Inflammatory Skin Lesions in Three SARS-Cov-2 Swab-Negative

Article Inﬂammatory Skin Lesions in Three SARS-CoV-2 Swab-Negative Adolescents: A Possible COVID-19 Sneaky Manifestation?

Giuseppe Ingravallo 1,* , Francesco Mazzotta 2, Leonardo Resta 1 , Sara Sablone 3 , Gerardo Cazzato 1 , Antonietta Cimmino 1, Roberta Rossi 1, Anna Colagrande 1, Beniamino Ferrante 2, Teresa Troccoli 2 and Ernesto Bonifazi 4

1 Department of Emergency and Organ Transplantation, Section of Pathology, University of Bari Aldo Moro, 11, 70124 Bari, Italy; [email protected] (L.R.); [email protected] (G.C.); [email protected] (A.C.); [email protected] (R.R.); [email protected] (A.C.) 2 Pediatric Dermatology and Surgery Outpatients Department, Azienda Sanitaria Locale Barletta-Andria-Trani, 76123 Andria, Italy; [email protected] (F.M.); [email protected] (B.F.); [email protected] (T.T.) 3 Section of Forensic Medicine, Department of Interdisciplinary Medicine, University of Bari Aldo Moro, 70124 Bari, Italy; [email protected] 4 Pediatric Dermatology, University of Bari Aldo Moro, 11, 70124 Bari, Italy; [email protected] * Correspondence: [email protected] Abstract: Coronavirus disease 19 (COVID-19), caused by severe acute respiratory syndrome coro- Citation: Ingravallo, G.; Mazzotta, F.; navirus 2 (SARS-CoV-2), is associated with various clinical manifestations, including skin lesions. Resta, L.; Sablone, S.; Cazzato, G.; In particular, during the COVID-19 pandemic lock-down period numerous chilblain-like lesions, Cimmino, A.; Rossi, R.; Colagrande, mainly located on the feet, were observed in adolescents. The latter were often asymptomatic or A.; Ferrante, B.; Troccoli, T.; et al. associated with very mild respiratory symptoms. Here, we report three cases of acral nodular lesions Inflammatory Skin Lesions in Three in SARS-CoV-2 swab-negative adolescents with histological findings of chronic immune-mediated SARS-CoV-2 Swab-Negative inflammation and immunohistochemical evidence of SARS-CoV-2 spike glycoproteins in endothe- Adolescents: A Possible COVID-19 Sneaky Manifestation? lial cells and eccrine sweat glands. In one of these cases, the virus presence was confirmed by Pediatr. Rep. 2021, 13, 181–188. electron microscopy. https://doi.org/10.3390/ pediatric13020025 Keywords: SARS-CoV-2; skin; granuloma annulare; chilblains

Academic Editor: Maurizio Aricò

Received: 12 March 2021 1. Introduction Accepted: 7 April 2021 During the COVID-19 pandemic, increasing numbers of skin lesions have been de- Published: 9 April 2021 scribed in positive individuals [1–4]. Various reports [1–6] have offered clinical documen- tation of cutaneous lesions in adults, such as Erythema Pernio, Erythema Multiforme, Publisher’s Note: MDPI stays neutral Chilblains, Urticated Erythema, Morbilliform, Varicelliform, Chickenpox-like exanthem, with regard to jurisdictional claims in etc. In the pediatric population, the incidence of skin manifestations reported by vari- published maps and institutional affil- ous authors is low: 0.25% [7,8]. Biopsies from skin lesions in children with confirmed iations. or suspected COVID-19 have rarely been described in the literature [8]. Most of the histopathological descriptions were of isolated cases or small series, and some types of lesions have been biopsied only in adults. In general, the histopathological patterns were similar to chilblains [9,10], maculopapular eruptions [11,12], Erythema Multiforme [13,14], Copyright: © 2021 by the authors. and purpuric and livedoid forms [15–17]. Licensee MDPI, Basel, Switzerland. In this brief article, we present three cases of SARS-CoV-2 swab-negative adolescents, This article is an open access article with no flu-like symptoms in the previous months, who developed nodular lesions of the distributed under the terms and feet reminiscent of deep granuloma annulare. A thorough histopathological analysis was conditions of the Creative Commons carried out in these cases. Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Pediatr. Rep. 2021, 13, 181–188. https://doi.org/10.3390/pediatric13020025 https://www.mdpi.com/journal/pediatrrep Pediatr. Rep. 2021, 13, FOR PEER REVIEW 2 Pediatr. Rep. 2021, 13 182

