Crowley, B.

Herra, Celine M

Laoi, B

Norris, S.

English

Arrow@TUDublin

Technological University Dublin ARROW@TU Dublin Articles School of Biological Sciences 2007-4 Hepatitis B Virus Resistance to Adefovir in a Nucleotide Naive Patient with Chronic Hepatitis B Virus Infection B Laoi Celine M. Herra Dr Technological University Dublin, celine.herra@tudublin.ie S. Norris B. Crowley Follow this and additional works at: https://arrow.tudublin.ie/scschbioart  Part of the Medicine and Health Sciences Commons Recommended Citation Laoi, B., Herra, C., Norris, S. & Crowley, B. (2007). Hepatitis B virus resistance to adefovir in a nucleotide naive patient with chronic hepatitis B virus infection. Journal of Antimicrobial Chemotheraphy,, 59(4), pp.807-809. doi:10.1093/jac/dkm026 J Antimicrob Chemother. 2007 Apr;59(4):807-9 This Article is brought to you for free and open access by the School of Biological Sciences at ARROW@TU Dublin. It has been accepted for inclusion in Articles by an authorized administrator of ARROW@TU Dublin. For more information, please contact yvonne.desmond@tudublin.ie, arrow.admin@tudublin.ie, brian.widdis@tudublin.ie. This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 License showed , 70% similarity (Figure 1) and were considered unre-lated. Multilocus sequence typing (MLST) analysis showed that iso-lates of the outbreak strain had an identical allelic profile, ST17belonging to the clonal complex 17 (CC-17). Four E. faecium iso-lates were used as controls in the MLST analysis, two vancomycin-susceptible isolates from HGS had the ddl2 allele, instead ofthe ddl1 of the vanB2 isolates, and belonged to ST16; andtwo other vanB producers from other hospitals were ST18 andST132.Infections caused by VanB vancomycin-resistant E. faeciumbelonging to CC-17 are still relatively uncommon in Europe.In two hospitals in Zaragoza (Spain), a clonal relationshipwas demonstrated between seven vanB2-containing E. faeciumisolates.4 The first VanB E. faecium isolated in a Spanishhospital was described in 2004.5 In Sweden, a nosocomial out-break of VanB2 E. faecium with the purK allele 1 has beenreported.6An enhanced infection control programme was introduced inthe HGS during 2006. Patients infected by VanB enterococciwere isolated in single rooms whenever possible, and theserooms were mechanically cleaned each day. The outbreak ofVanB E. faecium finished in August 2006, with no further casesdetected.In conclusion, to the best of our knowledge, we describe thefirst outbreak in Spain of a vanB2 E. faecium strain belongingto CC-17 and which was multiply resistant to vancomycin,ampicillin and fluorquinolones.AcknowledgementsThis study was supported by research grant from Instituto deSalud Carlos III, Fondo de Investigaciones Sanitarias, Ministryof Health, Spain (Red Espan˜ola de Investigacio´n en Patologı´aInfecciosa, REIPI; reference RD06/0008/0023), and by theDireccio´n General de Salud Pu´blica, Ministry of Health, Spain(reference 1429/05-11).Silvia Garcı´a-Cobos is a recipient of a fellowship of theISCIII (reference 05/0033).Transparency declarationsNone to declare.References1. Dahl KH, Simonsen GS, Olsvik et al. Heterogeneity in the vanBgene cluster of genomically diverse clinical strains of vancomycin-resistant enterococci. Antimicrob Agents Chemother 1999; 43:1105–10.2. Dahl KH, Lundblad EW, Rokenes TP et al. Genetic linkage of thevanB2 gene cluster to Tn5382 in vancomycin-resistant enterococci andcharacterization of two novel insertion sequences. Microbiol 2000; 146:1469–79.3. Carias LL, Rudin SD, Donskey CJ et al. Genetic linkage andcotransfer of a novel, vanB-containing transposon (Tn5382) and a low-affinity penicillin-binding protein 5 gene in a clinical vancomycin-resistant Enterococcus faecium isolate. J Bacteriol 1998; 180:4426–34.4. Torres C, Escobar S, Portillo A et al. Detection of clonally relatedvanB2-containing Enterococus faecium strains in two Spanish hospi-tals. J Med Microbiol 2006; 55: 1237–43.5. Lorenzo-Dı´az F, Delgado T, Reyes-Darias JA et al. Characterizationof the first vanB vancomycin-resistant Enterococcus faecium isolated in aSpanish hospital. Current Microbiol 2004; 48: 199–203.6. Granlund M, Carlsson C, Edebro H et al. Nosocomial outbreak ofvanB2 vancomycin-resistant Enterococcus faecium in Sweden. J HospInfect 2006; 62: 254–6.Journal of Antimicrobial Chemotherapydoi:10.1093/jac/dkm026Advance Access publication 1 March 2007Hepatitis B virus resistance to adefovir in anucleotide naive patient with chronic hepatitis Bvirus infectionB. Ni Laoi1,2, C. Herra3, S. Norris4 and B. Crowley1,5*1Department of Microbiology, St James’s Hospital, James’sStreet, Dublin 