|DERMATOLOGY PRACTICE DURING COVID-19 PANDEMIC - ORIGINAL ARTICLES
|Year : 2021 | Volume
| Issue : 1 | Page : 12-19
Management of pemphigus vulgaris and bullous pemphigoid in times of COVID-19 pandemic
Tejaswini Salunke, Vinay Kulkarni, Dhanashree Bhide, Pradyumna Vaidya
Department of Dermatology and STD, Deenanath Mangeshkar Hospital, Pune, Maharashtra, India
|Date of Submission||23-Jun-2020|
|Date of Decision||08-Sep-2020|
|Date of Acceptance||23-Nov-2020|
|Date of Web Publication||19-Feb-2021|
Department of Dermatology and STD, Deenanath Mangeshkar Hospital and Research Centre, Pune - 411 004, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: The pandemic the world is facing today started in the city of Wuhan, central China, in December 2019, and presented as unexplained lower respiratory infections. On February 11, 2020, the World Health Organization announced that the disease caused by the severe acute respiratory syndrome coronavirus 2 virus be called “corona virus disease 2019 (COVID-19).” Since the beginning of the pandemic there has been quite some debate regarding starting or continuing the treatment of diseases requiring immunosuppression. The major issues arise with management of dermatological diseases, such as vesiculobullous disorders, moderate-to-severe psoriasis, acute severe drug reactions, and connective tissue diseases which would require high level of immune-suppression or immune-modulation as treatment. In this article, we discuss the management strategies adopted for the management of pemphigus vulgaris (PV) and bullous pemphigoid (BP) as the prototypical examples. We will discuss the algorithm we evolved in our department for managing such cases during COVID-19 pandemic to give optimum benefit for our patients along with decreasing their risk for increased vulnerability for severe COVID-19 infection. The same principle can be used for other diseases requiring immunosuppressive or immunomodulatory therapies. Objectives: The management of the patients suffering from BP and PV that came to our department during the national lockdown during COVID-19 pandemic. Materials and Methods: It was a prospective study of 30 consecutive patients that came to our outpatient department either previously diagnosed or as fresh cases of PV or BP. Results: Treating patients with BP or PV during the pandemic has been a challenge. Analyzing the risk versus benefit and making a custom-based treatment helped us minimize the risk for severe COVID-19 infection as well as the side effects due to the primary drugs that are used in BP/PV. We were successful in keeping the primary disease in control. None of our patients was infected with symptomatic COVID-19 infection. Conclusion: With rapidly changing scenario of the pandemic it is very difficult to assess the optimal management of complex diseases such as PV and BP. With contradictory data available whether suppressing the immunity will result in a milder form of COVID 19 with less complications or render the patient at higher risk of complicated COVID-19 is unknown. Thus we, as dermato-physicians, need to keep ourselves and our patients updated, educated, and choose the best possible option where in the risk for severe COVID-19 infection minimized while keeping their primary disease in control.
Keywords: Bullous pemphigoid, coronavirus disease 2019, pemphigus vulgaris, vesiculobullous diseases
|How to cite this article:|
Salunke T, Kulkarni V, Bhide D, Vaidya P. Management of pemphigus vulgaris and bullous pemphigoid in times of COVID-19 pandemic. Clin Dermatol Rev 2021;5:12-9
|How to cite this URL:|
Salunke T, Kulkarni V, Bhide D, Vaidya P. Management of pemphigus vulgaris and bullous pemphigoid in times of COVID-19 pandemic. Clin Dermatol Rev [serial online] 2021 [cited 2021 Mar 1];5:12-9. Available from: https://www.cdriadvlkn.org/text.asp?2021/5/1/12/309750
| Introduction and Background|| |
The pandemic the world is facing today started in the city of Wuhan, central China, in December 2019, and presented as unexplained lower respiratory infections. On February 11, 2020, the World Health Organization announced that the disease caused by the virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus be called “corona virus disease 2019 (COVID-19),” which is the acronym of “COVID-19.”
As of June 17, 2020, more than 8,339,268 people have tested positive for the virus and 448,388 have died (case fatality rate of about 5.7%) all over the world. Although majority of infected people have mild or moderately severe symptoms; those with more severe disease have severe respiratory failure resulting in pneumonitis and death. In India, the first case of COVID-19 was detected on January 30, 2020.
