Prevalence of the K65R Resistance ReverseTranscriptase Mutation in Different HIV-1Subtypes in Israel

Dan Turner,1* Eduardo Shahar,2 Eugene Katchman,1 Eynat Kedem,2 Natasha Matus,1

Michal Katzir,1 Gamal Hassoun,2 Shimon Pollack,2 Rivka Kessner,1

Mark A. Wainberg,3 and Boaz Avidor1

1Kobler AIDS Center, Infectious Diseases Unit, Tel-Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel-AvivUniversity, Tel-Aviv, Israel2AIDS Institute, Rambam Medical Center and Rappaport Faculty of Medicine, Haifa, Israel3McGill University AIDS Centre, Jewish General Hospital, Montreal, Quebec, Canada

The K65R mutation in HIV-1 reverse transcriptase(RT) can be selected by the RT inhibitors tenofovir(TDF), abacavir (ABC), and didanosine (DDI).Recently, in vitro studies have shown that K65Ris selected in tissue culture more rapidly withsubtype C than subtype B viruses. The prevalenceof K65R in viruses sequenced at the Tel-Aviv AIDSCenter was evaluated. This study analyzed retro-spectively sequences from 1999 to 2007 inpatients treated with TDF, ABC, and/or DDI andcompared rates of mutational prevalence be-tween subtypes. Fisher’s exact test was used todetermine statistical significance. Forty-foursequences from patients treated with the threeabove-cited drugs were analyzed. Subtypes A(n¼1), CRF01_AE (n¼4), CRF02_AG (n¼2), B(n¼21), C (n¼11), D (n¼ 1), F (n¼ 3), and G(n¼1) were represented. Seven non-B viruseshad the K65R mutation, which was only found inone subtype B virus. Of these seven samples fourwere subtype C, one was subtype CRF01_AE, andtwo were subtype CRF02_AG. None of the eightviruses with K65R harbored thymidine analoguemutations. In this study, non-subtype B virusespossessed the K65R mutation at higher incidencethan subtype B viruses. Subtype C viruses may beespecially prone to develop this mutation. Largerstudies are needed to confirm these data. Effortsshould be intensified to understand better differ-ences in drug resistance between various HIVsubtypes. J. Med. Virol. 81:1509–1512,2009. ª 2009 Wiley-Liss, Inc.

KEY WORDS: HIV-1;subtypes;resistance;K65R

INTRODUCTION

The K65R mutation in reverse transcriptase (RT) isassociated with resistance to tenofovir (TDF), abacavir

(ABC), and didanosine (DDI) [Gu et al., 1994, 1995;Wainberg et al., 1999; Stone et al., 2004] and may beresponsible for high rates of virological failure [Waterset al., 2008]. A recent study described an overall inci-dence of K65R of 2.8 cases per 100 person-years. When anon-nucleoside reverse transcriptase (NNRTI) was usedtogether with TDF, the incidence was 2.59 cases per 100person-years, 2.36 for DDI, 0.36 for ABC. The riskdecreased with the use of protease inhibitors andincreased to 41.46 when TDF, DDI, and ABC were usedtogether without other drugs [Waters et al., 2008].

K65R occurs rarely in combination with thymidineanalogue mutations (TAMs) due to antagonism of theNRTI monophosphate excision activity of RT [Parikhet al., 2007]. Although K65R is relatively uncommon,there has been an increase in its prevalence in northerncountries as a result of the widespread clinical use ofTDF [Gianotti et al., 2004; Valer et al., 2004; Winstonet al., 2004; Wainberg, 2005; Gallant et al., 2006].Approximately 5,000 individuals with HIV who wereliving in Israel at the end of 2007, with a prevalence of0.07%. (http://www.health.gov.il). There are severalHIV subtypes in Israel, and TDF is one of the main drugsbeingprescribed nownationwide.According toonestudyon drug-na€ıve patients, the presence of different HIV-1subtypes in the country was approximately 11%for A, 11% CRF01_AE, 16% B, and 57% C [Grossmanet al., 2005].

A recent in vitro study showed that K65R is selectedmore rapidly in tissue culture with subtype C than

*Correspondence to: Dan Turner, Kobler AIDS Center, Infec-tious Diseases Unit, Tel-Aviv Sourasky Medical Center, WeizmannStreet, Tel-Aviv 64239, Israel.E-mail: dan.turner@tasmc.health.gov.il

Accepted 27 April 2009

DOI 10.1002/jmv.21567

Published online in Wiley Interscience(www.interscience.wiley.com)

Journal of Medical Virology 81:1509–1512 (2009)

ª 2009 WILEY-LISS, INC.

subtype B viruses [Brenner et al., 2006]. Other studieshave now indicated a higher prevalence of K65R intreated patients in countries harboring subtype C virus-es [Doualla-Bell et al., 2006; Orrell et al., 2008; Pillayet al., 2008]. The availability of antiretroviral drugs,including TDF, in low-income countries and the expan-sion of non-B subtypes in western countries has estab-lished the need to evaluate the preference of K65R inthese subtypes in comparison to subtype B; this was theprimary purpose of this study. A secondary objective wasto compare the time to therapeutic failure betweenpatients with and without the K65R mutation. Theincidence of K65R among samples genotyped at the Tel-Aviv Kobler AIDS Center (Israel) in patients who hadbeen treated with TDF, ABC, or DDI was analyzed.Study samples were obtained from patients treated atthe Kobler AIDS Center in Tel-Aviv as well as frompatients treated at the Rambam Medical Center(Haifa, Israel).

