Albuquerque, AS

Atkinson, C

Breuer, J

Brown, JR

Brown, L-AK

Burns, SO

Clark, I

Cudini, J

Dervisevic, S

Karim, MY

Lowe, DM

Miller, J

Moore, J

Moreira, F

Neild, P

Patel, SY

Roy, S

Ruis, C

Seneviratne, SL

Toumpanakis, C

Williams, CA

Workman, S

English

UCL Discovery

Letter to the EditorA comprehensive characterization ofchronic norovirus infection in immu-nodeficient hostsTo the Editor:Chronic norovirus infection (CNI) can be devastating inpatients with immunodeficiency.1 We sought to investigateimmunologic and virologic factors associated with chronicinfection and document outcomes from a structured treatmentalgorithm.We identified 10 patients with CNI (1 referred from OxfordUniversity Hospitals), which was defined as compatible symp-toms (diarrhea, weight loss, and vomiting) or malabsorptionwith stool samples with positive PCR results for norovirus formore than 3 months. We also recruited control groups (see theMethods section in this article’s Online Repository at www.jacionline.org).Nine patients had CVID, and 1 had chronic lymphocyticleukemia and secondary antibody deficiency. Comorbiditiesand concurrent infections are detailed in Table E1 in this arti-cle’s Online Repository at www.jacionline.org. All patientshad diarrhea, 3 had significant vomiting, and most had weightloss. The majority had low vitamin E, folate, or B12 levels,and patients A and H had low zinc levels; therefore all 10 pa-tients had evidence of malabsorption. Three required paren-teral nutrition.In comparison with other patients with CVID from ourcenter, those with CNI (n 5 8) had lower long-term averageCD31 (median, 0.61 vs 1.12 3 109/L; P 5 .009) and CD191(median, 0.07 vs 0.18 3 109/L; P 5 .017) cell counts (Fig 1).From the earliest available data, 6 patients with CNI were clas-sified as Freiburg group 1A, and 3 were classified as Freiburggroup 1B (all of whom eventually met the criteria for group 1Abecause of a subsequent increase in CD212 B-cell counts).T-cell proliferation with 8 mg/mL PHA was impaired in allpatients (median, 8.1% [5.2% in 6 patients undergoing immu-nosuppression and 9.5% in 4 patients off immunosuppression];range, 0.5% to 33.7% of contemporaneous control; see TableE4 in this article’s Online Repository at www.jacionline.org).In 7 of 8 patients, we observed a possible temporal relationshipbetween decreasing CD31 and CD191 cell counts and diag-nosis or earliest date of norovirus acquisition (see Fig E1 inthis article’s Online Repository at www.jacionline.org). Oftenthis correlated with iatrogenic immunosuppression forinflammatory or autoimmune disease: 2 patients receivedhigh-dose corticosteroids, and 1 received corticosteroidsand rituximab. The patient from Oxford UniversityHospitals was receiving prednisolone and mycophenolatemofetil at diagnosis. Four patients (D, E, I, and J) receivedcorticosteroids with or without infliximab for ‘‘CVIDenteropathy’’ before norovirus diagnosis.We further characterized peripheral blood lymphocytes inpatients with CVID through flow cytometry. Versus healthy 2019 The Authors. Published by Elsevier Inc. on behalf of the American Academy ofAllergy, Asthma & Immunology. This is an open access article under the CC BY li-cense (http://creativecommons.org/licenses/by/4.0/).control subjects, patients with CNI had significantly reducedCD271 B-cell counts, including switched memory cells andCD801 and CXCR51 B-cell percentages but increasedCD212 (of which 68.6% were CXCR52) and transitionalB-cell percentages (see Fig E2, A, in this article’s OnlineRepository at www.jacionline.org). There was no differencein numbers of plasmablasts and CD51 or CD861 B cells.Patients with CVID without CNI (n 5 8) also demonstrateda significant (but smaller) increase in CD212 B-cellpercentages versus control subjects but no difference in otherpopulations (see Fig E2, B).Norovirus-infected patients had significantly increasednumbers of CD41 follicular helper T (CXCR51PD11) cells(9.5% 6 6.3% of total CD41 T cells) and overall programmedcell death protein 1 (PD-1)1CD41 cells (36.7%6 22.4% of totalCD41 T cells), with a trend toward decreased naive(CD45RA1CD62L1) cell percentages versus control subjects(see Fig E2, A). Patients with CVID without norovirus alsodemonstrated increased follicular helper T-cell counts but not to-tal PD-11CD41 T-cell percentages.Table E2 in this article’s Online Repository at www.jacionline.org summarizes