both these viruses. We have, however, mentioned in the discussion / limitations that this does represent a substantial limitation in our findings (page 18, line 406). It has similarly been clarified in the methods.

15-I found very interesting the evaluation of previous typical ZIKV symptoms mainly for its moderate Positive Predictive Value for GBS diagnosis. The PPV could be calculated.

JJS: While a PPV can technically be calculated, this is a case-control study, with the number of cases and controls purposefully selected by the study investigators; a PPV calculated from this study has no relation to a PPV applicable to the general population. However, of 25 patients with one of the typical Zika symptoms, 16 were cases (PPV=64%). Of the 11 patients with a Zika symptom and Zika+ by PCR or IgM, 7 were cases (PPV = 64%).

Submitted filename: Response to Reviewers_final.docx

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24 Feb 2020

Zika Virus infection and Guillain-Barré syndrome in Northeastern Mexico: a case-control study

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Zika Virus infection and Guillain-Barré syndrome in Northeastern Mexico: a case-control study

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