2.2. CaseCase PresentationPresentation CaseCase 1:1:An An 11-year-old11-year-old boyboy waswasobserved observedfor formultiple multiple swelling swelling of of the the feet feet associated associated withwith mildmild burning.burning. HisHis familyfamily andand personalpersonal historyhistory waswas negativenegative forfor connectiveconnective tissue,tissue, autoimmuneautoimmune oror otherother noteworthynoteworthy diseases.diseases. AA physicalphysical examinationexamination showedshowed 7–87–8 lesions lesions (Figure(Figure1 )1) on on the the lateral lateral surface surface of of the the feet, feet, that that were were slightly slightly erythematous erythematous and and of of a a hard- hard- elasticelastic consistency.consistency. The The nodules nodules were were not not painful painful onon palpation,palpation, mobilemobile onon thethe deepdeep plane,plane, withwith aa diameterdiameter rangingranging betweenbetween 66 andand 1212 mm,mm, andand hadhad appearedappeared aboutabout 2020 daysdays earlier.earlier. DueDue toto thethe uncertainuncertain clinicalclinical features,features, wewe decideddecided toto performperform aa biopsy,biopsy, afterafter molecular molecular testingtesting for for SARS-CoV-2 SARS-CoV-2 resulted resulted negative. negative. A A histological histological examination examination (Figure (Figure2 a)2a) showed showed acralacral skinskin withwith mildmild hypergranulosis,hypergranulosis, acanthosis,acanthosis, andand focalfocal vacuolizationvacuolization ofof thethe basalbasal keratinocytes.keratinocytes. BelowBelow thethe dermo-epidermaldermo-epidermal junctionjunction therethere was was ectasia ectasia of of the the vessels vessels of of the the superﬁcialsuperficial capillarycapillary plexus,plexus, thatthat presentedpresented aa narrow narrow lumen lumen with with protruding protruding endothelial endothelial cellscells (hobnail).(hobnail). AccentuatedAccentuated dermo-hypodermicdermo-hypodermic mucinosismucinosis coexistedcoexisted with with fragmentation fragmentation oror eveneven dissolutiondissolution of of the the collagencollagen and and elasticelastic ﬁbers fibers (Figure (Figure2 b).2b). The The nodules nodules were were treated treated withwith topical topical corticosteroids corticosteroids and and subsided subsided in in about about 4 4 months. months. A A serological serological test test carried carried out out afterafter 33 andand aa halfhalf monthsmonths was was positive positive for for IgG. IgG.

FigureFigure 1.1. AsymptomaticAsymptomatic firm ﬁrm nodules nodules measuring measuring 2–3 2–3 cm cm in in diameter diameter on on the the lateral lateral aspect aspect of ofthe the leftleft foot. foot.

CaseCase 2:2: A 14-year-old 14-year-old boy, boy, already already known known since since he he suffers suffers from from persistent persistent atopic atopic der- dermatitis,matitis, was was observed observed during during the pandemic the pandemic due to due exacerbation to exacerbation of his dermatitis. of his dermatitis. During Duringthe examination the examination we observed we observed the presence the presence of asymptomatic of asymptomatic nodules, nodules,clinically clinicallycompara- comparableble to those toof thosecase 1, of which case 1, ha whichd appeared had appeared about 20 about days 20earlier. days earlier. Therefore,Therefore, we we performed performed a puncha punch biopsy. biopsy. A histological A histological examination examination showed showed orthoker- or- atosis and mild acanthosis. Immediately below the dermal-epidermal junction, the vessels thokeratosis and mild acanthosis. Immediately below the dermal-epidermal junction, the of the superficial capillary plexus presented a narrow lumen with protruding endothelial vessels of the superficial capillary plexus presented a narrow lumen with protruding en- cells (hobnail), sometimes completely occluding the vessel lumens. In the middle and deep dothelial cells (hobnail), sometimes completely occluding the vessel lumens. In the middle dermis an ample area of edema, with extensive and marked fragmentation or even dissolu- and deep dermis an ample area of edema, with extensive and marked fragmentation or tion of the collagen and elastic fibers, was evident, as well as thrombotic foci (Figure2c) of even dissolution of the collagen and elastic fibers, was evident, as well as thrombotic foci the small vessels, which showed sclero-hyalinosis of the wall and hemorrhagic suffusion. (Figure 2c) of the small vessels, which showed sclero-hyalinosis of the wall and hemor- There were also fibroblasts of a likely reactive significance around extensive, widespread rhagic suffusion. There were also fibroblasts of a likely reactive significance around ex- dermal mucinosis. A serological test carried out after 4 months was positive for IgG. tensive, widespread dermal mucinosis. A serological test carried out after 4 months was Case 3: An 11-year-old girl, with no notable medical history, developed acro-localized positive for IgG. nodular lesions similar to those of cases 1 and 2 during the pandemic period. We again Case 3: An 11-year-old girl, with no notable medical history, developed acro-local- performed a punch biopsy. A histological examination showed acanthosis and mucinosis ized nodular lesions similar to those of cases 1 and 2 during the pandemic period. We