SARS-CoV-2 belongs to the same family of viruses that caused SARS epidemic in 2002 and Middle East respiratory syndrome in 2018. It is a zoonotic infection primarily affecting bats. It is mostly spread via droplet infection. Another less common mode of transmission is through surface to humans. It causes a milder form or asymptomatic presentation in healthy individuals. However, in individuals with comorbidities, it may cause severe illness and acute respiratory distress syndrome (ARDS) that may lead to death. The overall case fatality rate is variable in different countries from 8% in Italy to 1%–2% in India.
The risk factors for severe illness and mortality have been identified as being of old age, obese, male gender, and presence of comorbidities (hypertension, diabetes, chronic respiratory diseases, and cardiovascular problems). The pathogenesis, pathology, and understanding of how the virus attacks the immune system is still evolving. It is generally believed that in early course it is the viral infection that dictates the clinical picture while in the later part it is due to overwhelming immunological response by the host manifesting as cytokine storm. Comorbidities and other risk factors could be contributing to this type of immunological response.
COVID-19 is not primarily a dermatotrophic virus. Skin manifestations have been described in minority of patients such as fever with urticarial, exanthematous, or varicella such as vesicular rashes and COVID toes. Other cutaneous manifestations include features resembling Kawasaki disease. It has been postulated that diseases with epidermal barrier interruption could enhance the virus acquisition through indirect contact. Hence, dermatology patients might be at an increased risk for acquiring the infection. However, this has not yet been established. By and large dermatologists are not the frontline health workers dealing with COVID-19 pandemic and are not involved in the management of these cutaneous manifestations due to COVID-19.
In these difficult times, dermatology outpatient (OPD) practice is grossly affected and footfall is reduced due to state enforced lockdowns as well as fear in the minds of patients. It has become necessary for dermatologists to revise the strategies for treating patients with various immunosuppressive agents as well as have modified protocols for performing various dermatosurgical and esthetic procedures. The major issues arise with the management of dermatological diseases, such as vesiculobullous disorders (VBD), moderate-to-severe psoriasis, acute severe drug reactions and connective tissue diseases which would require high level of immune-suppression or immune-modulation as treatment. If these patients on immunosuppressive drugs get COVID-19, they are thought to be more likely to have severe illness and a possibility of worse outcomes. Since the beginning of the pandemic, there has been quite some debate regarding starting or continuing the treatment of diseases requiring immune-suppression. These cases have also been the ones that have sought emergency care even during the lockdown periods.
In this article, we discuss the management strategies adopted for the management of pemphigus vulgaris (PV) and bullous pemphigoid (BP) as the prototypical examples. We present the algorithm we evolved in our department for managing such cases during COVID-19 pandemic. During the nationwide lockdown period, these were the most common auto-immune VBD seen in our outpatient department. Although BP is a milder disease as compared to PV, which requires higher level of immunosuppression, it usually affects relatively older population. This age group is at a higher risk of having severe COVID-19 and immunosuppression can add to the risk. The older age group majority of the patients are also likely to have other comorbidities, such as diabetes mellitus (DM), hypertension, ischemic heart disease; thereby increasing their vulnerability. The treatment of VBD revolves around immune suppression with systemic corticosteroids (SCS) often combined with azathioprine (Aza), cyclophosphamide (CP), and mycophenolate mofetil (MMF), methotrexate (MTX), or immune-modulators such as dapsone, tetracyclines, nicotinamide, and intravenous immunoglobulin G (IVIG). In recent, past Rituximab (RTX) has emerged as a first-line regimen in treating pemphigus.
