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• Guillain-Barré syndrome and COVID-19: an observational multicentre study from two Italian hotspot regions; response to Keddie et al.
  Massimiliano Filosto, Stefano Cotti Piccinelli, Stefano Gazzina, Alessandro Padovani and Antonino Uncini
  Published on: 24 November 2020
• Further research required to support a causative association between GBS and COVID-19
  Stephen Keddie, Julia Pakpoor, Aisling Carr and Michael P Lunn
  Published on: 12 November 2020

• Published on: 24 November 2020

  Guillain-Barré syndrome and COVID-19: an observational multicentre study from two Italian hotspot regions; response to Keddie et al.

  • Massimiliano Filosto, Neurologist Department of Clinical and Experimental Sciences, University of Brescia, Italy
  • Other Contributors:
    • Stefano Cotti Piccinelli, MD
    • Stefano Gazzina, Neurologist
    • Alessandro Padovani, Neurologist
    • Antonino Uncini, Neurologist

  We read with interest the comments of Keddie and Colleagues who suggested caution in accepting a causation link between SARS-CoV-2 infection and Guillain-Barré syndrome (GBS) and in interpreting results from our study “Guillain-Barrè syndrome and COVID-19: an observational multicentre study from two Italian hotspot regions" (1).
  We believe they have misinterpreted the message of our paper and have drawn conclusions that was not our intention to draw.
  Their first consideration is that our paper cannot demonstrate a causation link between COVID-19 and GBS. Of course, we agree. In fact, we did not talk about any causal nexus. It is well known that, in statistics, “causation” indicates a relationship between two events where one event is affected by the other. In order to demonstrate “causation”, prospective studies are needed. Our study is based on retrospective findings and identified an increased rate of GBS cases concomitantly with the COVID-19 spread in our regions. On this basis, we could not (and indeed we did not) conclude for a definite causative relationship but we suggested a pathogenic link for which COVID-19 could represent a trigger for GBS, as already suggested by other authors (2).
  Keddie et al. claimed some possible methodological biases. Part of them is obviously related to the retrospective nature of the study and have been listed as limitations of the study at the end of our paper. They calculated the 95% confidence intervals of the...