gastrointestinal findings. Duodenal biopsy spec-imens demonstrated villous atrophy and intraepithelial lympho-cytosis in 7 patients (see Fig E3, A, in this article’s OnlineRepository at www.jacionline.org); both patients with ‘‘normal’’duodenal histology had profound small bowel villous atrophyobserved endoscopically (see Fig E3, B). Therefore all 9 patientswith mucosal assessment had macroscopic or histologic evidenceof villous atrophy.Immunohistochemical staining in 6 biopsy specimens revealedincreased intraepithelial CD31 lymphocytes, predominantlyCD81. CD41 lymphocyte frequencies varied. Patients withCVID had absent or severely reduced numbers of CD191 lym-phocytes. All patients had granzyme B–positive cells, with per-forin staining in 3: their distribution was identical to CD81 Tcells, suggesting that epithelial cell injury is mediated in part bycytotoxic cells.We performed norovirus whole-genome sequencing2 toexclude cross-transmission. Patient sample consensus sequenceswere used with worldwide surveillance reference strains toconstruct phylogenetic trees (see Fig E4 in this article’s OnlineRepository at www.jacionline.org). Patients A, B, and E were in-fected with the GII.4 variant New Orleans 2009, and patients Dand J were infected with viruses clustering between GII.4US95/96 and GII.4 Osaka 2007, but the viruses separated in thetrees (see Fig E5 in this article’s Online Repository at www.jacionline.org). Other patients were infected with distinct geno-types or variants (see Table E3 in this article’s Online Repositoryat www.jacionline.org), and therefore no evidence supportedtransmission within the cohort.We estimated patient-specific infection intervals by calculatingdivergence and ancestor dates (see Table E3). Patient F was likelyinfected within the 5 months before diagnosis, but intervals forother patients suggested years of antecedent infection.Sequencing also demonstrated relapses with the same strain afterlong periods of negative PCR results (up to 11months in patient E[see below] and 14months in patient F), whichwas far longer thanhas been described previously.31FIG 1. Long-term average (from 2000 [or date of CVID diagnosis, if later] to2018) CD31 and CD191 values for 8 patients with CVID with CNI versus 113patients with CVID without CNI. Lines represent medians. P values are fromMann-Whitney tests.J ALLERGY CLIN IMMUNOLnnn 20192 LETTER TO THE EDITORViral diversity (number of nucleotide differences betweenreads at a specific site)4,5 was extremely high in our cohortcompared with that in the control groups (see Fig E6, A, inthis article’s Online Repository at www.jacionline.org). This di-versity is rapidly generated, for example, in patient A within6 months of the most recent common ancestor (see Fig E6, B)and then fluctuates over time (see Fig E6, C). The diversitywithin patient C, whose virus had GII.P7 RdRp, was lowerthan in those with GII.P4 RdRp, which is consistent with a lowermutation rate with the GII.P7 polymerase.6 Diversity or esti-mated infection duration did not correlate with drug treatmentoutcome, but notably, only patient C has achieved sustained viralclearance.Dietary interventions were occasionally of benefit (Fig 2); 3of 8 patients improved symptomatically with a lactose-freediet, and 2 of 10 patients improved symptomatically with agluten-free diet. High-dose intravenous immunoglobulin (1 g/kg per week for 4 to 6 weeks) had no clear effect. Patient Dreported modest symptomatic benefit with Pentaglobin (animmunoglobulin product containing IgA and IgM in additionto IgG; Biotest AG, Dreieich, Germany).Nitazoxanide (500 mg administered twice daily for a minimumof 4 weeks) was tried in all patients. Patients B and F appeared toclear norovirus after treatment (with symptomatic improvementin patient B, whereas patient F’s symptoms worsened duringtherapy). However, both later relapsed (after 6 and 14 months,respectively). Patient B restarted nitazoxanide and had negativePCR results for 5 months until relapsing after high-dose steroidsand rituximab for hemolytic anemia. Patient G symptomaticallyimproved with nitazoxanide but did not clear norovirus (see FigE7 in this article’s Online Repository at www.jacionline.org),whereas patient A symptomatically deteriorated; other patientssaw no effect.Seven patients received ribavirin therapy (initial dose, 200 mgin the morning/400 mg in the evening and titrated according