In the COVID-19 era choosing the right molecule in the newly diagnosed cases as well as continuing existing medicines in already diagnosed cases is a difficult decision for the treating dermatologist. A balanced approach by weighing the risks against the benefits is necessary. Inadequate or subtherapeutic doses can take longer time to achieve disease remission. Impaired skin barrier makes these patients prone to secondary bacterial infection and septicemia. This itself can be the cause of increased morbidity and mortality. In addition, the number of visits to OPD as well as possibility of indoor admission can increase and this can be an additional factor for increased exposure to COVID-19 environment.
| Materials and Methods|| |
Since the beginning of the pandemic, we treated 30 patients of VBD. (20 PV, 10 BP), of which 6 patients required indoor care [Table 1]. All patients that came to our department were given treatment on basis of their disease activity irrespective of age, gender, disease distribution, severity, or morbidity. There was no exclusion criteria.
|Table 1: Tabular presentation of demographic details, comorbidities, diagnosis and treatment and treatment outcomes at our department|
Click here to view
Every case was carefully evaluated on the following principles:
- In all new cases, diagnosis was achieved with histopathological examination and direct immunofluorescence testing. In case of PV dsg1 and dsg3 titers to check the activity of the disease were obtained
- Severity was scored using autoimmune bullous skin disorder intensity score and classified as mild, moderate, or severe
- Laboratory investigations – Hemogram, liver function tests, renal function tests, blood sugars, triple test (anti-HIV1 and 2, Hepatitis C Virus and Hepatitis B surface antigen), Montoux test, chest X-ray, ultrasonogram, etc., were done to evaluate other comorbidities and level of fitness of the patient
- Based on the severity treatment was planned
- Detailed discussion and counseling of the patient and the family regarding the pros and cons of the said plan in this pandemic era was done and treatment plan decision was taken jointly
- Special emphasis was given on adequate personal protection and need to stay indoors all the time
- Treatment was started after informed consent was obtained
- If the patient stabilized and responded to treatment, visits to the hospital were deferred and online/virtual consultations were advised
- Laboratory reports and follow-up clinical photographs were viewed over email or WhatsApp®.
During this pandemic and lockdown period, we came across five scenarios:
- Patients who were treated with RTX (Rheumatoid Arthritis [RA] protocol) >1 year ago with or without flare
- Patients who were treated with RTX (RA protocol) <1 year with or without flare
- Patients who had not completed their RTX (RA protocol) regimen
- Previously treated patients who were not primarily on RTX-based treatment with or without flare
- Newly diagnosed patients.
Our patients (n = 30) were in the age group of 7–78 years with most of them suffering from DM, hypertension, and cardiovascular issues. Seventeen of them were in the high risk category for COVID-19 infection.
| Results|| |
Treating patients with BP or PV during the pandemic was a challenge. Analyzing the risk versus benefit and making a custom-based treatment helped us minimize the risk for COVID-19 infection as well as side effects due to the primary drugs that are used in BP/PV.
We have so far been successful in keeping the primary disease in control in all cases. None of our patients have so far been infected with symptomatic COVID-19 infection. Table 1 shows the treatment outcomes of all the patients treated during the lockdown period of COVID-19.
| Discussion|| |
In pre COVID-19 era chimeric anti-CD20 monoclonal antibody – RTX had evolved as a first-line treatment at our department as it required less adjunctive therapy and minimum use of SCS with faster relief. We deferred treatment with RTX during this period. Although a single RTX infusion does not seem to impair memory responses against known pathogens, it does exert a defective immune reaction against new pathogens. In general, immune response against cytopathic viruses is heterogeneous and an efficient intervention of innate and cellular immunity is fundamental to combat the early phase of the virus. The characteristics of immune response to COVID-19 is not well understood and the risk of knocking down B-cell immunity completely and for prolonged period is not recommended in the present scenario. Reasonable risk of viral activation has been previously described with RTX. RTX has its immunosuppressive effects that lasts for at least a year and the same is irreversible rendering the patient highly susceptible to many infections including COVID-19.
Weighing the risk-benefit ratio, we decided to stick to our traditional treatment with SCS. In contrast to PV, BP may remit spontaneously and can often be treated with lower doses of immunosuppressant. We preferred to avoid the usual pulse regimen which used methylprednisolone 1 g for 3–5 days in moderate-to-severe cases and used methylprednisolone 500 mg for 3–5 days based on case to case analysis. The principle that we followed was to use short-acting non-biological immunosuppressants like SCS to initially control the activity of the diseases instead of using large molecules like RTX which would have prolonged and irreversible pan-immunosuppression. The use of algorithmic approach [Figure 1] and [Figure 2] helped us categorize the severity and activity of the disease, associated risks, comorbidities etc., and give maximum results to the patient without increasing the risk ratio. SCS definitely improve the prognosis, reduce activity of the disease but in the long run increase risk to infections and also have a list of its own systemic side effects., SCS on one hand inhibit the immune response and delay the clearance of pathogen while on the other hand they suppress the host inflammatory response, which in the case of viral infection of respiratory tract is the mostly responsible for lung damage and occurrence of ARDS. Our strategy was to immune suppress the patient only as much to control the activity of the disease with minimum side effects and risk reduction for COVID-19.
