Antiviral Research 111 (2014) 36–41
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Resistance of herpes simplex viruses to acyclovir: An update from a ten-year survey in France Emilie Frobert a,b,⇑, Sonia Burrel c,d, Sophie Ducastelle-Lepretre e, Geneviève Billaud a, Florence Ader f, Jean-Sébastien Casalegno a,b, Viviane Nave a, David Boutolleau c,d, Mauricette Michallet e, Bruno Lina a,b, Florence Morfin a,b a
Laboratoire de Virologie, Centre de Biologie et Pathologie Est, Hospices Civils de Lyon, Lyon, France Virologie et Pathologie Humaine, Université Lyon 1, EA 4610, Faculté de Médecine RTH Laënnec, Lyon, France Sorbonne Universités, UPMC Univ Paris 06, CR7, Centre d’Immunologie et des Maladies Infectieuses (CIMI-Paris), INSERM U1135, Paris, France d AP-HP, Hôpitaux Universitaires Pitié-Salpêtrière – Charles Foix, Service de Virologie, Paris, France e Service d’Hématologie Clinique, Centre Hospitalier Lyon Sud, Lyon, France f Service de Maladies Infectieuses et Tropicales, Hôpital de la Croix-Rousse, Lyon, France b c
a r t i c l e
i n f o
Article history: Received 1 July 2014 Revised 30 July 2014 Accepted 25 August 2014 Available online 8 September 2014 Keywords: Herpes simplex virus Survey Antiviral drug resistance Hematopoietic stem cell transplant patients UL23 thymidine kinase UL30 DNA polymerase
a b s t r a c t The widespread use of acyclovir (ACV) and the increasing number of immunocompromised patients have raised concern about an increase in ACV-resistant herpes simplex virus (HSV). ACV resistance has traditionally been a major concern for immunocompromised patients with a frequency reported between 2.5% and 10%. The aim of this study was to reassess the status of HSV resistance to ACV in immunocompetent and immunocompromised patients over a ten year period, between 2002 and 2011. This was done by retrospectively following 1425 patients. In immunocompetent patients, prevalence of resistance did not exceed 0.5% during the study period; whereas in immunocompromised patients, a significant increase was observed, rising from 3.8% between 2002 and 2006 (7/182 patients) to 15.7% between 2007 and 2011 (28/178) (p = 0.0001). This sharp rise in resistance may largely be represented by allogeneic hematopoietic stem cell transplant patients, in which the prevalence of ACV resistance rose similarly from 14.3% (4/28) between 2002 and 2006 to 46.5% (26/56) between 2007 and 2011 (p = 0.005). No increase in ACV resistance was detected in association with other types of immune deficiencies. Genotypic characterization of HSV UL23 thymidine kinase and UL30 DNA polymerase genes revealed 11 and 7 previously unreported substitutions, respectively. These substitutions may be related to potential polymorphisms, drug resistance, or other mutations of unclear significance. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Repeated and long-term acyclovir (ACV) use associated with clinical and immunological features may lead to the development of herpes simplex virus (HSV) resistant to this drug. HSV resistance to ACV is primarily a concern for immunocompromised patients whereas the prevalence remains low in immunocompetent patients. Since the commercialisation of ACV in the 1980s, resistance to this drug in immunocompetent patients has been estimated at 0.5% (Christophers et al., 1998; Fife et al., 1994; Nugier et al., 1992). After twenty years of use, the prevalence did not ⇑ Corresponding author at: Laboratoire de Virologie, Centre de Biologie et Pathologie Est, Hospices Civils de Lyon, 59 boulevard Pinel, 69 500 Bron, France. Tel.: +33 4 72 12 96 53; fax: +33 4 72 12 95 00. E-mail address: [email protected] (E. Frobert). http://dx.doi.org/10.1016/j.antiviral.2014.08.013 0166-3542/Ó 2014 Elsevier B.V. All rights reserved.
increase (Danve-Szatanek et al., 2004; Stranska et al., 2005). Nevertheless, the recurrent and widespread use of ACV and its prodrug Val-ACV, including FDA-approved short-course and high dose regimens to treat HSV recurrence, may be contributing factors towards the emergence of resistance (Cunningham et al., 2012). One recent Chinese study reported an unexpectedly high prevalence of ACV-resistant HSV of 4% in immunocompetent children with oral herpetic lesions (Wang et al., 2011). Further, some cases of recurrent herpetic keratitis have been found to be associated with ACV-resistant virus with a prevalence of 6.4% in immunocompetent patients (Duan et al., 2009). Additionally, immunocompetent patients with genital herpes have a higher prevalence of ACV-resistant HSV (Kriesel et al., 2005). With regards to immunocompromised patients, the prevalence of ACV-resistant HSV is largely higher and varies between 3.5% and 10%, depending on the type of immunosuppression. HSV resistant to ACV can induce large,
E. Frobert et al. / Antiviral Research 111 (2014) 36–41
extensive and ulcerative cutaneous infections, but also esophagitis, pneumonia and encephalitis, which can be fatal without clinical improvement. Among immunocompromised patients, the prevalence of resistance was reported between 3.5% and 7% in HIV patients, 2.5% and 10% in solid organ transplant patients, and with the greatest prevalence witnessed at 4.1% and 10.9% in hematopoietic stem cell transplant (HSCT) patients (Danve-Szatanek et al., 2004; Piret and Boivin, 2011). The prevalence has even been observed as high as 25% in allogeneic HSCT patients (Chakrabarti et al., 2000; Morfin et al., 2004). 95% of resistant cases are attributed to mutations on the UL23 gene encoding for thymidine kinase and 5% are attributed to mutations on the UL30 gene encoding for viral DNA polymerase (Piret and Boivin, 2011, 2014). Antiviral drug resistance screening using genotypic techniques are an efficient approach to make prompt virological detection to adapt antiviral treatment. Nevertheless, genotypic characterization of UL23 and UL30 genes have revealed many natural polymorphisms in both genes, which can complicate the interpretation of genetic sequences, particularly when detected substitutions have not been previously described or characterized (Burrel et al., 2010). The objectives of the present study were: (i) to reassess the frequency of ACV resistance in immunocompetent and immunocompromised patients from a ten-year survey; (ii) to describe new potential polymorphisms and resistance mutations, in the UL23 and UL30 genes, based on 22 ACV-sensitive and 40 ACV-resistant HSV.
37
and FOS (Astra, France), as previously described (Langlois et al., 1986). The effective concentration 50 (EC50) cut-off values for resistance to ACV were 6.5 lM for HSV1 and 13.5 lM for HSV2, and the EC50 cut-off value for resistance to FOS was 350 lM for both HSV1 and HSV2 (Nugier et al., 1992). 2.3. Statistical analysis Student’s t-test and Person v2-test were used to assess intergroup differences between the two timelines 2002–2006 versus 2007–2011. The division of the 10-year study period was made with regards to resistance evolution. Statistical analyses were performed on EpiInfo software (V 3.5.1 CDC). Odds ratio (OR) and 95% confidence interval (CI) were calculated to determine the likelihood of detecting ACV-resistant HSV during the two periods. A test was significant when the p value was less than 0.05. 2.4. Genotypic analysis of antiviral resistance
2. Materials and methods
Genotypic analysis of ACV-resistant HSV1 and HSV2 was performed directly on clinical samples by sequencing the UL23 and UL30 genes, which encodes for thymidine kinase and DNA polymerase, respectively, as previously described (Burrel et al., 2010; Frobert et al., 2008). When a sensitive HSV was previously isolated from a patient harboring a resistant virus, the UL23 gene of the sensitive strain was also sequenced. Nucleotide sequences were compared with reference strains SC16 (HSV1) and 333 (HSV2) (GenBank accession numbers X03764 and V00466, respectively) using SeqmanII software (DNAStar Inc.).
