We report the first use of whole viral genome sequencing to identify nosocomial transmission of varicella zoster virus with fatal outcome. The index case, nursed in source isolation, developed disseminated zoster with rash being present for one day prior to being transferred to the Intensive Care Unit. Two patients who had received renal transplants while inpatients in an adjacent ward, developed chickenpox and one died. In neither case was there direct contact with the index patient

Breuer, J

Brown, J

Depledge, DP

Macanovic, J

Underhill, G

UCL Discovery

The Journal of Infectious DiseasesB R I E F R E P O R TViral Genome Sequencing ProvesNosocomial Transmission of FatalVaricellaDaniel P. Depledge,1 Julianne Brown,2 Jasna Macanovic,3 Gill Underhill,4 andJudith Breuer11Division of Infection and Immunity, University College London, 2Great Ormond Street HospitalVZV Reference Laboratory, Great Ormond Street Hospital, London, 3Wessex Renal andTransplant Services, and 4Departments of Clinical Microbiology, Pathology Centre, QueenAlexandra Hospital, Cosham, Portsmouth, United KingdomWe report the ﬁrst use of whole viral genome sequencing toidentify nosocomial transmission of varicella-zoster virus withfatal outcome. The index case patient, nursed in source isola-tion, developed disseminated zoster with rash present for 1day before being transferred to the intensive care unit (ICU).Two patients who had received renal transplants while inpa-tients in an adjacent ward developed chickenpox and 1 died;neither patient had direct contact with the index patient.Keywords. fatal varicella; nosocomial transmission; wholegenome sequencing.Varicella-zoster virus (VZV) causes both varicella and, afterlatency in sensory ganglia, herpes zoster. Universal vaccina-tion of children has reduced the incidence of varicella andits complications in the United States, and vaccination againstzoster is available for persons aged >60 years in the UnitedStates or >70 years in the United Kingdom. Neither vaccineis licensed for use in immunocompromised patients, forwhom both chickenpox and herpes zoster are potentiallyfatal. Because transmission is thought to arise mainly throughaerosolized virus from skin lesions, current advice in hospitalsettings is to isolate patients with diagnoses of chickenpox orwidespread shingles to prevent nosocomial spread to suscepti-ble contacts [1]. Viral genotyping provides a potential tool forlinking cases where transmission is not certain [2–4]. Howev-er, where viruses are of the same clade [5], current genotypingmethods cannot distinguish directly transmitted viruses fromthose that are unrelated or freely circulating [2]. Here wereport the use of a novel method for sequencing whole viralgenomes directly from residual diagnostic samples to conﬁrmnosocomial transmission of fatal varicella in a renal transplantrecipient.METHODSPatient 1A 55-year-old woman with end-stage renal failure caused byrenovascular disease received a cadaveric renal transplant(MM110; donor negative/recipient positive for human cyto-megalovirus [HCMV]), followed by basiliximab inductionand triple therapy with tacrolimus, mycophenolate mofetil,and prednisolone. After receiving the transplant, she was aninpatient in a 4-bed bay for 9 days. Fourteen days after dis-charge she was readmitted into isolation with abdominalpain, a macular rash, and abnormal liver function tests. Therash vesiculated within 24 hours and a clinical diagnosis ofvaricella was made. She was treated with intravenous acyclovir(10 mg/kg 3 times daily), antibiotics, and cessation of immu-nosuppression. Three days after admission she was transferredto the ICU, where she received inotropic and ventilatorsupport. She died 3 days later. Varicella pneumonitis wasdiagnosed, and it was noted that the patient had been VZV im-munoglobulin (Ig) G negative before transplantation and hadnot received varicella vaccine. She had had no known contactwith persons with varicella or zoster (Figure 1A; Supplemen-tary Figure 1).Patient 2A 61-year-old male patient with advanced renal failure, due toIgA nephropathy with secondary focal segmental glomerulo-sclerosis, received a preemptive live donor renal transplant(MM121; donor negative/recipient negative for HCMV). Hereceived