|Figure 1: Algorithm for management of pemphigus vulgaris in times of corona virus disease 2019 pandemic|
Click here to view
|Figure 2: Algorithm for management of bullous pemphigoid in times of corona virus disease 2019 pandemic|
Click here to view
Other steroid sparing agents were used as an adjunctive therapy to SCS. We decided the same group of drug and increased or decreased the dosage as per requirement. They too were tapered off on basis of individual patient response. Studies show that steroid sparing agents when used in combination lead to a reduction in risk of relapse. There are various clinical trials studying antiviral actions against COVID-19 of MMF. Interestingly, MMF could act as a synergistic drug to potentiate the anti-viral effects of other drugs, through the depletion of the endogenous GTP pools but there is no data on in-vivo trials. There is no clear data on safety of Aza, MTX in COVID-19 times but Queiroz et al. in their study say that there is no evidence of increased risk of COVID-19 infection and that there are no short-term benefits of stopping these medications as they take months to clear their immunosuppressing effect. There is a hypothesis that if at all a strategy using CP is found effective in mitigating/preventing SARSCoV-2-associated ARDS, it is plausible that it could be put into immediate global use.
Doxycycline was used in combination with topical corticosteroids (TCS) in mild cases of BP. Various studies show that tetracyclines have an anti-inflammatory and possible neuroprotective properties that prove useful in BP. There is also a hypothesis where doxycycline in combination with Dapsone can reduce cytokine storm in COVID-19-infected patients.
TCS from the super-potent group like clobetasol propionate are one of the first line therapies for treating localized BP which reduced the burden of SCS and steroid sparing agents.
Paraffin-embedded tulle nets that minimize accidental shearing were used for dressing for patients with large denuded areas, and helps re-epithelization.
Maintaining hygiene of mucosal surfaces is of prime importance to avoid any secondary infections. Intralesional corticosteroid injections can be useful in cases with persistent localized skin lesions. Burn hoods can be used over denuded areas so as to avoid peeling of skin every time the patient changes position or due to friction of clothes and bedding. Co-care with physician in cases where comorbidities such as DM and hypertension exist was highly essential.
All patients were advised to follow all the COVID-19 preventive precautions. They were advised to keep a watch for any fever, cough, breathlessness, and flu-like symptoms and report to the nearest emergency department.
As regards approach deciding higher risk individuals with respect to COVID-19, there are two schools of thought where in the Centers for Disease Control and Prevention considers immunocompromised patients such as those on cancer treatment, HIV-infected persons, transplant patients and those who are on long-term corticosteroids belong to the high risk population. On the other hand, Mehta et al. consider hyperinflammatory syndrome and other cytokine storm syndromes (secondary hemophagocytic lymphohistiocytosis) characterized by increased levels of releases interleukin (IL)-1β as the major cause of morbidity and mortality. Other pro-inflammatory cytokines are also secreted such as IL-6, IL-7, IL-10, interferon gamma, monocyte chemoattractant protein 1, inducible protein-10, granulocyte-colony stimulating factor, and tumor necrosis factor. Thus, this hypercytokinemia with multi-organ failure is thought to be main pathophysiology for high fatality rate due to COVID-19. In fact, she believes that immunosuppression is likely to be beneficial and can reduce or minimize the adverse effects of COVID-19 infection. A study carried out by Fadel et al. on 213 hospitalized patients with COVID-19 concluded that early short course methylprednisolone reduced escalation of care and improved clinical outcomes. There are studies that suggest that immunosuppressed patients are at not at increased risk of severe complications compared with general population. However, we feel that jury is still out, and clearer picture would emerge as the epidemic matures. We have adopted a more conservative approach.