2.1. Patients and specimens
3. Results
In this single-centre study, during the 2002–2011 period, 1529 samples from 1425 patients had tested HSV-positive, by culture on MRC-5 cells. The University-Hospital of Hospices Civils of Lyon is the regional reference centre of 5500 beds and serves a population of 1.7 million. Some patients were sampled more than once but each sample was considered as a distinct episode of herpetic infection. 70% of patients were adults (1064 clinical samples were obtained from 1005 adults, median age of 50 (range: 18–75)) and 30% were children (465 clinical samples were obtained from 420 children, median age of 6.5 (range: 1–17)). HSV1 was detected in 1337 clinical samples (429 oropharyngeal, 191 nasopharyngeal, 200 cutaneous, 252 bronchoalveolar lavages, 185 genital and 80 ocular samples) and HSV2 in 192 clinical samples (116 genital, 2 nasopharyngeal, 70 cutaneous, 1 bronchoalveolar lavage, and 3 ocular samples) by specific immune fluorescence. CSF and aqueous humor were excluded as they could not be cultured. In the 1064 clinical adult samples, one was from a patient whose immune status was unavailable, 703 were from immunocompetent patients, and 360 were from immunosuppressed patients including 90 hematopoietic stem cell transplants (HSCT) (87 allogeneic and 3 autologous), 37 HIV, 68 haemopathies, 37 non-haemopathy malignancies, 94 solid organ transplants (SOT), and 34 other sources of immunosuppression. From the 465 children samples, 324 were from immunocompetent children and 141 were from immunosuppressed children including 30 allogeneic HSCT, 36 haemopathies, 12 non-haemopathy malignancies, 38 SOT, 4 HIV and 21 other sources of immunosuppression.
3.1. Frequency of ACV-resistant HSV over 2002–2011
2.2. Phenotypic diagnosis of antiviral resistance Screening for antiviral resistance was systematically performed on the 1529 HSV-positive culture using a neutral red dye-uptake assay to determine its sensitivity to ACV (GlaxoWellcome, France)
3.1.1. Adults One case of ACV-resistant HSV was detected in an immunocompetent patient (0.14%) and another case of resistance was detected in a patient whose immune status was unavailable (Table 1). Among 360 immunocompromised patients, 35 cases of resistance were detected (9.7%) with 30 cases from allogeneic HSCT patients (30/87, 34.5%), 3 cases from HIV patients (3/37, 8.1%), 1 case from a patient with haemopathy (1/68, 1.5%) and 1 case from a SOT patient (1/94, 1.1%). 3.1.2. Children In immunocompetent children, no cases of resistance were detected; but in immunocompromised children, 7 cases were detected (7/141, 5%) with 5 cases coming from allogeneic HSCT patients (5/30, 16.7%), 1 case from a child with haemopathy (1/ 36, 2.8%) and 1 case from a SOT patient (1/38, 2.6%). 3.2. Resistance evolution 3.2.1. Adults Over the 2002–2011 period, the frequency of ACV resistance has increased from 1.4% in 2002 to 5.2% in 2011, while the number of clinical samples testing HSV-positive remained relatively consistent year over year (Fig. 1). A significant increase was observed in 2007, prompting the division of the study period to be made between 2002–2006 versus 2007–2011 (Fig. 1). The overall prevalence of ACV-resistant HSV in adults increased significantly between 2002–2006 and 2007–2011, from 1.4% to 6% (OR = 4.22; p = 0.00006) (Table 1). In immunocompetent adults, the prevalence of resistance remained under 0.5%, throughout the years (p = 0.59) (Table 1). In immunocompromised patients, a significant increase
6.3 28.6
4
15 10 13 12 4.8
3.9 6.3
was observed from 3.8% in 2002–2006 to 15.7% in 2007–2011 (OR = 4.67; p = 0.0001). The substantial rise in resistance for immunocompromised patients can be primarily attributed to allogeneic HSCT patients where the prevalence rose from 14.3% in 2002–2006 to 46.5% in 2007–2011 (OR = 4.72; p = 0.005). Analysis of other immune deficiencies did not reveal significant increases in resistance, as shown by the p-value: HIV patients (p = 0.52), patients with haemopathies (p = 0.53), patients with non-haemopathy malignancies (p undefined), SOT patients (p = 0.35), and for other cases of immunosuppression (p undefined) (Table 1).
ACV: acyclovir; HSCT: hematopoietic stem cell transplant; HIV: human immunodeficiency virus; SOT: solid organ transplant. a One adult had a non-documented immune status. b Were included in other malignancies: cancer of bronchus, pancreas and breast, adenocarcinoma, osteosarcoma. c Were included in other: patients under immunosuppression treatment and/or corticoids for Crohn disease, Wegener’s granulomatosis, sarcoidosis, Horton disease or pemphigus.
2.6
2.8
5 16.7
p = 0.40 Undefined p = 0.40 p = 0.13 Undefined p = 0.58 p = 0.58 p = 0.66 Undefined 1.60 [0.33–8.67] Undefined 1.64 [0.35–7.63] 5.66 [0.6–157.44] Undefined Undefined Undefined Undefined Undefined 1.5
465 324 141 30 4 36 12 38 21 212 148 64 14
253 176 77 16 4 21 2 25 9 3 0 3 1 0 1 0 1 0 Children Immunocompetent Immunocompromised Allogeneic HSCT HIV Haemopathies Other malignanciesb SOT Otherc
1.2
4 0 4 4 0 0 0 0 0
1.9
7 0 7 5 0 1 0 1 0
1.1 3
37 68 37 94 34 7 36 23 33 19 30 32 14 61 15 3 0 0 0 0
10
0 1 0 1 0
15.7 46.5
2.8
3 1 0 1 0
8.1 1.5
p = 0.00006 p = 0.59 p = 0.0001 p = 0.005 Undefined p = 0.52 p = 0.53 Undefined p = 0.35 Undefined 1064 703 360 873 464 286 178 563 600 417 182 280 9a 1 7 40
Adults Immunocompetent Immunocompromised Allogeneic HSCT Autologous HSCT HIV Haemopathies Other malignanciesb SOT Otherc
1.4 0.2 3.8 14.3
28 0 28 260
6
37 1 35 300
3.5 0.14 9.7 34.5
Odd ratio % of resistance Nb of samples Nb of resistance
Total
% of resistance Nb of samples Nb of resistance
2007–2011
% of resistance Nb of samples Nb of resistance
2002–2006
Table 1 Frequency of ACV resistance between 2002–2006 versus 2007–2011.