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  We read with interest the comments of Keddie and Colleagues who suggested caution in accepting a causation link between SARS-CoV-2 infection and Guillain-Barré syndrome (GBS) and in interpreting results from our study “Guillain-Barrè syndrome and COVID-19: an observational multicentre study from two Italian hotspot regions" (1).
  We believe they have misinterpreted the message of our paper and have drawn conclusions that was not our intention to draw.
  Their first consideration is that our paper cannot demonstrate a causation link between COVID-19 and GBS. Of course, we agree. In fact, we did not talk about any causal nexus. It is well known that, in statistics, “causation” indicates a relationship between two events where one event is affected by the other. In order to demonstrate “causation”, prospective studies are needed. Our study is based on retrospective findings and identified an increased rate of GBS cases concomitantly with the COVID-19 spread in our regions. On this basis, we could not (and indeed we did not) conclude for a definite causative relationship but we suggested a pathogenic link for which COVID-19 could represent a trigger for GBS, as already suggested by other authors (2).
  Keddie et al. claimed some possible methodological biases. Part of them is obviously related to the retrospective nature of the study and have been listed as limitations of the study at the end of our paper. They calculated the 95% confidence intervals of the incidence rates as 0.08 per 100,000 per month in 2019 (95% C.I.: 0.04-0.15) and 0.2 per 100,000 per month in 2020 (C.I.: 0.14-0.28) and concluded for an overlap between confidence intervals. Actually the 95% confidence intervals based on the numbers of cases and population we reported in our study were 0.077 per 100,000 per month in 2019 (C.I.: 0.041-0.132) and 0.202 per 100,000 per month in 2020 (C.I. 0.140-0.282) (MedCalc, MedCalc Software Ltd, Belgium). Thus, there is no overlap of confidence intervals. Moreover, judging the significance of differences by examining the overlap between two confidence intervals has been debated and discouraged by the statistical community (3).
  As a second point, Keddie et al. suggested that the denominator (COVID-19 positive cases) could be underestimated because testing was limited to symptomatic subjects. We clearly acknowledged in the paper that the real number of COVID-19-positive patients was likely higher than that in the official data and this could cause an overestimation of the GBS incidence in the COVID-19-positive population. On the other hand, it should be stressed that also the numerator can be biased. During the Italian outbreak, many Neurological Units were closed in order to provide medical staff for COVID-19 Units and many neurological patients (COVID-19 positive and negative) could not be admitted. Thus, it is likely that GBS cases (either COVID-19 positive or negative) were not recognized. This might also justify the lower number of COVID-19 negative GBS observed in 2020.
  Similar problems arose in evaluating the incidence of GBS in Latin America and Caribbean countries during the 2015-20216 Zika virus epidemic because only about 25% of subjects infected by Zika virus showed clinical manifestations and difficulties were encountered in certainty of laboratory diagnosis in regions where other flavivirus are endemic. Nonetheless, a recent meta-analysis of observational studies concluded that GBS increased 2.6 times over background rates during Zika outbreak (4).
  Keddie et al. claimed that the denominator for rate calculation of GBS in COVID-19 patients should be substituted with the seroprevalence in the same population (630,000, based on a 7.5% prevalence in Lombardy reported by ISTAT) (5). Again, we agree that the real denominator is difficult to obtain, as we reported in the limitations of the study. However, it should be noted that the reported SARS-CoV-2 seroprevalence tests were performed from May 25, 2020 to July 15, 2020, i.e., in a period subsequent to the months considered in our study and disclosed in August 2020 (6). We do not know and cannot enucleate the seroprevalence in the months of March and April and, for this reason, we preferred to work on the real data of nasopharyngeal swabs rather than on estimated data referring to a period subsequent to that considered in our study.
  Findings about duration of seropositivity are discordant (7,8) and whether levels of IgG specific for SARS-CoV-2 antigen persist or decay remains a debated issue (7). We also want to point out that many asymptomatic subjects are seropositive and this raises further questions: 1) Is it correct to investigate a COVID-19/GBS relationship in seropositive patients without active or recent infectious signs? 2) Do we know if asymptomatic patients with positive nasopharyngeal swab are prone to develop para- or post-infectious complications? To date, our answers are no.
  Obviously, we cannot exclude that some COVID-19 positive GBS patients represented a casual association and we never excluded this possibility. However, the fact remains that GBS was 2.6 time increased in the same hospitals between 2019 and 2020 and that the incidence of GBS in March and April 2019 (0.077/100 000/month; estimated rate 0.93/100 000/year) was consistent with a previous epidemiological study showing a GBS incidence of 0.75–1.09/100 000/year in Lombardy (9), therefore suggesting a true increased incidence of GBS during the pandemic peak in Northern Italy.
  We acknowledge as a limitation of the study the fact that, in many cases, was not possible to exclude other infective antecedents and this is true also for most of reported cases of GBS associated with SARS-CoV2 infection from all over the world (10). Again, this was due to the severe and exceptional health emergency we suffered in Northern Italy during the first epidemic wave.
  About the pathogenesis of COVID-19 related GBS, it is untrue that we used data from modelling studies suggesting interaction of the SARS-CoV-2 spike protein with ganglioside GM1 to support a causative link (11). Actually, in our discussion we just mentioned this hypothesis as a “possibility” (“an immune cross-reaction between epitopes within the spike-bearing gangliosides and sugar residues of surface peripheral nerve glycolipids is also possible”) without claiming any causative link.
  We also were far from wanting to prove that GBS associated with SARS-CoV-2 infection fulfilled the Witebsky’s criteria for autoimmune disease, that as far as we know in the field of GBS, have been fulfilled only for the acute motor axonal neuropathy variant with anti-GM1 antibodies (12).
  In conclusion, isolated or small series of GBS cases associated with SARS-CoV 2 infection were described from all over the world in the first pandemic months (10) and have reached to November 19th 2020 a total of 139 cases especially reported from Italy (38.1%). Our study showed an increased incidence of GBS during COVID-19 outbreak in Northern Italy. Despite the limitations mentioned in the paper and inherent to a retrospective study we deem that these results suggest a relationship.
  Keddie et al. reported in their disclosures that they have a similar paper due to be published in a larger cohort and reaching different conclusion. We do not have any detail about their study but different reasons can be hypothesized to explain the differences in results including that the circulating SARS-CoV-2 in Northern Italy and England during the first pandemic wave might have been genetically different with eventually a different capability to trigger GBS (13).
  Anyway, currently we are conducting a follow-up analysis on GBS incidence for the months following April 2020 and ISTAT data will be certainly taken into account and evaluated in order to obtain further findings on this debated area.