Host response and immunity is an important contributor to disease process in any infection. In this respect, dysregulated and excessive immune responses appear to be important drivers of tissue damage during COVID-19 infection. These aspects may be relevant when it comes to infection of an immunocompromised host, potentially protected by a weaker immune response against the infection. None of our patients have so far been infected with COVID-19 yet. However, when times comes, depending on the severity and through decision tree steroid sparing immunomodulation may be stopped, and patients continued on SCS. Effective as adjuvant treatment in both PV and COVID-19, IVIG could be considered in such a scenario.
| Conclusion|| |
With rapidly changing scenario of the COVID-19 pandemic, it is very difficult to assess the optimal management of complex diseases such as PV and BP. They are chronic autoimmune inflammatory disorders that carry a higher risk of infection than the general population. However, poor control of the primary disease would pose as an even greater risk of disease flare with super added secondary infections. We strongly recommend avoiding or postponing the use large molecules like RTX that cause pan-immune suppression and turn to SCS as a bridge therapy in doses required as per the disease severity. When healthy immunocompetent host also can have a severe form of infection and unpredictable outcomes, it is very difficult to assess the risk coefficient in immunosuppressed patients. With contradictory data available whether suppressing the immunity will result in a milder form of COVID-19 with less complications or render the patient at higher risk of complicated COVID-19 is unknown. Thus, we as dermato-physicians need to keep ourselves and our patients updated, educated, and choose the best possible option where in the risk factors for COVID-19 infection is minimum along with keeping their primary disease in control.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form the patient (s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initial s will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
NCBI Bookshelf. Service of the National Library of Medicine, National Institutes of Health;2020.
Tao J, Song Z, Yang L, Huang C, Feng A, Man X. Emergency management for preventing and controlling nosocomial infection of the 2019 novel coronavirus: implications for the dermatology department. Br J Dermatol.2020;182(6):1477-78.
Singhal T. A review of coronavirus disease-2019 (COVID-19). Indian J Pediatr 2020;87:281-6.
Yang J, Zhang Y, Gou X, Pu K, Chen Z, Guo Q, Ji R Wang H, Wang Y, Zhou Y. Prevalence of comorbidities and its effects in patients infected with SARS-CoV-2: a systematic review and meta-analysis. Int J Infect Dis 2020;94:91-95.
Sachdeva M, Gianotti R, Shah M, Bradanini L, Tosi D, Veraldi S, et al. Cutaneous manifestations of COVID-19: Report of three cases and a review of literature. J Dermatol Sci. 2020;98(2):75-81.
Tao J, Song Z, Yang L, Huang C, Feng A, Man X. Emergency management for preventing and controlling nosocomial infection of the 2019 novel coronavirus: Implications for the dermatology department. Br J Dermatol 2020;182:1477-8.
Liu YD, Wang YH, Ye YC, Zhao WL, Li L. Prognostic factors for mortality in patients with bullous pemphigoid: A meta-analysis. Arch Dermatol Res 2017;309:335-47.
Joly P, Maho-Vaillant M, Prost-Squarcioni C, Hebert V, Houivet E, Calbo S, et al
. First-line rituximab combined with short-term prednisone versus prednisone alone for the treatment of pemphigus (Ritux 3): A prospective, multicentre, parallel-group, open-label randomised trial. Lancet 2017;389:2031-40.
Cho A, Bradley B, Kauffman R, Priyamvada L, Kovalenkov Y, Feldman R, et al
. Robust memory responses against influenza vaccination in pemphigus patients previously treated with rituximab. JCI Insight. 2017;2(12):e93222. 6doi:https://10.1172/jci.insight.93222. PMID: 28614800
Kasperkiewicz M, Eming R, Behzad M, Hunzelmann N, Meurer M, Koops SH, et al
. Efcacy and safety of rituximab in pemphigus: Experience of the German registry of autoimmune diseases. J Dtsch Dermatol Ges 2012;10:727-33.
Khurana A, Pandhi D. IADVL position statement and recommendations on post lockdown dermatology practice amidst the Covid-19 pandemic(IADVL Academy and IADVL Executive Committee). Indian Dermatol Online J 2020;11:520-5. [Full text]
Gupta L, Misra DP, Agarwal V, Balan S, Agarwal V. Management of rheumatic diseases in the time of covid 19 pandemic:perspectives of rheumatology practionera from India. Ann Rheum Dis.2021;80(1):e1. Doi: https://10.1136/annrheumdis-2020-217509
Cooper SM, Wojnarowska F. Treatments of choice for bullous pemphigoid. Skin Therapy Lett 2002;7:4-6.