4.22 [1.97–9.03] Undefined 4.67 [1.98–10.99] 4.72 [1.46–15.33] Undefined Undefined Undefined Undefined Undefined Undefined
E. Frobert et al. / Antiviral Research 111 (2014) 36–41
p value
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3.2.2. Children No increase in resistance was observed in immunocompetent or immunocompromised children during the two study periods (p = 0.40). Likewise, there was no increase in ACV-resistant HSV in children undergoing allogeneic HSCT (p = 0.13). 3.3. Genotypic analysis of HSV 3.3.1. Genotypic characterization of drug sensitive HSV clinical isolates 22 ACV-sensitive HSV1 have been previously isolated among patients with ACV-resistant HSV1. The UL23 sequencing revealed 7 natural polymorphisms that have not been previously reported: D76N, I100T, H142Y, D215H (D215N has been previously described (Wang et al., 2011)), P268L (P268T and P268S has been previously described (Kudo et al., 1998; van Velzen et al., 2013)), A299S and L341R. 3.3.2. Genotypic characterization of drug resistant HSV clinical isolates 3.3.2.1. New UL23 gene mutations not previously described (Table 2). In ACV-resistant HSV1, 3 previously unreported substitutions were detected: L242P (Patient No. 34), L249P (Patient No. 10) and E371D (Patient No. 35). In ACV-resistant HSV2, 1 previously unreported substitution was detected: M129I (Patient No. 39). 3.3.2.2. New UL30 gene mutations not previously described (Table 2). In ACV-resistant HSV1, 5 novel mutations were found: E860K (Patient No. 6), E671K (Patient No. 20), T931A (Patient No. 31), A874T (Patient No. 38) and A910T (Patient No. 7) (A910V has been previously described (Saijo et al., 2005)). In ACV-resistant HSV2, 2 novel mutations were detected: K485N (Patient No. 39) and G506D (Patient No. 44). 3.3.3. Distribution of UL23 and UL30 mutations in drug resistant HSV Among the 44 drug resistant HSV, 40 HSV were analyzed for UL23 and UL30 mutations as 4 samples were depleted (Table 3, supplementary data). ACV resistance was due to mutations on the UL23 gene in 85% of the cases (34/40). These mutations include 10 substitutions (10/40; 25%), 3 stop codons (3/40; 7.5%), 11 nucleotide insertions (11/40; 27.5%), and 10 nucleotides deletions (10/40; 25%). It should be noted that additions/deletions represent 62% (21/34) of mutations involving the UL23 gene. 7.5% of resistance cases (3/40) involved mutations on the UL30 gene encoding DNA polymerase. Two HSV had drug resistant mutations on both the UL23 and UL30 genes (Patients No. 25 and 41) and one strain had mutations on both respective genes, but with unclear significance (Patients No. 39). One ACV-resistant HSV had no mutation on the UL23 and UL30 genes (Patient No. 21). 4. Discussion The widespread use of ACV since its commercialization in the 1980s supports the need for a continued surveillance of HSV resistance, in immunocompetent and immunocompromised patients. The last surveys published on this topic date back to 2004
E. Frobert et al. / Antiviral Research 111 (2014) 36–41
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Fig. 1. Evolution of ACV resistance over 2002–2011.
(Danve-Szatanek et al., 2004) and 2005 (Stranska et al., 2005). There was a 4-year survey done in 2013 that described HSV resistance to antivirals, but only in patients with suspected clinical resistance (Burrel et al., 2013). In the 10-year retrospective study presented herein, 1529 HSV-positive samples were analyzed from 1425 patients. Consistent with prior statistics, ACV resistance in immunocompetent patients has not increased in recent years and the prevalence remains below 0.5%. In France and a majority of other countries, systemic ACV therapy is regulated by medical prescription. A future with potentially greater access to antivirals provides more reasons for sustained surveillance in immunocompetent populations for resistance. In immunocompromised patients, our survey showed a significant increase in ACV resistance from 2007 that was exclusively linked to HSCT patients. With improved protocols for bone marrow transplantation, the number of HSCTs performed has risen in recent years. New chemotherapy regimens in preparation for HSCT now induce longer and deeper immunosuppression. Since 2006, the Haematology Department in Lyon, France, has been using the FLAMSA-based high-dose sequential chemotherapy regimen before allogeneic hematopoietic stem cell transplantation for refractory patients with high risk myelodysplastic syndrome or secondary acute myeloid leukaemia (Saure et al., 2011; Schmid et al., 2005). This protocol is particularly aggressive and exposes patients to a longer period of neutropenia than in myeloablative or reduced intensity treatments. The type of HSCT (ie stem cells from bone marrow harvest, from leukapheresis or from cord blood cell) may also be a risk factor in the emergence of infectious diseases (Gilis et al., 2014). Thus, multivariate analyses are needed at a national and international level to understand why and how HSV resistance to antiviral drugs has increased over these last years in HSCT patients. It has been conventionally reported that 95% of ACV resistance cases are a consequence of mutations in the UL23 gene (reviewed in Piret and Boivin, 2011, 2014), of which additions/deletions account for half of the mutations (Gaudreau et al., 1998; Morfin et al., 2000). The sequence analysis of this study revealed that 85% of ACV-resistant strains were due to UL23 gene mutations, of which 62% were nucleotide additions/deletions. Recently, Burrel et al. have reported an even higher proportion addition/deletions accounting for 80% of UL23 gene mutations conferring ACV resistance (Burrel et al., 2013). This increased trend of UL23 gene mutations caused by nucleotide additions/deletions would facilitate the interpretation of genotypic results. In this study, UL30 gene
mutations were involved in 7.5% of resistance cases. Interestingly, 7.5% of HSV samples carried mutations in both UL23 and UL30 genes, which is not conventionally seen in ACV-resistant and FOS-sensitive strains. The genotypic profile of drug resistant HSV must be taken into consideration to optimize the detection of resistant HSV, particularly for the purposes of molecular-based assays. The natural polymorphism of UL23 and UL30 genes has been largely described before (Bohn et al., 2011; Burrel et al., 2010) and drug resistance can be induced by concomitant mutations (Frobert et al., 2008). Classifying uncharacterized mutations as either new polymorphism or conferring drug resistance continues to be a difficult task. Concerning new UL23 gene mutations detected from our survey of ACV-resistant HVS1 (L242P, L249P, E371D), L249P and E371D may likely confer ACV resistance as no concurrent mutation was found on the UL30 gene. The L242P substitution remains of unclear significance, as it was observed in conjunction with D162N, an already known drug resistance mutation (Chibo et al., 2004; Malartre et al., 2012). In ACV-resistant HSV2, the sole M129I substitution found on UL23 gene remains of unclear significance, as it was observed in conjunction with a novel K485N substitution on UL30, which has never been previously reported. Concerning new UL30 gene mutations in ACV-resistant HSV1 (E860K, E671K, T931A, A874T, A910T), E860K may potentially confer resistance as no concurrent mutation was uncovered in the UL23 gene. E860K may also have a role in FOS resistance, but this remains to be determined as it was observed in conjunction with R700G, a known FOS and ACV resistance conferring mutation (Andrei et al., 2000; Gibbs et al., 1988). E671K may be a UL30 polymorphism, as a ‘‘+1G’’ nucleotide insertion on the UL23 gene is presumably the more likely factor in ACV resistance. Further, a proximate mutation, E675A, has been previously described as a natural UL30 polymorphism (Burrel et al., 2010). The amino acid change A910T could be responsible for FOS resistance as A910V has been attributed to an FOS-resistant but ACV-sensitive phenotype (Saijo et al., 2005). Prior studies have yielded conflicting results concerning the role of UL30’s D672N substitution in ACV resistance (Burrel et al., 2010; Sauerbrei et al., 2010), but one of our ACV-resistant and FOS-sensitive strain had no mutation on the UL23 and UL30 genes except D672N. UL30 substitutions, T931A and A874T, do not induce FOS resistance and they likely correspond to natural polymorphism, as they were detected in conjunction with UL23 mutations known to confer ACV resistance (Duan et al., 2009). UL30 substitutions, A605V and A719V, have
+1G 440
G200D
M129I
L242P E371D
G240E G240E N78D L140F G39E
L249P
+1G 430 +1G 436 D162N
G200S
G240E Q89R G240E C6G G251C V267L P268T D286E N376H
737 334 125 151 125 125 92 92 152 79 20 35 53 41 20 26 9 24 48 155 1 1 1 1 1 1 1 1 2 2 2005 2005 2007 2008 2011 2011 2011 2011 2002 2011 Throat Throat Throat BAL Throat Throat Throat Throat Cutaneous Genital HSCT HSCT HSCT HSCT HSCT HSCT HSCT HSCT NA HSCT 6 7 10 20 31 34 35 38 39 44
BAL: bronchoalveolar lavage; HSCT: hematopoietic stem cell transplant; HIV: human immunodeficiency virus; IC: immunocompetent patient; SOT: solid organ transplant. The EC50 cutoff values for ACV resistance were 6.5 lM for HSV1 and 13.5 for HSV2, and the EC50 cutoff values for FOS resistance was 350 lM for both HSV1 and HSV2. EC50 in bold: drug resistance. The genotypic characterization of UL23 and UL30 genes from the 44 drug resistant clinical samples is available as a supplementary data (Table 3). a Insertions and deletions are referred in nucleotides. b Previously described resistance mutations. c Not previously characterized as polymorphism or associated with resistance.