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mucinosis as in case 2. The vessels showed a narrow lumen with protruding endothelial cells andas there in case was 2. also The sclero-hyalinosis vessels showed of a narrowthe wall lumen and hemorrhagic with protruding suffusion. endothelial cells and Methods.there was The also samples sclero-hyalinosis were fixed ofin the 20% wall formalin and hemorrhagic for light microscopy suffusion. and after processing, Methods.paraffin-embeddingThe samples and were microtom fixed ine cutting, 20% formalin sections for were light prepared microscopy for and after routineprocessing, staining with paraffin-embedding Hematoxylin-Eosin. and microtome cutting, sections were prepared for routine Then,staining 5 µm with thick Hematoxylin-Eosin. sections of all cases were prepared for immunostaining with anti- SARS-CoV-2Then, Spike 5glycoproteinµm thick sections S1 monoclonal of all cases antibody were prepared(ThermoFisher) for immunostaining for Coronavirus with anti- (MA5-36247),SARS-CoV-2 with 100 Spike µg at glycoprotein 1 mg/mL concentration, S1 monoclonal isotype: antibody IgG. (ThermoFisher) Appropriate negative for Coronavirus and positive(MA5-36247), controls were with included 100 µg at in 1 mg/mLthe procedure concentration, (shown in isotype: Supplementary IgG. Appropriate Material). negative In andaddition, positive a small controls portion were of included the sample in the of procedure.only one case was post-fixed with 1% In addition, a small portion of the sample of only one case was post-fixed with 1% osmium tetroxide in PBS for 2 h at 4 °C for electronic◦ microscopy. The fixed specimens were processedosmium for tetroxide embedding in PBS in Epoxy-Resi for 2 h at 4n-AralditeC for electronic (M) CY212 microscopy. (TAAB, Aldermaston, The fixed specimens UK). Semi-thinwere processed (two micron for embedding thick) sections in Epoxy-Resin-Araldite were stained with toluidine (M) CY212 blue. (TAAB, Ultrathin Aldermaston, sectionsUK). were Semi-thin mounted (twoon formvar-coated micron thick) nick sectionsel grids were and stained routinely with stained toluidine with blue.uranyl Ultrathin acetate sectionsand lead were citrate. mounted Images on of formvar-coated the semi-thin sections nickel grids were and captured routinely using stained a Nikon with uranyl photomicroscopeacetate and equipped lead citrate. with Imagesa Nikon ofDigital the semi-thin sight DS-U1 sections camera were (Nikon captured Instruments using a Nikon SpA, Calenzano,photomicroscope Italy). equippedThe ultra-thin with asections Nikon Digitalwere observed sight DS-U1 using camera a transmission (Nikon Instruments electronSpA, microscope Calenzano, (Morgagni Italy). The 268 ultra-thin, FEI Company, sections Naples, were observedItaly). using a transmission electron microscope (Morgagni 268, FEI Company, Naples, Italy). 3. Results 3. Results Immunohistochemical analysis was done in all three cases using the SARS-CoV-2 Immunohistochemical analysis was done in all three cases using the SARS-CoV-2 anti- anti-spike proteins antibody, yielding positive results in the eccrine sweat glands (Figure spike proteins antibody, yielding positive results in the eccrine sweat glands (Figure3. 3.

(a)

(b)

Figure 2. Cont.

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(b)

(c)