toserum levels). Patient E saw symptomatic improvement (see FigE8 in this article’s Online Repository at www.jacionline.org) andcleared norovirus from stool. However, discontinuation after11 months of negative results resulted in rapid relapse and wasalso associated with inflammatory arthritis. Ribavirin reintroduc-tion (with concomitant corticosteroid therapy for arthritis) did notclear norovirus, and the patient had treatment-related anemia. Pa-tients D and J also had anemia while receiving ribavirin. Patient Chad an initial virologic response but relapsed despite continuoustherapy. She eventually cleared norovirus spontaneously,2 months after discontinuing ribavirin. Patient J’s symptomsimproved with a small viral load decrease before having anemia.Conversely, patient G’s symptoms symptomatically deterioratedon ribavirin, whereas other patients saw no clear effect. PatientD was treated experimentally with favipiravir, as we havedescribed.7Despite the negative effect of significant immunosuppres-sion, low-dose prednisolone improved patient E’s symptoms(see Fig E8). Other patients receiving low-dose prednisolonehave not noted detrimental effects. Patient A with chronic lym-phocytic leukemia experienced dramatic symptomaticimprovement after receiving ibrutinib to attempt to achieve im-mune reconstitution.Cumulatively, more than 1000 days of antibiotics wereadministered during the study period, with no consistent effecton viral load or gastrointestinal symptoms despite evidence thatnorovirus cellular invasion requires bacteria.8In summary, CNI in patients with CVID is associated withalmost complete absence of CD271 B cells (includingswitched memory B cells), B-cell dysfunction characterizedby low expression of CD80 and CXCR5, a marked increasein numbers of transitional and CD212 B cells, poor T-cellproliferation, and high PD-1 expression on CD41 T cells.High numbers of CD212CXCR52 B cells and PD-11 T cellsboth imply immunologic ‘‘exhaustion.’’ Diagnosis might beheralded by decreasing lymphocyte counts, which are oftendriven by iatrogenic immunosuppression (which can alsoprecipitate relapse). The gut mucosa exhibits intraepithelialcytotoxic T-cell lymphocytosis and, with thorough assessment,Patient IDEffect on symptoms Effect on norovirus clearanceLactose-free dietGluten-free dietHigh-dose IVIGNitazox-anide Ribavirin Other OtherNitazox-anide Ribavirin OtherAIbrutinib (CLL) Antibiotics (various)BHigh dose steroids; rituximabLow dose prednisolone; mycophenolate mofetilHigh dose steroids; rituximabC**D* Favipiravir Initial M. avium treatment with azithromycin, amikacin and ethambutol*** FavipiravirELow dose prednisolone; abatacept*** High dose steroidsFAntibiotic treatment for concomitant Campylobacter infectionGLow dose prednisoloneHLow dose prednisoloneIJLow dose prednisolone ***FIG 2. Effect of interventions on symptoms and norovirus clearance. Effect on symptoms: green,improvement; orange, no clear effect; red, deterioration; and white, not attempted. Effect on norovirusclearance: green, sustained decrease in viral load or multiple negative samples; orange, no clear effect; red,relapse or increase in viral load; and white, not attempted. *Symptomatically improved with Pentaglobinbut no viral clearance. **Apparent initial response but subsequent relapse during treatment. Furtherepisode of infection 2 years later resolved rapidly with ribavirin therapy. ***Evidence of likely treatment-related anemia.J ALLERGY CLIN IMMUNOLVOLUME nnn, NUMBER nnLETTER TO THE EDITOR 3invariably demonstrates villous atrophy (unlike recent re-ports9); malabsorption is universal. Cross-transmission withinimmunodeficiency services appears rare, but we neverthelessadvocate strict infection control. Infections last years, withlate relapses and high viral diversity. Available treatmentsare variably effective and require further research.We acknowledge the staff of the Medical Research Council and UCL/UCLH Biomedical Research Centre–funded Pathogen Genomics Unit fortheir expert technical assistance.Li-An K. Brown, MRCPa,bChristopher Ruis, PhDc,dIan Clark, MB BSeSunando Roy, PhDcJulianne R. Brown, PhDfAdriana S. Albuquerque, PhDgSmita Y. Patel, PhD, FRCPathhJoanne Miller, FRCPathiMohammed Yousuf Karim, FRCPathi,jSamir Dervisevic, FRCPathkJennifer Moore, MRCPkCharlotte A. Williams, BSccJuliana Cudini, MSccFernando Moreira, BSclPenny Neild, MD, FRCPmSuranjith L. Seneviratne, DPhil, FRCPathlSarita