basiliximab induction, followed by tacrolimus, my-cophenolate mofetil, and prednisolone. Despite a history ofchickenpox in childhood he was noted to be IgG VZV nega-tive before transplantation. He remained on the transplantward for 7 days. Eighteen days after discharge, he was read-mitted to a different ward with a 4-day history of thoracic ve-sicular rash, fever, and abnormal liver function. Disseminatedshingles was diagnosed, and the presence of VZV DNA wasconﬁrmed by polymerase chain reaction. The patient wasimmediately isolated and treated with intravenous acyclovir(10 mg/kg 3 times daily). He completed 7 days of intravenousacyclovir, followed by 1 week of oral acyclovir. Five weeksafter the original presentation, he had further reactivationof VZV in the L1 dermatome and received 2 further weeksof oral acyclovir and 3 months of prophylactic treatment.He made a full recovery and retained good graft function(Figure 1A; Supplementary Figure 1). Importantly, a retro-spective review indicated that disseminated shingles was aReceived 17 May 2016; accepted 22 August 2016; published online 28 August 2016.Correspondence: D. P. Depledge, Division of Infection and Immunity, University College Lon-don, Cruciform Building, Gower Street, WC1E6BT, London, UK (d.depledge@ucl.ac.uk).The Journal of Infectious Diseases® 2016;214:1399–402© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society ofAmerica. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestrictedreuse, distribution, and reproduction in any medium, provided the original work is properlycited. DOI: 10.1093/infdis/jiw398BRIEF REPORT • JID 2016:214 (1 November) • 1399 at University College London on December 2, 2016http://jid.oxfordjournals.org/Downloaded from misdiagnosis and that recurrent varicella should have beendiagnosed.Patient 3 (Index Case Patient)A 67-year-old man was admitted into respiratory isolation onthe transplant ward, with cough and deteriorating renal func-tion after dual cadaveric renal transplant 4 weeks previously.He was isolated immediately and treated with antibiotics.Low-grade HCMV viremia (3.92 × 102/mL) was treated withvalgancyclovir (450 mg/d). On day 10 after admission the pa-tient was noted to have a papulovesicular rash on his rightleg. A clinical diagnosis of shingles was conﬁrmed, and treat-ment was started with intravenous acyclovir (10 mg/kg 3times daily). Within 24 hours, the rash became generalized, re-spiratory failure developed, and the patient was transferred tothe ICU. He recovered with a fully functioning graft. Becausethis patient was isolated and did not initially have a rashwhile patient had received a diagnosis of shingles, nosocomialtransmission was not suspected. However, further investigationafter the death of patient 1 identiﬁed a period when all 3 pa-tients had been inpatients on the same ward, albeit physicallyseparated from patient 3. To investigate whether the 3 caseswere linked, we genotyped and sequenced samples from all 3patients and 2 patients with unrelated cases that occurred inthe hospital at the same time (Figure 1A, SupplementaryFigure 1).GenotypingResidual DNA samples were extracted using the QIAampDNA Mini Kit (Qiagen) according to the manufacturer’sinstructions. Ampliﬁcation and capillary sequencing of a447–base pair region of open reading frame 22, containing 4single-nucleotide polymorphisms (SNPs) that discriminatebetween clades 1/3, 2, 4, and 5 (SNPs 37902, 38055, 38081,and 38177) was carried out as described elsewhere [6]. In ad-dition, the highly variable repeat region, origin of replication(OriS), which also discriminates clade 1 from other clades, wasampliﬁed and capillary sequenced using conditions publishedelsewhere [2].Whole-Genome Sequencing and Assembly of VZVSequencing libraries were constructed as described elsewhere[7, 8] and sequenced on an Illumina MiSeq Sequencer. DataFigure 1. A, Timeline showing hospital admissions and clinical histories for patients 1, 2, and 3. B, Neighbor-joined phylogeny (100 bootstraps) comprising the 3 linked cases(patients 1, 2, and 3) and 2 unlinked cases (patients 3 and 4), together with 35 varicella-zoster virus database sequences obtained from GenBank. Abbreviations: Dx, diagnosis;ICU, intensive care unit; Pt, patient; Tx, treatment.1400 • JID 2016:214 (1 November) • BRIEF REPORT at University College London on December 2, 2016http://jid.oxfordjournals.org/Downloaded from sets were trimmed and aligned against VZV strain Dumas(NC_001348.1) using BWA (version 0.7.12) [9] and SAMToolssoftware (version 1.0) [10].Duplicate reads were removed, and aconsensus sequence for each data set generated using in-housePERL scripts. Iterative repeat regions (R1, R2, R3, R4, and R5)and the terminal repeat region were excluded from analyses.OriS sequences were successfully captured within the readdata and used to verify polymerase chain reaction–based resultsfor all samples. Consensus sequences were aligned using MAFFTsoftware (version 6) [11], and a neighbor-joined phylogenetictree, based on the Tamura-Nei model, was inferred usingMEGA software (version 6.06) [12]. Genome sequences are avail-able in GenBank (accession Nos. KP771912, KP771912, and 3more awaiting assignment by GenBank as of 16 May 2016).RESULTSVirus from all 3 renal patients and 2 “control” patients weretyped as clade 1 by SNP genotyping, and all 5 had identicalOriS sequences. Whole-genome sequencing showed the 2 puta-tively linked patients to be 100% identical to the index case pa-tient, patient 3 (Figure 1B) but not the control case patients,whose sequences contained 21 (patient 4) and 11 (patient 5)SNP differences across the genome. Phylogenetic analysis con-ﬁrmed the clustering of the linked cases, whereas the 2 unlinkedclade 1 viruses recovered from other patients in the hospitalclustered separately from the linked cases and from eachother (Figure 1B).DISCUSSIONWe report the use of whole viral genome sequencing to conﬁrman unusual case of nosocomial VZV transmission that occurreddespite source isolation of the index case patient. Although the 3patients were in adjacent rooms for more than a week, no im-mediate connection between them was suspected, partly be-cause shingles was not diagnosed in the index case patientuntil just before his transfer to the ICU and partly becausethere was no direct contact between patients. Conventional gen-otyping conﬁrmed all 3 patients to be infected with clade 1 vi-ruses, the most common genotype in European populations [7].However, SNP genotyping failed to differentiate the putativelylinked viruses from 2 epidemiologically unrelated viruses sam-pled from other patients at about the same time. Sequencing ofhighly variable regions, such as the R1 tandem repeat located inopen reading frame 11 and the OriS, has also been used to in-vestigate nosocomial transmission [2, 8]. In the cases we report,both linked case patients and controls had identical OriS se-quences, and identical R1 sequences in unrelated viruses havealso been observed [2, 8].Although amplicon sequencing to track nosocomial andother transmission events is well established for small RNA vi-ruses including human immunodeﬁciency virus and hepatitis Cvirus [9, 13], whole-genome sequencing is required for largerDNA viruses, because these are genetically less variable.To this end, we have developed a method to sequence wholepathogen genomes directly from clinical material [10]. Ourdata show, unequivocally, the identity between the viruses re-covered from the index case patient and 2 contact case patients.The putatively linked viruses were phylogenetically distinctfrom other clade 1, viruses including 1 recovered contempora-neously in the same hospital.Although airborne and environmental transmission of virusfrom cases of zoster to distant contacts have previously been re-ported [4], it has not hitherto been possible to conﬁrm nosoco-mial spread, other than in rare cases where a unique signaturesequence was present in an amplicon used for SNP typing [4].Airborne transmission despite source isolation has been de-scribed previously, particularly where viral loads in the indexcase patient are high (eg, in immunosuppressed patients andthose with hemorrhagic varicella). Thus, the need for strictrespiratory isolation of such patients, preferably in negativepressure rooms, is paramount [4].Our case report also underlines 2 important clinical issues.First, primary varicella can be fatal in immunosuppressed pa-tients despite appropriate early treatment and support. VZV an-tibody testing and immunization