  References
  1. Filosto M, Cotti Piccinelli S, Gazzina S, et al. Guillain-Barré syndrome and COVID-19: an observational multicentre study from two Italian hotspot regions. J Neurol Neurosurg Psychiatry 2020; jnnp-2020-324837
  2. Rahimi K. Guillain-Barre syndrome during COVID-19 pandemic: an overview of the reports. Neurol Sci 2020; 41: 3149-3156
  3. Schenker N, Gentleman JF. On judging the significance of differences by examining the overlap between confidence intervals. The American Statistician 2001; 55:3, 182-186
  4. Capasso A, Ompad DC, Vieira DL, Wilder-Smith A, Tozan Y. Incidence of Guillain-Barre´ Syndrome (GBS) in Latin America and the Caribbean before and during the 2015–2016 Zika virus epidemic: A systematic review and meta-analysis. PLoS Negl Trop Dis 2019; 13: e0007622
  5. ISTAT, Ministero della Salute. Primi risultati dell’indagine di sieroprevalenza sul SARS-CoV-2. 2020; https://www.istat.it/it/files/2020/08/ReportPrimiRisultatiIndagineSiero.pdf
  6.Ministero della Salute, http://www.salute.gov.it/portale/news/p3_2_1_1_1.jsp?lingua=italiano&men...
  7.Isho B, Abe KT, Zuo M et al. Persistence of serum and saliva antibody responses to SARS-CoV-2 spike antigens in COVID-19 patients. Science Immunology 2020; 52: eabe5511
  8. Escribano P, Álvarez-Uría A, Alonso R, Catalán P, Alcalá L, Muñoz P, Guinea J. Detection of SARS-CoV-2 antibodies is insufficient for the diagnosis of active or cured COVID-19. Sci Rep 2020; 10: 19893
  9. Beghi E, Bogliun G. The Guillain-Barrè syndrome (GBS). implementation of a register of the disease on a nationwide basis. Italian GBS Study Group. Ital J Neurol Sci 1996; 17 :355–6
  10. Uncini A, Vallat J-M, Jacobs BC. Guillain-Barré syndrome in SARS-CoV-2 infection: an instant systematic review of the first six months of pandemic. J Neurol Neurosurg Psychiatry 2020; 91:1105–10
  11. Fantini J, Di Scala C, Chahinian H, et al. Structural and molecular modelling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against SARS-CoV-2 infection. Int J Antimicrob Agents 2020; 55: 105960
  12. Yuki N. Ganglioside mimicry and peripheral nerve disease Muscle Nerve 2007; 35: 691-711
  13. Pachetti M., Marini B., Benedetti F., Giudici F., Mauro E., Storici P. et al. Emerging SARS-CoV-2 mutation hot spots include a novel RNA-dependent-RNA polymerase variant. J Transl Med 2020; 18: 17

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  Conflict of Interest:
  None declared.

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• Published on: 12 November 2020

  Further research required to support a causative association between GBS and COVID-19

  • Stephen Keddie, Neurology SpR and Clinical Research Fellow Centre for neuromuscular disease, National Hospital for Neurology and Neurosurgery, UCLH, London.
  • Other Contributors:
    • Julia Pakpoor, Neurology SpR
    • Aisling Carr, Neurology Consultant
    • Michael P Lunn, Neurology consultant

  To the Editor

  We were interested to read the study of Filosto et al [1] concluding a significant link between Guillain-Barre Syndrome (GBS) and COVID-19 infection in Northern Italy at the peak of the 1st wave SARS-CoV2 pandemic. We urge caution in accepting such a causative conclusion using a retrospective observational study; causation is not conclusively proven and is drawn from potentially biased data and small case numbers of a rare condition, and a rate calculation without confidence intervals to infer uncertainty.