Gheisari M, Faraji Z, Dadras MS, Nasiri S, Robati RM, Moravvej H, et al
. Methylprednisolone pulse therapy plus adjuvant therapy for pemphigus vulgaris: An analysis of 10 years' experience on 312 patients. Dermatol Ther 2019;32:e13057.
Shahidi-Dadras M, Pishgahi M, Tabary M, Kheradmand Z, Araghi F, Dadkhahfar S, et al. Cardiac function in pemphigus vulgaris patients before and after steroid pulse therapy. J Dermatolog Treat. 2020 Jan 3:1-5. doi:
. PMID: 31868046.
Tang NL, Chan PK, Wong CK, To KF, Wu AK, Sung YM, et al
. Early enhanced expression of interferon-inducible protein -10(CXCL-10) and other chemokiniespredicts adverse outcome in acute severe respiratory syndrome. Clin Chem 2005;51:2333-40.
Atzmony L, Hodak E, Leshem YA, Rosenbaum O, Gdalevich M, Anhalt GJ, et al
. The role of adjuvant therapy in pemphigus: A systematic review and meta-analysis. J Am Acad Dermatol 2015;73:264-71.
Perriconea C, Triggianeseb P, Bartolonia E, Cafaroa G, Bonifacioa AF, Bursia R, et al. The anti-viral facet of anti-rheumatic drugs: Lessons from COVID-19. J Autoimmunity 2020;1:111. Doi: https://10.1016/j.jaut.2020.102468
Neyts J, Andrei G, DeClercq E. The novel immunosuppressive agent mycophenolate mofetil markedly potentiates the antiherpesvirus activities of acyclovir, ganciclovir, and penciclovir in vitro
and In vivo
. Antimicrob Agents Chemother 1998;42:216-22.
Queiroz NS, Barros LL, Azevedo MF, Oba J, Sobrado CW, Carlos AS, et al
. Management of inflammatory bowel diseases patinets in the COVID 19 pandemic era: A Brazilian tertiary referral center guidance. Clin 2020;75:e1909.
Revannasuddaiah S, Kumar Devadas S, Palassert R, Kumar Pant N, Maka VV. A potential role for cyclophosphamide in the mitigation of acute respiratory distress syndrome among patients with SARS-CoV-2. Med Hypotheses. 2020;144:109850. Doi: https://10.1016/j.mehy.2020.109850
Fivenson DP, Breneman DL, Rosen GB, Hersh CS, Cardone S, Mutasim D. Nicotinamide and tetracycline therapy of bullous pemphigoid. Arch Dermatol 1994;130:753-8.
Farouk A, Salman S. Dapsone and doxycycline could be potential treatment modalities for COVID-19. Med Hypotheses 2020;140:109768. Doi https://1016/j.mehy.2020.109768
Mutalik Sharad D, Yashashree R. Paraffin-embedded tulle nets as a simple dressing technique for patients with pemphigus. J Am Acad Dermatol Int 2020;1:11-2.
Mehta P, McAuley DF, Brown M, Sanchez E, Tattersall RS, Manson JJ, et al
. COVID-19: Consider cytokine storm syndromes and immunosuppression. Lancet 2020;395:1033-4.
Fadel R, Morrison AR, Pharm D, Vahia A, Smith ZR, Pharm D, et al. COVID 10 management task force, early hort course corticosteroids in hospitalized patients with COVID-19. Clin Infect Dis 2020;71(16):2114-20.
D'Antiga L. Coronaviruses and immunosuppressed patients: The facts during the third epidemic, wileysonlinelibrary. Liver Transpl 2020;26:832-4.
Shakshouk H, Daneshpazhooh M, Murrell DF, Lehman JS. Treatment considerations for patients with pemphigus during the COVID 19 pandemic, J Am Acad Dermatol 2020;82(6):e235-e236. Doi: https://10.1016/j.jaad.2020.04.005
[Figure 1], [Figure 2]