8, Ireland and 2Department of ClinicalMicrobiology, University of Dublin, Trinity College, Dublin 2,Ireland and 3School of Biological Sciences, Dublin Instituteof Technology, Kevin Street, Dublin 8, Ireland and4Department of Hepatology, St James’s Hospital, James’sStreet, Dublin 8, Ireland; 5National Virus ReferenceLaboratory, University College Dublin, Belfield, Dublin 4,IrelandKeywords: lamivudine treatment, primary adefovir resistance,HBV*Corresponding author. Tel: þ 353-1-4162966; Fax: þ353-1-4738902; E-mail: bcrowley@stjames.ieSir,Antiviral therapy of chronic hepatitis B (CHB) remains amajor clinical challenge. The new orally active nucleos(t)ideanalogues, such as lamivudine, adefovir and entecavir, havebeen shown to be efficacious in controlling viral replication,decreasing inflammatory activity within the liver and preventingthe progression of CHB infection to liver cirrhosis and hepato-cellular carcinoma.1 However, due to the spontaneous error rateof the hepatitis B virus (HBV) polymerase gene and persistenceof HBV in infected individuals, long-term antiviral treatment isneeded in most individuals and therefore increases the risk ofselecting drug-resistant mutants.Drug resistance is associated with the development ofpolymerase gene mutations, followed by a rise in HBV viralload and a subsequent increase in alanine transaminase levelswith exacerbation of liver disease. Genotypic resistance occursin  20% of individuals per year, but at a lower rate in thosetreated with adefovir (3% at year 2 and 29% at year 5 oftreatment).2 The major lamivudine resistance mutations inHBV are located in the highly conserved reverse transcriptase(rt) domain of the polymerase gene, specifically in thetyrosine-methionine-aspartate-aspartate (YMDD) motif in the Cdomain, at position rtM204I/V. Additional compensatorymutations are selected downstream in the B domain at positionsrtL180M and rtV173L, which augment drug resistance andenhance viral replication efficiency in vitro.3 Adefovir resistanceCorrespondence807 at Trinity College Dublin on March 12, 2016http://jac.oxfordjournals.org/Downloaded from is associated with mutations located in the B and D domains,at positions rtA181T/V and rtN236T, respectively.2 Adefoviris effective in suppressing lamivudine-resistant HBV and,therefore, is most commonly used in patients withlamivudine-resistant HBV infection.2 While acquired resistanceto adefovir is important, primary resistance to this nucleotideanalogue was reported recently.4 A variant of HBV was detectedin three non-responders even before they commenced treatmentwith adefovir. The associated mutation rtI233V significantlyincreased resistance to adefovir in vitro. This variant occurs in 2% of individuals with CHB.4In this report, molecular evidence is presented of the emer-gence of primary adefovir resistance in a patient on prolongedlamivudine treatment for chronic HBV infection.A 40-year-old lady presented to St James’s Hospital, Dublin,with hepatitis Be antigen (HBeAg)-positive chronic HBV infec-tion in April 1999. A liver biopsy performed in July 1999showed bridging fibrosis consistent with early cirrhosis. She wasgiven interferon monotherapy 5 MU daily but this was stoppedafter 16 weeks because of severe side effects. She remainedHBeAg-positive at the end of interferon treatment. No HBVDNA levels were determined before or after interferon treatment.In July 2000 she was started on lamivudine monotherapy(100 mg/day). She became HBeAg-negative in April 2003. HBVDNA was undetectable at this time by hybridization assay(Digene HCII) (limit of detection 1.4  105 IU/mL). She con-tinued on lamivudine monotherapy and remained stable untilJuly 2006 when alanine transaminase levels were noted to beraised at 46 IU/L (normal range , 35 IU/L). HBV DNA levelsmeasured by realtime PCR were 5.2Eþ 02 IU/mL (HBV PCRkit, Artus, Hamburg, Germany) and 3.8Eþ 03 IU/mL (CobasAmpliprep/Cobas Taqman HBV test, Roche Diagnostics,Mannheim, Germany) in July 2004 and June 2006, respectively.Resistance to lamivudine was suspected. Nucleic acid sequen-cing of the HBV polymerase gene from sera collected in 2004and June 2006 was performed as described previously.5Mutations associated with lamivudine resistance were detectedat positions rtL180M and rtM204V in both samples, with thertV173L mutation appearing in serum collected from 2006(Table 1). The latter mutation did not appear to affect the repli-cative capacity of HBV since there was no biologicallysignificant difference in HBV DNA levels in July 2004 andJune 2006. Surprisingly, a mutation associated with adefovirresistance was identified at position rtA181V