  Only 34 cases of GBS, of whom 30 were COVID-19 positive, are reported over a 2-month period, with a denominator population of 8,400,107. We calculated the 95% confidence intervals of the incidence rates as 0.08 per 100,000 per month (95% C.I.: 0.04-0.15) in 2019 and 0.2 per 100,000 per month (95% C.I.: 0.14-0.28) in 2020. The overlapping confidence intervals do not support a statistically significant increase in GBS rates from 2019 to 2020. Furthermore, the simple multiplication of the monthly rate by 12 to create an approximate annualised incidence potentially amplifies the inaccuracy. We suggest that the 2.6-fold difference in GBS incidence from 2019 to 2019 is prone to meaningful statistical error.

  During the initial stages of the pandemic the denominator of COVID-19 positive cases will have been under-reported because testing was limited to the symptomatic and presenting populations. We are told that 62,679 inhabitants of the regi...

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  To the Editor

  We were interested to read the study of Filosto et al [1] concluding a significant link between Guillain-Barre Syndrome (GBS) and COVID-19 infection in Northern Italy at the peak of the 1st wave SARS-CoV2 pandemic. We urge caution in accepting such a causative conclusion using a retrospective observational study; causation is not conclusively proven and is drawn from potentially biased data and small case numbers of a rare condition, and a rate calculation without confidence intervals to infer uncertainty.

  Only 34 cases of GBS, of whom 30 were COVID-19 positive, are reported over a 2-month period, with a denominator population of 8,400,107. We calculated the 95% confidence intervals of the incidence rates as 0.08 per 100,000 per month (95% C.I.: 0.04-0.15) in 2019 and 0.2 per 100,000 per month (95% C.I.: 0.14-0.28) in 2020. The overlapping confidence intervals do not support a statistically significant increase in GBS rates from 2019 to 2020. Furthermore, the simple multiplication of the monthly rate by 12 to create an approximate annualised incidence potentially amplifies the inaccuracy. We suggest that the 2.6-fold difference in GBS incidence from 2019 to 2019 is prone to meaningful statistical error.

  During the initial stages of the pandemic the denominator of COVID-19 positive cases will have been under-reported because testing was limited to the symptomatic and presenting populations. We are told that 62,679 inhabitants of the region had positive COVID-19 swabs during the study, but this is unlikely to provide an accurate population-based rate of COVID-19 infection. More specifically, given that hospital admissions were the only ascertainment source for GBS cases in this study, we are not given comparative information on the rate of COVID-19 positivity in the hospital population, which was likely to have been very high as the Northern Italy healthcare system was reportedly overwhelmed. It is possible, or probable, that a large proportion of the hospitalised population were COVID-19 positive independent of other factors. It is thus likely that these data report a coincidental association between COVID-19 and GBS rather than a causative one.

  The COVID-19 seroprevalence rate in the Italian Instituto Nazionale di Statistica (ISTAT) study for Lombardy was the highest in Italy at 7.5%. [2] From this rate, the estimated number of COVID-19 cases would be approximately 630,000 across the seven cities, compared to the 62 679 reported from swabs in the Filosto study. Using seroprevalence as the denominator, we believe the reported calculated incidence reduces from 47.9 to approximately 4.76 per 100,000 COVID-19 infections. The 7.5% seroprevalence may also be an underestimate because of selection bias (under half the planned sample size were tested) or imperfect sensitivity of the assay. It is striking that the seroprevalence reported in London from the NHS Blood Transfusion Service at a similar time was 17.5%;[3] Northern Italy, recognised as one of the hardest hit populations worldwide, seemingly had a seroprevalence of less than half that. Without major differences in health care structure and economic status of these two European countries it is difficult to explain the discrepancy other than the recorded seroprevalence figures were lower than actual infection rates.