A9T P15S L60P
E671K T931A N425T V905M V905M V905M N425T V905M V905M
R700G D672N V905M
E860K A910T
Resistanceb Natural polymorphism
UL30 gene mutations
Unknownc Resistanceb,a UL23 gene mutations
Natural polymorphism FOS (lM) ACV (lM)
Phenotypic susceptibility HSV type Date Sample Clinical context Patient No.
Table 2 UL23 and UL30 genes previously unreported mutations.
A874T K485N G506D
E. Frobert et al. / Antiviral Research 111 (2014) 36–41
Unknownc
40
been previously characterized as conferring ACV and FOS resistance. These mutations were likewise detected in this study, however only the strain harboring A605V was concordant with the characterized mutant phenotype (Gibbs et al., 1988; Larder et al., 1987; Saijo et al., 2005). This discrepancy remains to be explored. Finally, concerning novel UL30 mutations from ACV-resistant HSV2 (K485N and G506D), it has been determined that K485N and G506D do not induce FOS resistance. However, the potential for K485N to confer ACV resistance needs to be further explored, as it was found in conjunction with an uncharacterized M129I mutation on the UL23 gene. G506D is likely a natural polymorphism, as a nearby Q507H substitution has been previously described as a polymorphism (Burrel et al., 2010). K592E substitution on UL30 is considered to confer FOS resistance (Burrel et al., 2013), but its role in ACV resistance remains unclear due to concomitant substitutions on the UL23 gene. In conclusion, the significant rise in ACV resistance in HSCT patients supports the need to pursue national and international surveillance, and for the recruitment of multicentric studies. The mechanisms and reasons behind the recent rise in HSV resistance increase is worth further examination. Moreover, genotyping drug resistant HSV strains should be undertaken, particularly with respect to immunocompromised patients. Current mutations with unclear significance must be classified in order to establish an HSV mutation database for the purpose of drug resistant HSV screening using reliable genotypic assays (Burrel et al., 2012; Frobert et al., 2007; Malartre et al., 2012; Sauerbrei et al., 2012). Acknowledgements The authors would like to thank Emilie Sochay for collecting clinical data, Doris Li for reading the English version, and all the technicians in the virus isolation sector for their excellent expertise. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.antiviral. 2014.08.013. References Andrei, G., Snoeck, R., De Clercq, E., Esnouf, R., Fiten, P., Opdenakker, G., 2000. Resistance of herpes simplex virus type 1 against different phosphonylmethoxyalkyl derivatives of purines and pyrimidines due to specific mutations in the viral DNA polymerase gene. J. Gen. Virol. 81, 639– 648. Bohn, K., Zell, R., Schacke, M., Wutzler, P., Sauerbrei, A., 2011. Gene polymorphism of thymidine kinase and DNA polymerase in clinical strains of herpes simplex virus. Antivir. Ther. 16, 989–997. Burrel, S., Aime, C., Hermet, L., Ait-Arkoub, Z., Agut, H., Boutolleau, D., 2013. Surveillance of herpes simplex virus resistance to antivirals: a 4-year survey. Antiviral Res. 100, 365–372. Burrel, S., Bonnafous, P., Hubacek, P., Agut, H., Boutolleau, D., 2012. Impact of novel mutations of herpes simplex virus 1 and 2 thymidine kinases on acyclovir phosphorylation activity. Antiviral Res. 96, 386–390. Burrel, S., Deback, C., Agut, H., Boutolleau, D., 2010. Genotypic characterization of UL23 thymidine kinase and UL30 DNA polymerase of clinical isolates of herpes simplex virus: natural polymorphism and mutations associated with resistance to antivirals. Antimicrob. Agents Chemother. 54, 4833–4842. Chakrabarti, S., Pillay, D., Ratcliffe, D., Cane, P.A., Collingham, K.E., Milligan, D.W., 2000. Resistance to antiviral drugs in herpes simplex virus infections among allogeneic stem cell transplant recipients: risk factors and prognostic significance. J. Infect. Dis. 181, 2055–2058. Chibo, D., Druce, J., Sasadeusz, J., Birch, C., 2004. Molecular analysis of clinical isolates of acyclovir resistant herpes simplex virus. Antiviral Res. 61, 83–91. Christophers, J., Clayton, J., Craske, J., Ward, R., Collins, P., Trowbridge, M., Darby, G., 1998. Survey of resistance of herpes simplex virus to acyclovir in northwest England. Antimicrob. Agents Chemother. 42, 868–872.