Figure 2. (a) The microphotograph shows orthokeratosis and mild acanthosis (acral skin), a mild Figure 2. (a) The microphotograph shows orthokeratosis(c )an d mild acanthosis (acral skin), a mild lymphomonocytelymphomonocyte infiltrate infiltrateand myxoid and stroma myxoid of stroma the dermis. of the Hematoxylin-Eosin dermis. Hematoxylin-Eosin staining, staining,origi- original nal magnificationFiguremagnification 2. (a )100×. The 100microphotograph(b)× In.( theb) In medial the medial deep shows deepdermis, orthokeratosis dermis, an ampl an amplee an aread mild areaof edema ofacanthosis edema is evident, is (a evident,cral with skin), with a mild extensive extensivelymphomonocyteand and accentuatedaccentuated infiltrate fragmentation and myxoid or or even even stroma disso dissolution oflution the dermis. of of the the collagen Hematoxylin-Eosin collagen and and elastic elastic fibers staining, fibers (the (theorigi- latter latter confirmednalconfirmed magnification by the by Orceina the 100×. Orceina (b staining),) In the staining), medial togethertogether deep with dermis, the with presence an the ampl presence ofe areathrombotic ofof edema thrombotic foci is in evident, the foci in with the small small vessels.extensivevessels. (Hematoxylin-Eosin and (Hematoxylin-Eosin accentuated fragmentation staining, staining, original original or even magnification, magnification, dissolution 400×, of 400 the× with ,collagen with orcein orcein and staining staining elastic in fibers in the (the insert). the insert).latter(c )( Detailcconfirmed) Detail of of a thrombosed bya thrombosed the Orceina blood blood staining), vessel vessel together together together with with with the the the presence presence presence of of fibroblasts ofthrombotic fibroblasts of foci a of likely ain the “reactive” likely "reactive"smallsignificance vessels. significance (Hematoxylin-Eosin around around extensive, extensive, widespread staining, widespread original dermal magnification,dermal mucinosis. mucinosis. Hematoxylin-Eosin 400×, Hematoxylin-Eosin with orcein staining, staining originalin staining, original magnification 400×. themagnification insert). (c) Detail 400× of. a thrombosed blood vessel together with the presence of fibroblasts of a likely "reactive" significance around extensive, widespread dermal mucinosis. Hematoxylin-Eosin staining, original magnification 400×.

(a)

Figure 3. Cont.

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(b)

FigureFigure 3. 3.(a)( aPresence) Presence of viral of viral spike spike prot proteinseins in the in cytoplasm the cytoplasm of epithelial of epithelial cells of cells the of secretory the secretory portionportion of ofeccrine eccrine sweat sweat glands glands (brown (brown color). color). Immunostaining Immunostaining for SARS-CoV-2, for SARS-CoV-2, spike spike proteins. proteins. OriginalOriginal magnification magniﬁcation 200×. 200× (b.()b Presence) Presence of ofviral viral spike spike protei proteinsns in in the the eccrine eccrine sweat sweat glands glands (brown (brownstain). stain). Immunostaining Immunostaining for SARS-CoV-2, for SARS-CoV-2 spike, proteins.spike proteins. Original Original magniﬁcation magnification 400×. 400×.

AnAn electron electron microscopy microscopy study study was was performed performed in only in only one onecase, case, showing showing marked marked hy- pertrophyhypertrophy of the of theendothelial endothelial cells, cells, the the nuclei nuclei being being characterized characterized by by finely finely distributed distributed chromatinchromatin and and retiform retiform nucleoli. nucleoli. The The cytoplas cytoplasmm contained contained very very few few organelles, organelles, with with few few smallsmall globular globular mitochondria. mitochondria. The The fibroblast fibroblastss were were remarkably remarkably elongated elongated and and surrounded surrounded byby very very short short collagen collagen fibers. fibers. Spherical Spherical struct structuresures ranging from 6565 toto 136136 nmnm were were visible visible in inthe the cytoplasm cytoplasm of of endothelial endothelial cells cells and and fibroblasts fibroblasts (Figure (Figure4), 4), characterized characterized by by a peripherala periph- eralelectron-dense electron-dense rim rim and and a a clearer clearer core. core. TheseThese structuresstructures were more more frequently frequently near near the the cellcell membrane membrane or or externally externally attached attached to to it. it. Aggregates Aggregates of of multiple multiple viral viral particles particles were were groupedgrouped together together close close to to phag phagosome-likeosome-like vesicles. vesicles. In In some some images, images, faintly faintly electron-dense electron-dense projectionsprojections of of different different sizes sizes (6–10 (6–10 nm)—corresponding nm)—corresponding to to the the virus virus spikes—were spikes—were visible, visible, depictingdepicting a asolar solar crown. crown.

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FigureFigure 4. OriginalOriginal magnification magnification 89,000× 89,000 fibroblast× fibroblast cytoplasm cytoplasm containing containing numerous numerous maturing maturing coronaviruses.coronaviruses. Fragmented collagen collagen fibers fibers are are visi visibleble in in the the interstice. interstice. In In the the box, box, 110,000×, 110,000 ×whole, whole virions are evident with spikes of different lengths and an electron-dense peripheral rim. virions are evident with spikes of different lengths and an electron-dense peripheral rim.