against VZV in patients forwhom renal transplantation is planned has been shown to pre-vent or attenuate varicella [14]. Second, widespread rash in animmunosuppressed patient with a history of chickenpox, as inour patient 2, cannot be assumed to be disseminated shingles.Identifying cases of recurrent chickenpox due to nosocomialtransmission is important for assuring infection control mea-sures. This case report illustrates how whole-genome sequenc-ing can contribute to this process.In summary, whole VZV genome sequencing directly fromclinical material provides a tractable method for monitoring nos-ocomial transmission. Applying automated methods directly toresidual diagnostic samples, we can generate results within 5days of sample receipt. The data provide support for the risk ofairborne transmission of VZV, especially when the viral load ishigh, and underline the vulnerability of immunocompromisedpatients to serious infection, irrespective of varicella history.Our report highlights the ongoing need for vaccines that canbe prevent viral reactivation in immunocompromised patientsas an important measure for control to prevent nosocomialspread.Supplementary DataSupplementary materials are available at http://jid.oxfordjournals.org.Consisting of data provided by the author to beneﬁt the reader, the postedmaterials are not copyedited and are the sole responsibility of the author, soquestions or comments should be addressed to the author.NotesFinancial support. This work was supported by the MRC Centre forMedical Molecular Virology, the Medical Research Foundation (NewBRIEF REPORT • JID 2016:214 (1 November) • 1401 at University College London on December 2, 2016http://jid.oxfordjournals.org/Downloaded from Investigator award to D. P. D.), the National Institute for Health Research(fellowship to J. Brown), and the NIHR Biomedical Resource Centre at Uni-versity College London Hospitals NHS Foundation Trust and UniversityCollege London (J. Breuer).Potential conﬂicts of interest. All authors: No potential conﬂicts of in-terest. All authors have submitted the ICMJE Form for Disclosure of Poten-tial Conﬂicts of Interest. Conﬂicts that the editors consider relevant to thecontent of the manuscript have been disclosed.References1. Public Health England. Shingles (herpes zoster): the green book, chapter 28a.https://www.gov.uk/government/publications/shingles-herpes-zoster-the-green-book-chapter-28a. Accessed September 2015.2. 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Genotyping of varicella-zoster virus andthe discrimination of Oka vaccine strains by TaqMan real-time PCR. J Clin Micro-biol 2006; 44:3911–4.7. Hawrami K, Breuer J. Analysis of United Kingdom wild-type strains of varicella-zoster virus: differentiation from the Oka vaccine strain. J Med Virol 1997;53:60–2.8. Hawrami K, Harper D, Breuer J. Typing of varicella zoster virus by ampliﬁcationof DNA polymorphisms. J Virol Methods 1996; 57:169–74.9. Li H, Stoddard MB, Wang S, et al. Single-genome sequencing of hepatitis C virusin donor-recipient pairs distinguishes modes and models of virus transmissionand early diversiﬁcation. J Virol 2015; 90:152–66.10. Depledge DP, Palser AL, Watson SJ, et al. Speciﬁc capture and whole-genome se-quencing of viruses from clinical samples. PLoS One 2011; 6:e27805.11. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evo-lutionary genetics analysis version 6.0. Mol Biol Evol 2013; 30:2725–9.12. Katoh K, Standley DM. MAFFT multiple sequence alignment softwareversion 7: improvements in performance and usability. Mol Biol Evol 2013;30:772–80.13. Ou CY, Ciesielski CA, Myers G, et al. Molecular epidemiology of HIV transmis-sion in a dental practice. Science 1992; 256:1165–71.14. Zamora I, Simon JM, Da Silva ME, Piqueras AI. Attenuated varicella virus vaccinein children with renal transplants. Pediatr Nephrol 1994; 8:190–2.1402 • JID 2016:214 (1 November) • BRIEF REPORT at University College London on December 2, 2016http://jid.oxfordjournals.org/Downloaded from 

Viral genome sequencing proves nosocomial transmission of fatal varicella.

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Viral genome sequencing proves nosocomial transmission of fatal varicella.

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