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an in vitro assay based on detection of monophosphate forms of acyclovir and thymidine using HPLC/DAD. Antiviral Res. 95, 224–228. Morfin, F., Bilger, K., Boucher, A., Thiebaut, A., Najioullah, F., Bleyzac, N., Raus, N., Bosshard, S., Aymard, M., Michallet, M., Thouvenot, D., 2004. HSV excretion after bone marrow transplantation: a 4-year survey. J. Clin. Virol. 30, 341–345. Morfin, F., Souillet, G., Bilger, K., Ooka, T., Aymard, M., Thouvenot, D., 2000. Genetic characterization of thymidine kinase from acyclovir-resistant and -susceptible herpes simplex virus type 1 isolated from bone marrow transplant recipients. J. Infect. Dis. 182, 290–293. Nugier, F., Colin, J.N., Aymard, M., Langlois, M., 1992. Occurrence and characterization of acyclovir-resistant herpes simplex virus isolates: report on a two-year sensitivity screening survey. J. Med. Virol. 36, 1–12. Piret, J., Boivin, G., 2011. Resistance of herpes simplex viruses to nucleoside analogues: mechanisms, prevalence, and management. Antimicrob. Agents Chemother. 55, 459–472. Piret, J., Boivin, G., 2014. Antiviral drug resistance in herpesviruses other than cytomegalovirus. Rev. Med. Virol. 24, 186–218. Saijo, M., Suzutani, T., Morikawa, S., Kurane, I., 2005. Genotypic characterization of the DNA polymerase and sensitivity to antiviral compounds of foscarnetresistant herpes simplex virus type 1 (HSV-1) derived from a foscarnetsensitive HSV-1 strain. Antimicrob. Agents Chemother. 49, 606–611. Sauerbrei, A., Deinhardt, S., Zell, R., Wutzler, P., 2010. Phenotypic and genotypic characterization of acyclovir-resistant clinical isolates of herpes simplex virus. Antiviral Res. 86, 246–252. Sauerbrei, A., Liermann, K., Bohn, K., Henke, A., Zell, R., Gronowitz, S., Wutzler, P., 2012. Significance of amino acid substitutions in the thymidine kinase gene of herpes simplex virus type 1 for resistance. Antiviral Res. 96, 105–107. Saure, C., Schroeder, T., Zohren, F., Groten, A., Bruns, I., Czibere, A., Galonska, L., Kondakci, M., Weigelt, C., Fenk, R., Germing, U., Haas, R., Kobbe, G., 2011. Upfront allogeneic blood stem cell transplantation for patients with high-risk myelodysplastic syndrome or secondary acute myeloid leukemia using a FLAMSA-based high-dose sequential conditioning regimen. Biol. Blood Marrow Transplant. 18, 466–472. Schmid, C., Schleuning, M., Ledderose, G., Tischer, J., Kolb, H.J., 2005. Sequential regimen of chemotherapy, reduced-intensity conditioning for allogeneic stemcell transplantation, and prophylactic donor lymphocyte transfusion in highrisk acute myeloid leukemia and myelodysplastic syndrome. J. Clin. Oncol. 23, 5675–5687. Stranska, R., Schuurman, R., Nienhuis, E., Goedegebuure, I.W., Polman, M., Weel, J.F., Wertheim-Van Dillen, P.M., Berkhout, R.J., van Loon, A.M., 2005. Survey of acyclovir-resistant herpes simplex virus in the Netherlands: prevalence and characterization. J. Clin. Virol. 32, 7–18. van Velzen, M., Missotten, T., van Loenen, F.B., Meesters, R.J., Luider, T.M., Baarsma, G.S., Osterhaus, A.D., Verjans, G.M., 2013. Acyclovir-resistant herpes simplex virus type 1 in intra-ocular fluid samples of herpetic uveitis patients. J. Clin. Virol. 57, 215–221. Wang, Y., Wang, Q., Zhu, Q., Zhou, R., Liu, J., Peng, T., 2011. Identification and characterization of acyclovir-resistant clinical HSV-1 isolates from children. J. Clin. Virol. 52, 107–112.
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Resistance of herpes simplex viruses to acyclovir: An update from a ten-year survey in France Emilie Frobert a,b,⇑, Sonia Burrel c,d, Sophie Ducastelle-Lepretre e, Geneviève Billaud a, Florence Ader f, Jean-Sébastien Casalegno a,b, Viviane Nave a, David Boutolleau c,d, Mauricette Michallet e, Bruno Lina a,b, Florence Morfin a,b a
Laboratoire de Virologie, Centre de Biologie et Pathologie Est, Hospices Civils de Lyon, Lyon, France Virologie et Pathologie Humaine, Université Lyon 1, EA 4610, Faculté de Médecine RTH Laënnec, Lyon, France Sorbonne Universités, UPMC Univ Paris 06, CR7, Centre d’Immunologie et des Maladies Infectieuses (CIMI-Paris), INSERM U1135, Paris, France d AP-HP, Hôpitaux Universitaires Pitié-Salpêtrière – Charles Foix, Service de Virologie, Paris, France e Service d’Hématologie Clinique, Centre Hospitalier Lyon Sud, Lyon, France f Service de Maladies Infectieuses et Tropicales, Hôpital de la Croix-Rousse, Lyon, France b c
a r t i c l e
i n f o
Article history: Received 1 July 2014 Revised 30 July 2014 Accepted 25 August 2014 Available online 8 September 2014 Keywords: Herpes simplex virus Survey Antiviral drug resistance Hematopoietic stem cell transplant patients UL23 thymidine kinase UL30 DNA polymerase
a b s t r a c t The widespread use of acyclovir (ACV) and the increasing number of immunocompromised patients have raised concern about an increase in ACV-resistant herpes simplex virus (HSV). ACV resistance has traditionally been a major concern for immunocompromised patients with a frequency reported between 2.5% and 10%. The aim of this study was to reassess the status of HSV resistance to ACV in immunocompetent and immunocompromised patients over a ten year period, between 2002 and 2011. This was done by retrospectively following 1425 patients. In immunocompetent patients, prevalence of resistance did not exceed 0.5% during the study period; whereas in immunocompromised patients, a significant increase was observed, rising from 3.8% between 2002 and 2006 (7/182 patients) to 15.7% between 2007 and 2011 (28/178) (p = 0.0001). This sharp rise in resistance may largely be represented by allogeneic hematopoietic stem cell transplant patients, in which the prevalence of ACV resistance rose similarly from 14.3% (4/28) between 2002 and 2006 to 46.5% (26/56) between 2007 and 2011 (p = 0.005). No increase in ACV resistance was detected in association with other types of immune deficiencies. Genotypic characterization of HSV UL23 thymidine kinase and UL30 DNA polymerase genes revealed 11 and 7 previously unreported substitutions, respectively. These substitutions may be related to potential polymorphisms, drug resistance, or other mutations of unclear significance. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Repeated and long-term acyclovir (ACV) use associated with clinical and immunological features may lead to the development of herpes simplex virus (HSV) resistant to this drug. HSV resistance to ACV is primarily a concern for immunocompromised patients whereas the prevalence remains low in immunocompetent patients. Since the commercialisation of ACV in the 1980s, resistance to this drug in immunocompetent patients has been estimated at 0.5% (Christophers et al., 1998; Fife et al., 1994; Nugier et al., 1992). After twenty years of use, the prevalence did not ⇑ Corresponding author at: Laboratoire de Virologie, Centre de Biologie et Pathologie Est, Hospices Civils de Lyon, 59 boulevard Pinel, 69 500 Bron, France. Tel.: +33 4 72 12 96 53; fax: +33 4 72 12 95 00. E-mail address: [email protected] (E. Frobert). http://dx.doi.org/10.1016/j.antiviral.2014.08.013 0166-3542/Ó 2014 Elsevier B.V. All rights reserved.
increase (Danve-Szatanek et al., 2004; Stranska et al., 2005). Nevertheless, the recurrent and widespread use of ACV and its prodrug Val-ACV, including FDA-approved short-course and high dose regimens to treat HSV recurrence, may be contributing factors towards the emergence of resistance (Cunningham et al., 2012). One recent Chinese study reported an unexpectedly high prevalence of ACV-resistant HSV of 4% in immunocompetent children with oral herpetic lesions (Wang et al., 2011). Further, some cases of recurrent herpetic keratitis have been found to be associated with ACV-resistant virus with a prevalence of 6.4% in immunocompetent patients (Duan et al., 2009). Additionally, immunocompetent patients with genital herpes have a higher prevalence of ACV-resistant HSV (Kriesel et al., 2005). With regards to immunocompromised patients, the prevalence of ACV-resistant HSV is largely higher and varies between 3.5% and 10%, depending on the type of immunosuppression. HSV resistant to ACV can induce large,
E. Frobert et al. / Antiviral Research 111 (2014) 36–41
extensive and ulcerative cutaneous infections, but also esophagitis, pneumonia and encephalitis, which can be fatal without clinical improvement. Among immunocompromised patients, the prevalence of resistance was reported between 3.5% and 7% in HIV patients, 2.5% and 10% in solid organ transplant patients, and with the greatest prevalence witnessed at 4.1% and 10.9% in hematopoietic stem cell transplant (HSCT) patients (Danve-Szatanek et al., 2004; Piret and Boivin, 2011). The prevalence has even been observed as high as 25% in allogeneic HSCT patients (Chakrabarti et al., 2000; Morfin et al., 2004). 95% of resistant cases are attributed to mutations on the UL23 gene encoding for thymidine kinase and 5% are attributed to mutations on the UL30 gene encoding for viral DNA polymerase (Piret and Boivin, 2011, 2014). Antiviral drug resistance screening using genotypic techniques are an efficient approach to make prompt virological detection to adapt antiviral treatment. Nevertheless, genotypic characterization of UL23 and UL30 genes have revealed many natural polymorphisms in both genes, which can complicate the interpretation of genetic sequences, particularly when detected substitutions have not been previously described or characterized (Burrel et al., 2010). The objectives of the present study were: (i) to reassess the frequency of ACV resistance in immunocompetent and immunocompromised patients from a ten-year survey; (ii) to describe new potential polymorphisms and resistance mutations, in the UL23 and UL30 genes, based on 22 ACV-sensitive and 40 ACV-resistant HSV.