4.4. Discussion The nodular lesions lesions described described in in the the present present report report differ differ from from the thechilblain-like chilblain-like le- lesionssions related related to to COVID-19 COVID-19 due due to to their their frankly frankly nodular nodular appearance appearance and and chronic chronic course. course. However,However, theythey recall recall the the chilblain-like chilblain-like lesions lesions that that were were reported reported with with increasing increasing frequency fre- duringquency theduring pandemic the pandemic period [period18,19], [18,19], in regards in regards to the localizationto the localization on the on feet, the the feet, lack the of respiratorylack of respiratory manifestations manifestations and negative and negative molecular molecular tests, tests, histological histological aspects aspects of vasculitis of vas- andculitis small and vessels small vessels thrombotic thrombotic phenomena, phenomena, and above andall above thepresence all the presence of glyco-spike of glyco-spike proteins inproteins endothelial in endothelial cells [20 cells–22]. [20–22]. These hadThese been had detectedbeen detected in previous in previous studies studies of chilblain- of chil- likeblain-like lesions lesions [12], both[12], inboth endothelial in endothelial cells andcells inand eccrine in eccrine sweat sweat glands, glands, similar similar to those to describedthose described in the in present the present report. report. There There is still is still considerable considerable debate debate on on the the use use of of electron elec- microscopytron microscopy in the in course the course of COVID-19 of COVID-19 infection, infection, as the as initialthe initial confirmations confirmations of SARS-CoV- of SARS- 2CoV-2 in chilblain-like in chilblain-like lesions lesions [12 ][12] were were subsequently subsequently disavowed disavowed [ 23[23].]. This method method seems seems toto provide further further proof proof in in the the presence presence of ofother, other, more more credible credible elements, elements, rather rather than thanof- offeringfering proof proof in initself. itself. In any In anycase, case, epidemiologic epidemiologic data dataallow allow us to ushypothesize to hypothesize that both that bothacute acuteerythematous-edematous erythematous-edematous chilblain-like chilblain-like lesions and lesions chronic and nodular chronic lesions nodular reminis- lesions reminiscentcent of deep of granuloma deep granuloma annulare annulare may be may the be expression the expression of Coronavirus of Coronavirus contagion contagion in inyoung young subjects subjects who who are are particularly particularly reactive reactive to tothe the virus. virus. Some Some authors authors have have recently recently demonstrateddemonstrated the presence of Myxovirus Myxovirus resist resistanceance protein protein A, A, a a recognized recognized tissue tissue marker marker ofof interferoninterferon 1 activity, in the epidermis, endothelial cells, cells, and and inflammatory inflammatory infiltrate infiltrate of of cutaneouscutaneous chilblain-likechilblain-like lesions lesions [23 [23–25].–25]. This This finding finding supports supports the hypothesisthe hypothesis that inthat young in subjectsyoung subjects with an with energetic an energetic interferon interferon type 1 anti-virustype 1 anti-virus response, response, the latter the can latter on can the on one handthe one neutralize hand neutralize or minimize or mi thenimize symptoms the symptoms of viral of infection viral infection and justify and thejustify negativity the neg- of theativity molecular of the molecular tests, and tests, on the and other on the hand, other as hand, a side as effect,a side effect, induce induce microangiopathy, microangiop- as occursathy, as in occurs genetic in interferonopathiesgenetic interferonopathies and in systemic and in systemic lupus erythematosus. lupus erythematosus.

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5. Conclusions Overall, we describe a hybrid pattern of dermatologic lesions likely related to SARS- CoV-2, conﬁrmed only by EM and immunohistochemistry assay. In our opinion, the acral nodular lesions described herein may be related more to immune responses secondary to the viral infection than to the direct cytopathic effect of the virus. Many varied skin manifestations have been described in these months of the pandemic, sometimes not correlated with histopathological and immunohistochemical data. A careful evaluation of the time lapse since the presumed contact with SARS-CoV-2 is of fundamental importance to reconstruct the clinical history of patients with COVID-related skin manifestations. Further studies are needed to fully explore this issue and also to gain a more in-depth understanding of the spectrum of skin manifestations that can be caused by SARS-CoV-2.

Author Contributions: Conceptualization, G.C. and A.C. (Antonietta Cimmino); methodology, G.I. and T.T.; investigation, F.M.; resources, L.R.; data curation, G.I.; writing—original draft preparation, G.C.; R.R. and E.B.; writing—review and editing, G.I.; S.S. and E.B.; visualization, L.R.; B.F.; supervi- sion, A.C. (Anna Colagrande), G.I., F.M. and E.B. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: This does not applicable. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: This does not applicable. Acknowledgments: The authors thank Pragnell Mary Victoria Candace for her helpful language revision of the manuscript and editorial assistance. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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