  Importantly, no account is given for the dramatic 3.3-fold decline in non-COVID GBS to 0.29/100000 per year either. One potential reason for the apparent decline is the mis-attribution of GBS causation, where COVID-19 causation is applied to every COVID-19 positive GBS case. The authors do not explore the possibility of alternative causes of GBS and incidental COVID-19 infection. For example, four of the COVID-19 cases had gastrointestinal symptoms but causation was still attributed to COVID-19 despite a gastroenteritis being the commonest precursor of GBS and occurring less frequently as a COVID-19 specific feature. The authors have presented no data on the exclusion of other causes of GBS such as Campylobacter serology etc. and this is of relevance, as without excluding other causes as far as possible the possibility of misattribution again exists.

  Part of the justification Filosto et al make for the causative link is published data to support an interaction of the SarCoV2 spike protein with ganglioside GM1, referencing in silico modelling studies. [4] These studies model shape and energy efficient interactions of the Receptor Binding Domain (RBD) of the spike (S-)protein of SARS-CoV2 with the ACE2 Receptor. Convincing in silico predictive data of the subsequent available N-Terminal Domain of the S-Protein also indicate energy efficient potential binding sites for two GM1 molecules. However, these are models and interactions have not been shown to occur in or ex-vivo. Furthermore, there are no data to suggest that ACE2 receptors are present in peripheral nerve axons or myelin to provide the basis of binding to a colocalised ganglioside.

  GBS is a para- or more likely post-infectious autoimmune neuropathy, although acute polyradiculoneuropathies indistinguishable from classical Campylobacter jejuni enteritis associated GBS may occur by direct infection in Zika- and West Nile viruses and others. These cases of GBS occur with short latency whereas classical GBS occurs one to three (and possibly up to six) weeks after infection after an immune response induction. In the series of Filosto et al their median time from infection to GBS was 23 days (IQR 16-34), almost all within the COVID-19 symptom period (5 cases started after COVID symptoms resolved and thus are most consistent with a post-infectious timing). Thus, if it proposed that covalent binding to gangliosides is a mechanism for direct infection of peripheral nerves this would be considered too long a latency, and if an immune response is the key, then COVID-19 spike protein binding to a ganglioside is possibly irrelevant to the pathogenesis. Clearly more work is required to establish whether a definitive temporal relationship exists between COVID-19 and GBS, and more so the pathogenic mechanism which links infection to neuropathy.

  The Witebsky Postulates, with some minor modifications, have stood the test of time to provide support for autoimmune linkage of a pathogen to a disease.[5] None are really fulfilled here and we should be very cautious about drawing causative conclusions from observational studies of small numbers of patients where the prevalence of COVID-19 in the population is so high as to have affected very many people who may have been presenting to hospital anyway.
  Lastly, it is reassuring that the treatment and outcomes of patients are no different between COVID-19 positive and negative GBS patients and that given the lack of any spike in GBS presentations in Northern Italy (with the total incidence rate being 2.43/100000) we do not expect an additional pandemic of acute neuromuscular paralysis.

  Stephen Keddie, Julia Pakpoor, Aisling Carr, Michael P Lunn

  References

  1. Filosto M, Cotti Piccinelli S, Gazzina S, et al. Guillain-Barré syndrome and COVID-19: an observational multicentre study from two Italian hotspot regions. J Neurol Neurosurg Psychiatry 2020;0:jnnp-2020-324837. doi:10.1136/jnnp-2020-324837
  2. ISTAT, Ministero della Salute. Primi risultati dell’indagine di sieroprevalenza sul SARS-CoV-2. 2020;:10.
  3. Public Health England. Sero-surveillance of COVID-19. 2020.https://www.gov.uk/government/publications/national-covid-19-surveillanc... (accessed 13 Jul 2020).
  4. Fantini J, Di Scala C, Chahinian H, et al. Structural and molecular modelling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against SARS-CoV-2 infection. Int J Antimicrob Agents 2020;55. doi:10.1016/j.ijantimicag.2020.105960
  5. Witebsky E, Rose NR, Terplan K, et al. Chronic thyroiditis and autoimmunization. J Am Med Assoc 1957;164:1439–47. doi:10.1001/jama.1957.02980130015004

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  Conflict of Interest:
  We have a similar paper due to be published with larger numbers and have found opposing conclusions.

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