Workman, MSclChristos Toumpanakis, PhD, FRCPnClaire Atkinson, PhDgSiobhan O. Burns, PhDg,lJudith Breuer, MD, FRCPathc*David M. Lowe, PhD, FRCPg,l*From the Departments of aInfectious Diseases, eHistopathology, lClinical Immunology,and nGastroenterology, Royal Free London NHS Foundation Trust, London, UnitedKingdom; bthe Department of Microbiology, Whittington Health NHS Trust, London,United Kingdom; cthe Division of Infection and Immunity, University College Lon-don, London, United Kingdom; dthe Molecular Immunity Unit, Department of Med-icine, University of Cambridge, Medical Research Council (MRC)–Laboratory ofMolecular Biology, Cambridge, United Kingdom; fthe Department of Microbiology,Virology and Infection Prevention and Control, Great Ormond Street Hospital forChildren NHS Foundation Trust, London, United Kingdom; gthe Institute of Immu-nity and Transplantation, University College London, Royal Free Campus, London,United Kingdom; hthe Oxford University Hospitals NHS Trust and NIHR OxfordBiomedical Research Centre, Oxford, United Kingdom; iRoyal Surrey County Hospi-tal NHS Foundation Trust, Egerton Road, Guildford, Surrey, United Kingdom; jPa-thology, Sidra Medicine, Doha, Qatar; kNorfolk and Norwich University Hospital,Norwich, Norfolk, United Kingdom; and mSt George’s University Hospitals NHSFoundation Trust, London, United Kingdom. E-mail: d.lowe@ucl.ac.uk.*These authors contributed equally to this work.At the time of the study, J.R.B. was funded by a National Institute for Health Research(NIHR) doctoral fellowship (grant no NIHR-HCS-D12-03-15). C.R. and S.R. werefunded by a Wellcome Trust collaborative award (grant no. 203268/Z/16/Z; toJ.R.B.). J.R.B. and D.M.L. receive funding from the NIHR UCL/UCLH BiomedicalResearch Centre.Disclosure of potential conflict of interest: S. Workman has received an educationalgrant from CSL Behring, speakers’ fees from LFB, honorarium from Octapharma,and support to attend conferences from CSL Behring, Grifols, Biotest, Octapharma,LFB, Shire, and the Jeffrey Modell Foundation. S. O. Burns has received speakers’fees/honoraria from Immunodeficiency Canada/IAACI, CSL Behring, and BaxaltaUS. D. M. Lowe has received travel and subsistence costs for unpaid consultancywork from CSL Behring. The rest of the authors declare that they have no relevantconflicts of interest.REFERENCES1. Brown LK, Clark I, Brown JR, Breuer J, Lowe DM. Norovirus infection in primaryimmune deficiency. Rev Med Virol 2017;23:e1926.2. Brown JR, Roy S, Ruis C, Yara Romero E, Shah D, Williams R, et al. Noroviruswhole-genome sequencing by SureSelect target enrichment: a robust and sensitivemethod. J Clin Microbiol 2016;54:2530-7.J ALLERGY CLIN IMMUNOLnnn 20194 LETTER TO THE EDITOR3. Brown JR, Roy S, Tutill H, Williams R, Breuer J. Super-infections and relapsesoccur in chronic norovirus infections. J Clin Virol 2017;96:44-8.4. Nei M, Li WH. Mathematical model for studying genetic variation in terms of re-striction endonucleases. Proc Natl Acad Sci U S A 1979;76:5269-73.5. Depledge DP, Cudini J, Kundu S, Atkinson C, Brown JR, Haque T, et al. High viraldiversity and mixed infections in cerebral spinal fluid from cases of varicella zostervirus encephalitis. J Infect Dis 2018;218:1592-601.6. Bull RA, Eden JS, Rawlinson WD, White PA. Rapid evolution of pandemic norovi-ruses of the GII.4 lineage. PLoS Pathog 2010;6:e1000831.7. Ruis C, Brown LK, Roy S, Atkinson C, Williams R, Burns SO, et al. Mutagenesis innorovirus in response to favipiravir treatment. N Engl J Med 2018;379:2173-6.8. Baldridge MT, Nice TJ, McCune BT, Yokoyama CC, Kambal A, Wheadon M, et al.Commensal microbes and interferon-lambda determine persistence of enteric mu-rine norovirus infection. Science 2015;347:266-9.9. Rolfes MC, Sriaroon P, Davila Saldana BJ, Dvorak CC, Chapdelaine H, FerdmanRM, et al. Chronic norovirus infection in primary immune deficiency disorders:an international case series. Diagn Microbiol Infect Dis 2019;93:69-73.https://doi.org/10.1016/j.jaci.2019.07.036

A comprehensive characterization of chronic norovirus infection in immunodeficient hosts

https://discovery.ucl.ac.uk/10082321/1/Burns_A%20comprehensive%20characterization%20of%20chronic%20norovirus%20infection%20in%20immunodeficient%20hosts_AOP.pdf

A comprehensive characterization of chronic norovirus infection in immunodeficient hosts

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