in both samples,although the patient has never been treated with adefovir. Shehas now started treatment with tenofovir (300 mg/day).Genotypic primary resistance to adefovir can occur withoutprior adefovir treatment. In this report, the patient receivedlamivudine monotherapy for several years and developed thertA181V mutation which confers resistance to adefovir.2 Thisfinding supports a previous report of the emergence of thertA181T mutation in 4 of 23 patients on long-term lamivudinetherapy. Similar to the current case, the amino acid substitution atposition 181 appeared concomitantly with the appearance ofYMDD motif mutations.6 The results suggest that amino acid sub-stitutions at 181 of the HBV polymerase gene may confer resist-ance to the licensed nucleoside and nucleotide antiviral analogues,lamivudine and adefovir, respectively. Furthermore, while thertI233V mutation has been shown to cause primary adefovir resist-ance,4 there was no evidence of the mutation in this case. Theemergence of the rtA181V mutation in a patient with no prioradefovir treatment confirms the need to test for the presence ofadefovir resistance mutations in patients who develop lamivudineresistance.This report showed that development of primary adefovirresistance can be associated with long-term lamivudine mono-therapy. Furthermore, HBV polymerase gene sequencing shouldbecome part of the treatment monitoring algorithm to improvetreatment efficacy. The amino acid substitution at position 181may be important in conferring cross-resistance to anti-HBVdrugs and therefore should be investigated further.Transparency declarationsDr Brendan Crowley has received a financial contibution in theform of a research grant from Gilead Sciences, Foster City, CA,USA.References1. Liaw YF, Leung NW, Chang TT et al. Effects of extended lamivu-dine therapy in Asian patients with chronic hepatitis B. HepatitisLamivudine Study Group. Gastroenterology 2000; 119: 172–80.2. Locarnini S, Qi X, Arterburn S et al. Incidence and predictors ofemergence of HBV mutations associated with ADV resistance during 4years of adefovir therapy for patients with chronic HBV. J Hepatol2005; 42 Suppl 2: A36.Table 1. Amino acid mutations in the HBV polymerase gene detected by direct nucleic acid sequencingAssociated resistance Muation sites Wild-typePatient seraJuly 2004 June 2006LAM rt173 V V LLAM rt180 L M MADV rt181 A V VLAM rt204 M V VADV rt233 I I IADV rt236 N N NNote: mutations are shown in bold.LAM, lamivudine; ADV, adefovir.Correspondence808 at Trinity College Dublin on March 12, 2016http://jac.oxfordjournals.org/Downloaded from 3. Delaney WE IV, Yang H, Westland CE et al. The hepatitis Bvirus polymerase mutation rtV173L is selected during lamivudinetherapy and enhances viral replication in vitro. J Virol 2003; 77:11833–41.4. Schildgen O, Sirma H, Funk A et al. Variant of hepatitis Bvirus with primary resistance to adefovir. N Engl J Med 2006; 354:1807–12.5. Aye TT, Bartholomeusz A, Shaw T et al. Hepatitis B virus poly-merase mutations during antiviral therapy in a patient following livertransplantation. J Hepatol 1997; 26: 1148–53.6. Yeh CT, Chien RN, Chu CM et al. Clearance of the original hepa-titis B virus YMDD-motif mutants with emergence of distinctlamivudine-resistant mutants during prolonged lamivudine therapy.Hepatology 2000; 31: 1318–26.Journal of Antimicrobial Chemotherapydoi:10.1093/jac/dkl543Advance Access publication 6 February 2007Direct detection of vanB2 using the RocheLightCycler vanA/B detection assay to indicatevancomycin-resistant enterococcal carriage –sensitive but not specificH. L. Young1,2, S. A. Ballard3, P. Roffey2 andM. L. Grayson3,4,5*1Department of Microbiology, Austin Health, Melbourne,Australia and 2School of Biomedical Sciences, Charles SturtUniversity, N.S.W., Australia and 3Department of InfectiousDiseases, Austin Health, Melbourne, Australia and4Department of Epidemiology and Preventive Medicine,Monash University, Melbourne, Australia; 5Department ofMedicine, University of Melbourne, Melbourne, AustraliaKeywords: VRE, PCR, anaerobes, diagnostics*Correspondence address. Infectious Diseases Department,Austin Health, Studley Rd, Heidelberg, VIC, Australia 3084.Tel: þ613-9496-6676; Fax: þ613-9496-6677; E-mail: lindsay.grayson@austin.org.auSir,Vancomycin-resistant enterococci (VRE) are an infectioncontrol issue in many hospitals, such that rapid, accurate detec-tion has important clinical consequences. There are five geno-types responsible for acquired vancomycin resistance (vanA,vanB, vanD, vanE, vanG), with vanB the most prevalent inAustralia.1,2 Detection of VRE is generally by culture-basedmethods, with