37
and FOS (Astra, France), as previously described (Langlois et al., 1986). The effective concentration 50 (EC50) cut-off values for resistance to ACV were 6.5 lM for HSV1 and 13.5 lM for HSV2, and the EC50 cut-off value for resistance to FOS was 350 lM for both HSV1 and HSV2 (Nugier et al., 1992). 2.3. Statistical analysis Student’s t-test and Person v2-test were used to assess intergroup differences between the two timelines 2002–2006 versus 2007–2011. The division of the 10-year study period was made with regards to resistance evolution. Statistical analyses were performed on EpiInfo software (V 3.5.1 CDC). Odds ratio (OR) and 95% confidence interval (CI) were calculated to determine the likelihood of detecting ACV-resistant HSV during the two periods. A test was significant when the p value was less than 0.05. 2.4. Genotypic analysis of antiviral resistance
2. Materials and methods
Genotypic analysis of ACV-resistant HSV1 and HSV2 was performed directly on clinical samples by sequencing the UL23 and UL30 genes, which encodes for thymidine kinase and DNA polymerase, respectively, as previously described (Burrel et al., 2010; Frobert et al., 2008). When a sensitive HSV was previously isolated from a patient harboring a resistant virus, the UL23 gene of the sensitive strain was also sequenced. Nucleotide sequences were compared with reference strains SC16 (HSV1) and 333 (HSV2) (GenBank accession numbers X03764 and V00466, respectively) using SeqmanII software (DNAStar Inc.).
2.1. Patients and specimens
3. Results
In this single-centre study, during the 2002–2011 period, 1529 samples from 1425 patients had tested HSV-positive, by culture on MRC-5 cells. The University-Hospital of Hospices Civils of Lyon is the regional reference centre of 5500 beds and serves a population of 1.7 million. Some patients were sampled more than once but each sample was considered as a distinct episode of herpetic infection. 70% of patients were adults (1064 clinical samples were obtained from 1005 adults, median age of 50 (range: 18–75)) and 30% were children (465 clinical samples were obtained from 420 children, median age of 6.5 (range: 1–17)). HSV1 was detected in 1337 clinical samples (429 oropharyngeal, 191 nasopharyngeal, 200 cutaneous, 252 bronchoalveolar lavages, 185 genital and 80 ocular samples) and HSV2 in 192 clinical samples (116 genital, 2 nasopharyngeal, 70 cutaneous, 1 bronchoalveolar lavage, and 3 ocular samples) by specific immune fluorescence. CSF and aqueous humor were excluded as they could not be cultured. In the 1064 clinical adult samples, one was from a patient whose immune status was unavailable, 703 were from immunocompetent patients, and 360 were from immunosuppressed patients including 90 hematopoietic stem cell transplants (HSCT) (87 allogeneic and 3 autologous), 37 HIV, 68 haemopathies, 37 non-haemopathy malignancies, 94 solid organ transplants (SOT), and 34 other sources of immunosuppression. From the 465 children samples, 324 were from immunocompetent children and 141 were from immunosuppressed children including 30 allogeneic HSCT, 36 haemopathies, 12 non-haemopathy malignancies, 38 SOT, 4 HIV and 21 other sources of immunosuppression.
3.1. Frequency of ACV-resistant HSV over 2002–2011
2.2. Phenotypic diagnosis of antiviral resistance Screening for antiviral resistance was systematically performed on the 1529 HSV-positive culture using a neutral red dye-uptake assay to determine its sensitivity to ACV (GlaxoWellcome, France)
3.1.1. Adults One case of ACV-resistant HSV was detected in an immunocompetent patient (0.14%) and another case of resistance was detected in a patient whose immune status was unavailable (Table 1). Among 360 immunocompromised patients, 35 cases of resistance were detected (9.7%) with 30 cases from allogeneic HSCT patients (30/87, 34.5%), 3 cases from HIV patients (3/37, 8.1%), 1 case from a patient with haemopathy (1/68, 1.5%) and 1 case from a SOT patient (1/94, 1.1%). 3.1.2. Children In immunocompetent children, no cases of resistance were detected; but in immunocompromised children, 7 cases were detected (7/141, 5%) with 5 cases coming from allogeneic HSCT patients (5/30, 16.7%), 1 case from a child with haemopathy (1/ 36, 2.8%) and 1 case from a SOT patient (1/38, 2.6%). 3.2. Resistance evolution 3.2.1. Adults Over the 2002–2011 period, the frequency of ACV resistance has increased from 1.4% in 2002 to 5.2% in 2011, while the number of clinical samples testing HSV-positive remained relatively consistent year over year (Fig. 1). A significant increase was observed in 2007, prompting the division of the study period to be made between 2002–2006 versus 2007–2011 (Fig. 1). The overall prevalence of ACV-resistant HSV in adults increased significantly between 2002–2006 and 2007–2011, from 1.4% to 6% (OR = 4.22; p = 0.00006) (Table 1). In immunocompetent adults, the prevalence of resistance remained under 0.5%, throughout the years (p = 0.59) (Table 1). In immunocompromised patients, a significant increase
6.3 28.6
4
15 10 13 12 4.8
3.9 6.3
was observed from 3.8% in 2002–2006 to 15.7% in 2007–2011 (OR = 4.67; p = 0.0001). The substantial rise in resistance for immunocompromised patients can be primarily attributed to allogeneic HSCT patients where the prevalence rose from 14.3% in 2002–2006 to 46.5% in 2007–2011 (OR = 4.72; p = 0.005). Analysis of other immune deficiencies did not reveal significant increases in resistance, as shown by the p-value: HIV patients (p = 0.52), patients with haemopathies (p = 0.53), patients with non-haemopathy malignancies (p undefined), SOT patients (p = 0.35), and for other cases of immunosuppression (p undefined) (Table 1).
ACV: acyclovir; HSCT: hematopoietic stem cell transplant; HIV: human immunodeficiency virus; SOT: solid organ transplant. a One adult had a non-documented immune status. b Were included in other malignancies: cancer of bronchus, pancreas and breast, adenocarcinoma, osteosarcoma. c Were included in other: patients under immunosuppression treatment and/or corticoids for Crohn disease, Wegener’s granulomatosis, sarcoidosis, Horton disease or pemphigus.