confirmation of species and van genotype basedon molecular methods.2The Roche LightCycler VRE Detection Kit (RocheDiagnostics, Mannheim, Germany) has been developed torapidly assess for VRE colonization.3 This kit provides theprimers and the hybridization probes for the amplification andsequence-specific detection of vanA, vanB1 and vanB2/3, alongwith an internal control for detection of sample inhibition. It hasa processing time of approximately 1.5 h. Using this kit withDNA directly extracted from clinical specimens, detection ofVRE can be reduced to less than 24 h. However, when used inthis manner, it is important to note that the system detects vangenes rather than VRE per se. Thus, the presence of vanB innon-enterococcal species may influence the sensitivity andspecificity of this approach for detection of VRE.4 – 7We assessed the usefulness and accuracy of the LightCyclerVRE detection system in the rapid identification of vanB VREcolonization in 59 faecal samples previously obtained from hos-pital inpatients (53 haemodialysis, 6 non-haemodialysis patients)and stored at 280ºC. Twelve of these faecal specimens (from 6haemodialysis and 6 non-haemodialysis patients) were shownpreviously to contain vanB2 Enterococcus faecium, while 42specimens were repeatedly shown to be culture-negative forVRE.4 As we have previously reported, four specimens wereshown to be vanB2 anaerobe culture-positive, but were VREculture-negative (one specimen contained Clostridium bolteaeand Ruminococcus sp., two specimens contained Clostridiumhathewayi and one specimen contained an un-speciatedClostridium), while a further specimen contained a vanB2 anae-robe (C. hathewayi) and vanB2 E. faecium.4 Faecal specimenswere defrosted and DNA was extracted directly from 200–220mg of faeces using the QIAampw DNA Stool Mini Kit (QiagenGmbH, Germany) according to the manufacturer’s recommen-dations. In addition, 17 h enrichment cultures in antibiotic-freeEnterococcosel broth (BBL, Cockeysville, MD, USA) of thesefaecal samples were prepared and DNA extracted as describedpreviously.4 Control isolates were Enterococcus faecalis ATCC29212 (van A/B negative), E. faecalis ATCC 51299 (vanB posi-tive) and a clinical vanA E. faecium isolate obtained from ourinstitution. All DNA was stored at –20ºC then tested for the pre-sence of vanA or vanB using the LightCycler VRE DetectionKit (Roche Diagnostics, Catalogue no. 3 334 996) and theLightCycler instrument (Roche Diagnostics) with softwareversion 3.5, according to the manufacturer’s instructions. Thelimit of detection was determined by dilution of the plasmidDNA positive control (1000 copies per 5 mL) provided in thekit. Dilutions were performed in triplicate with the resultingstandard curve imported into the data analysis mode. The sensi-tivity and specificity for detection of VRE was calculated.Statistical differences in the rates of accurate VRE identificationand PCR inhibition were compared using x2/Fisher’s exact test.All 12 vanB2 E. faecium culture-positive samples were posi-tive for vanB by LightCycler on both the direct faecal specimenand the 17 h enrichment culture (100% sensitivity; Table 1).However, specificity for detecting VRE with LightCycler was36% and 30% using direct DNA extraction and 17 h enrichmentbroth culture, respectively. Of the four specimens known tocontain vanB anaerobes, three of these were positive byLightCycler by both methods (Table 1). The fourth specimenwas most likely below the limit of detection as this was onlypositive following 36 h enrichment broth culture (data notshown). In addition, the specimen that contained both a vanB2anaerobe (C. hathewayi) and vanB2 E. faecium was also positivefor vanB by LightCycler. Overall, the LightCycler demonstrateda lower detection limit of 40 genome equivalents per reaction(data not shown). The rate of assay inhibition using theLightCycler procedure was 0% for 17 enrichment broth culturesand 3% for direct detection, similar to that observed previouslyfor the Roche LightCycler VRE kit.3The Roche LightCycler VRE detection assay demonstrateshigh sensitivity (100%), but limited specificity (30–36%) for thedetection of vanB VRE in surveillance specimens of faeces. ThisCorrespondence809 at Trinity College Dublin on March 12, 2016http://jac.oxfordjournals.org/Downloaded from 

Hepatitis B Virus Resistance to Adefovir in a Nucleotide Naive Patient with Chronic Hepatitis B Virus Infection

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Hepatitis B Virus Resistance to Adefovir in a Nucleotide Naive Patient with Chronic Hepatitis B Virus Infection

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