2.6
2.8
5 16.7
p = 0.40 Undefined p = 0.40 p = 0.13 Undefined p = 0.58 p = 0.58 p = 0.66 Undefined 1.60 [0.33–8.67] Undefined 1.64 [0.35–7.63] 5.66 [0.6–157.44] Undefined Undefined Undefined Undefined Undefined 1.5
465 324 141 30 4 36 12 38 21 212 148 64 14
253 176 77 16 4 21 2 25 9 3 0 3 1 0 1 0 1 0 Children Immunocompetent Immunocompromised Allogeneic HSCT HIV Haemopathies Other malignanciesb SOT Otherc
1.2
4 0 4 4 0 0 0 0 0
1.9
7 0 7 5 0 1 0 1 0
1.1 3
37 68 37 94 34 7 36 23 33 19 30 32 14 61 15 3 0 0 0 0
10
0 1 0 1 0
15.7 46.5
2.8
3 1 0 1 0
8.1 1.5
p = 0.00006 p = 0.59 p = 0.0001 p = 0.005 Undefined p = 0.52 p = 0.53 Undefined p = 0.35 Undefined 1064 703 360 873 464 286 178 563 600 417 182 280 9a 1 7 40
Adults Immunocompetent Immunocompromised Allogeneic HSCT Autologous HSCT HIV Haemopathies Other malignanciesb SOT Otherc
1.4 0.2 3.8 14.3
28 0 28 260
6
37 1 35 300
3.5 0.14 9.7 34.5
Odd ratio % of resistance Nb of samples Nb of resistance
Total
% of resistance Nb of samples Nb of resistance
2007–2011
% of resistance Nb of samples Nb of resistance
2002–2006
Table 1 Frequency of ACV resistance between 2002–2006 versus 2007–2011.
4.22 [1.97–9.03] Undefined 4.67 [1.98–10.99] 4.72 [1.46–15.33] Undefined Undefined Undefined Undefined Undefined Undefined
E. Frobert et al. / Antiviral Research 111 (2014) 36–41
p value
38
3.2.2. Children No increase in resistance was observed in immunocompetent or immunocompromised children during the two study periods (p = 0.40). Likewise, there was no increase in ACV-resistant HSV in children undergoing allogeneic HSCT (p = 0.13). 3.3. Genotypic analysis of HSV 3.3.1. Genotypic characterization of drug sensitive HSV clinical isolates 22 ACV-sensitive HSV1 have been previously isolated among patients with ACV-resistant HSV1. The UL23 sequencing revealed 7 natural polymorphisms that have not been previously reported: D76N, I100T, H142Y, D215H (D215N has been previously described (Wang et al., 2011)), P268L (P268T and P268S has been previously described (Kudo et al., 1998; van Velzen et al., 2013)), A299S and L341R. 3.3.2. Genotypic characterization of drug resistant HSV clinical isolates 3.3.2.1. New UL23 gene mutations not previously described (Table 2). In ACV-resistant HSV1, 3 previously unreported substitutions were detected: L242P (Patient No. 34), L249P (Patient No. 10) and E371D (Patient No. 35). In ACV-resistant HSV2, 1 previously unreported substitution was detected: M129I (Patient No. 39). 3.3.2.2. New UL30 gene mutations not previously described (Table 2). In ACV-resistant HSV1, 5 novel mutations were found: E860K (Patient No. 6), E671K (Patient No. 20), T931A (Patient No. 31), A874T (Patient No. 38) and A910T (Patient No. 7) (A910V has been previously described (Saijo et al., 2005)). In ACV-resistant HSV2, 2 novel mutations were detected: K485N (Patient No. 39) and G506D (Patient No. 44). 3.3.3. Distribution of UL23 and UL30 mutations in drug resistant HSV Among the 44 drug resistant HSV, 40 HSV were analyzed for UL23 and UL30 mutations as 4 samples were depleted (Table 3, supplementary data). ACV resistance was due to mutations on the UL23 gene in 85% of the cases (34/40). These mutations include 10 substitutions (10/40; 25%), 3 stop codons (3/40; 7.5%), 11 nucleotide insertions (11/40; 27.5%), and 10 nucleotides deletions (10/40; 25%). It should be noted that additions/deletions represent 62% (21/34) of mutations involving the UL23 gene. 7.5% of resistance cases (3/40) involved mutations on the UL30 gene encoding DNA polymerase. Two HSV had drug resistant mutations on both the UL23 and UL30 genes (Patients No. 25 and 41) and one strain had mutations on both respective genes, but with unclear significance (Patients No. 39). One ACV-resistant HSV had no mutation on the UL23 and UL30 genes (Patient No. 21). 4. Discussion The widespread use of ACV since its commercialization in the 1980s supports the need for a continued surveillance of HSV resistance, in immunocompetent and immunocompromised patients. The last surveys published on this topic date back to 2004
E. Frobert et al. / Antiviral Research 111 (2014) 36–41
39
Fig. 1. Evolution of ACV resistance over 2002–2011.
(Danve-Szatanek et al., 2004) and 2005 (Stranska et al., 2005). There was a 4-year survey done in 2013 that described HSV resistance to antivirals, but only in patients with suspected clinical resistance (Burrel et al., 2013). In the 10-year retrospective study presented herein, 1529 HSV-positive samples were analyzed from 1425 patients. Consistent with prior statistics, ACV resistance in immunocompetent patients has not increased in recent years and the prevalence remains below 0.5%. In France and a majority of other countries, systemic ACV therapy is regulated by medical prescription. A future with potentially greater access to antivirals provides more reasons for sustained surveillance in immunocompetent populations for resistance. In immunocompromised patients, our survey showed a significant increase in ACV resistance from 2007 that was exclusively linked to HSCT patients. With improved protocols for bone marrow transplantation, the number of HSCTs performed has risen in recent years. New chemotherapy regimens in preparation for HSCT now induce longer and deeper immunosuppression. Since 2006, the Haematology Department in Lyon, France, has been using the FLAMSA-based high-dose sequential chemotherapy regimen before allogeneic hematopoietic stem cell transplantation for refractory patients with high risk myelodysplastic syndrome or secondary acute myeloid leukaemia (Saure et al., 2011; Schmid et al., 2005). This protocol is particularly aggressive and exposes patients to a longer period of neutropenia than in myeloablative or reduced intensity treatments. The type of HSCT (ie stem cells from bone marrow harvest, from leukapheresis or from cord blood cell) may also be a risk factor in the emergence of infectious diseases (Gilis et al., 2014). Thus, multivariate analyses are needed at a national and international level to understand why and how HSV resistance to antiviral drugs has increased over these last years in HSCT patients. It has been conventionally reported that 95% of ACV resistance cases are a consequence of mutations in the UL23 gene (reviewed in Piret and Boivin, 2011, 2014), of which additions/deletions account for half of the mutations (Gaudreau et al., 1998; Morfin et al., 2000). The sequence analysis of this study revealed that 85% of ACV-resistant strains were due to UL23 gene mutations, of which 62% were nucleotide additions/deletions. Recently, Burrel et al. have reported an even higher proportion addition/deletions accounting for 80% of UL23 gene mutations conferring ACV resistance (Burrel et al., 2013). This increased trend of UL23 gene mutations caused by nucleotide additions/deletions would facilitate the interpretation of genotypic results. In this study, UL30 gene
mutations were involved in 7.5% of resistance cases. Interestingly, 7.5% of HSV samples carried mutations in both UL23 and UL30 genes, which is not conventionally seen in ACV-resistant and FOS-sensitive strains. The genotypic profile of drug resistant HSV must be taken into consideration to optimize the detection of resistant HSV, particularly for the purposes of molecular-based assays. The natural polymorphism of UL23 and UL30 genes has been largely described before (Bohn et al., 2011; Burrel et al., 2010) and drug resistance can be induced by concomitant mutations (Frobert et al., 2008). Classifying uncharacterized mutations as either new polymorphism or conferring drug resistance continues to be a difficult task. Concerning new UL23 gene mutations detected from our survey of ACV-resistant HVS1 (L242P, L249P, E371D), L249P and E371D may likely confer ACV resistance as no concurrent mutation was found on the UL30 gene. The L242P substitution remains of unclear significance, as it was observed in conjunction with D162N, an already known drug resistance mutation (Chibo et al., 2004; Malartre et al., 2012). In ACV-resistant HSV2, the sole M129I substitution found on UL23 gene remains of unclear significance, as it was observed in conjunction with a novel K485N substitution on UL30, which has never been previously reported. Concerning new UL30 gene mutations in ACV-resistant HSV1 (E860K, E671K, T931A, A874T, A910T), E860K may potentially confer resistance as no concurrent mutation was uncovered in the UL23 gene. E860K may also have a role in FOS resistance, but this remains to be determined as it was observed in conjunction with R700G, a known FOS and ACV resistance conferring mutation (Andrei et al., 2000; Gibbs et al., 1988). E671K may be a UL30 polymorphism, as a ‘‘+1G’’ nucleotide insertion on the UL23 gene is presumably the more likely factor in ACV resistance. Further, a proximate mutation, E675A, has been previously described as a natural UL30 polymorphism (Burrel et al., 2010). The amino acid change A910T could be responsible for FOS resistance as A910V has been attributed to an FOS-resistant but ACV-sensitive phenotype (Saijo et al., 2005). Prior studies have yielded conflicting results concerning the role of UL30’s D672N substitution in ACV resistance (Burrel et al., 2010; Sauerbrei et al., 2010), but one of our ACV-resistant and FOS-sensitive strain had no mutation on the UL23 and UL30 genes except D672N. UL30 substitutions, T931A and A874T, do not induce FOS resistance and they likely correspond to natural polymorphism, as they were detected in conjunction with UL23 mutations known to confer ACV resistance (Duan et al., 2009). UL30 substitutions, A605V and A719V, have
+1G 440
G200D
M129I
L242P E371D
G240E G240E N78D L140F G39E
L249P
+1G 430 +1G 436 D162N
G200S
G240E Q89R G240E C6G G251C V267L P268T D286E N376H
737 334 125 151 125 125 92 92 152 79 20 35 53 41 20 26 9 24 48 155 1 1 1 1 1 1 1 1 2 2 2005 2005 2007 2008 2011 2011 2011 2011 2002 2011 Throat Throat Throat BAL Throat Throat Throat Throat Cutaneous Genital HSCT HSCT HSCT HSCT HSCT HSCT HSCT HSCT NA HSCT 6 7 10 20 31 34 35 38 39 44
BAL: bronchoalveolar lavage; HSCT: hematopoietic stem cell transplant; HIV: human immunodeficiency virus; IC: immunocompetent patient; SOT: solid organ transplant. The EC50 cutoff values for ACV resistance were 6.5 lM for HSV1 and 13.5 for HSV2, and the EC50 cutoff values for FOS resistance was 350 lM for both HSV1 and HSV2. EC50 in bold: drug resistance. The genotypic characterization of UL23 and UL30 genes from the 44 drug resistant clinical samples is available as a supplementary data (Table 3). a Insertions and deletions are referred in nucleotides. b Previously described resistance mutations. c Not previously characterized as polymorphism or associated with resistance.
A9T P15S L60P
E671K T931A N425T V905M V905M V905M N425T V905M V905M
R700G D672N V905M
E860K A910T
Resistanceb Natural polymorphism
UL30 gene mutations
Unknownc Resistanceb,a UL23 gene mutations
Natural polymorphism FOS (lM) ACV (lM)
Phenotypic susceptibility HSV type Date Sample Clinical context Patient No.
Table 2 UL23 and UL30 genes previously unreported mutations.
A874T K485N G506D
E. Frobert et al. / Antiviral Research 111 (2014) 36–41
Unknownc
40
been previously characterized as conferring ACV and FOS resistance. These mutations were likewise detected in this study, however only the strain harboring A605V was concordant with the characterized mutant phenotype (Gibbs et al., 1988; Larder et al., 1987; Saijo et al., 2005). This discrepancy remains to be explored. Finally, concerning novel UL30 mutations from ACV-resistant HSV2 (K485N and G506D), it has been determined that K485N and G506D do not induce FOS resistance. However, the potential for K485N to confer ACV resistance needs to be further explored, as it was found in conjunction with an uncharacterized M129I mutation on the UL23 gene. G506D is likely a natural polymorphism, as a nearby Q507H substitution has been previously described as a polymorphism (Burrel et al., 2010). K592E substitution on UL30 is considered to confer FOS resistance (Burrel et al., 2013), but its role in ACV resistance remains unclear due to concomitant substitutions on the UL23 gene. In conclusion, the significant rise in ACV resistance in HSCT patients supports the need to pursue national and international surveillance, and for the recruitment of multicentric studies. The mechanisms and reasons behind the recent rise in HSV resistance increase is worth further examination. Moreover, genotyping drug resistant HSV strains should be undertaken, particularly with respect to immunocompromised patients. Current mutations with unclear significance must be classified in order to establish an HSV mutation database for the purpose of drug resistant HSV screening using reliable genotypic assays (Burrel et al., 2012; Frobert et al., 2007; Malartre et al., 2012; Sauerbrei et al., 2012). Acknowledgements The authors would like to thank Emilie Sochay for collecting clinical data, Doris Li for reading the English version, and all the technicians in the virus isolation sector for their excellent expertise. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.antiviral. 2014.08.013. References Andrei, G., Snoeck, R., De Clercq, E., Esnouf, R., Fiten, P., Opdenakker, G., 2000. Resistance of herpes simplex virus type 1 against different phosphonylmethoxyalkyl derivatives of purines and pyrimidines due to specific mutations in the viral DNA polymerase gene. J. Gen. Virol. 81, 639– 648. Bohn, K., Zell, R., Schacke, M., Wutzler, P., Sauerbrei, A., 2011. Gene polymorphism of thymidine kinase and DNA polymerase in clinical strains of herpes simplex virus. Antivir. Ther. 16, 989–997. Burrel, S., Aime, C., Hermet, L., Ait-Arkoub, Z., Agut, H., Boutolleau, D., 2013. Surveillance of herpes simplex virus resistance to antivirals: a 4-year survey. Antiviral Res. 100, 365–372. Burrel, S., Bonnafous, P., Hubacek, P., Agut, H., Boutolleau, D., 2012. Impact of novel mutations of herpes simplex virus 1 and 2 thymidine kinases on acyclovir phosphorylation activity. Antiviral Res. 96, 386–390. Burrel, S., Deback, C., Agut, H., Boutolleau, D., 2010. Genotypic characterization of UL23 thymidine kinase and UL30 DNA polymerase of clinical isolates of herpes simplex virus: natural polymorphism and mutations associated with resistance to antivirals. Antimicrob. Agents Chemother. 54, 4833–4842. Chakrabarti, S., Pillay, D., Ratcliffe, D., Cane, P.A., Collingham, K.E., Milligan, D.W., 2000. Resistance to antiviral drugs in herpes simplex virus infections among allogeneic stem cell transplant recipients: risk factors and prognostic significance. J. Infect. Dis. 181, 2055–2058. Chibo, D., Druce, J., Sasadeusz, J., Birch, C., 2004. Molecular analysis of clinical isolates of acyclovir resistant herpes simplex virus. Antiviral Res. 61, 83–91. Christophers, J., Clayton, J., Craske, J., Ward, R., Collins, P., Trowbridge, M., Darby, G., 1998. Survey of resistance of herpes simplex virus to acyclovir in northwest England. Antimicrob. Agents Chemother. 42, 868–872.
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