METHODS

The presence of K65R in all sequences undertakenbetween years 1999 and 2007 was retrospectively as-sessed. Viral samples were from resistance tests carriedout in patients who had failed antiretroviral therapy andwho had received regimens that included TDF, ABC,and/or DDI. In order to avoid deselection of K65R, pa-tients who had received previously these drugs and werenot taking any of them at the time of the test wereexcluded. The duration of therapy with any of TDF, DDI,or ABC in order to compare the time to therapeuticfailure between patients with and without the K65Rmutation was also assessed. Sequencing was carried outusing TruGene� kits obtained from Siemens MedicalSolutions Diagnostics, Inc. (Tarrytown, NY). Sequenc-ing of both the RT and PR genes also permitted thesubtypes determination of the viral isolates (Stanforddatabase) (http://hivdb.stanford.edu/). Fisher’s exacttest was used to compare the prevalence of mutationsamong different subtypes. Comparison of treatmentduration by TDF, ABC, and/or DDI was by Mann–Whitney t-test (statistical analyses were performedusing GraphPad Prism version 5.00 for Windows,GraphPad Software, San Diego, CA, www.graphpad.com). The study was approved by relevant InstitutionalEthical Committees.

RESULTS

Forty-four sequences of viruses from patients treatedwith the above drugs were analyzed (one sequence perpatient). Subtypes A, CRF01_AE, CRF02_AG, B, C, D, F,and G were represented. Demographic and laboratorycharacteristics and as drug regimens are described inTable I. Patients originating from Ethiopia harboredsubtype C viruses and patients from Ukraine usuallyharbored CRF01_AE. The distribution of the differentsubtypes based on the presence of the K65R mutation isshown in Table II.

Eight of the 44 sequences examined harbored K65R(18.1%). Only one subtype B virus possessed K65Rcompared to seven non-B subtypes (P< 0.05): OneCRF01_AE, two CRF02_AG, and four subtype C virusescontained K65R. Over one-half of the viruses withoutK65RweresubtypeB(Table I).Twoof thesevirusesweremixtures of K65R, which could reflect non-adherence totherapy. Although the numbers are small, there was asignificant difference between the numbers of subtype Band C viruses with K65R (P< 0.05). The numbers ofpatients harboring B or non-B viruses who had receivedTDF were comparable, 8 and 11, respectively. Six of theeight (75%) patients with K65R were treated with TDF(among them the patient with the subtype B virus). Thetwo other (one patient subtype C and one CRF02_AG)were treated with a DDI-containing regimen. TAMswere not seen in viruses harboring the K65R mutation.TAMs were observed in 14 viruses in the non-K65Rgroup. One subtype B virus possessed the multi-NRTIresistance Q151M mutation.

The median duration of therapy with TDF, ABC, and/or DDI was 23 months for the subtype B samples withoutK65R compared with 6 months for non-B samples withK65R (P<0.05). Median time to acquisition of K65R innon-B samples without K65R was 12 months. Therewere no significant differences between the non-B sub-types with K65R and the B subtype without K65R.Because only one B virus harbored K65R, this subtypewas not included in the duration of treatment analysis(thispatienthadbeen in treatment foronly3monthsandhad confessed to being non-adherent to therapy).

DISCUSSION

These results show a higher prevalence of K65R innon-B subtypes compared to B viruses in patientstreated with TDF, DDI and/or ABC. Although most ofthe patients harboring viruses with K65R had receivedTDF, the distribution of the drug was comparable be-tween B and non-B subtypes. The distribution of TAMsbetween B and non-B subtypes was also comparable;previous regimens apparently did not influence theseresults. The treatment of patients who failed regimensthat lead to K65R has been intensified and changed toinclude ZDV. The viral load in most of the patientsdecreased significantly and most of them were clinicallystable (data not shown).

A recent in vitro study showed that K65R can beselected more rapidly in cell culture in subtype C thansubtype B viruses [Brenner et al., 2006]. One of theexplanations is that subtype C isolates contain uniquepolymorphisms in RT codons 64 (AAG ! AAA), 65(AAA ! AAG), and 66 (AAA ! AAG), absent in othersubtypes [Brenner et al., 2006]. This could explain ourobservation that time to treatment failure in the K65Rgroup in the non-B subtype was relatively shorter thanthat in the subtype B group without the mutation. Only afew clinical studies have described the rates of K65R innon-B subtypes. In Botswana [Doualla-Bell et al., 2006],patients with subtype C treated with DDI showed a

1510 Turner et al.

J. Med. Virol. DOI 10.1002/jmv

relatively high prevalence of K65R, that is, 7 of 15patients who failed therapy of a total of 23 treatedpatients harbored this mutation. A study from SouthAfrica showed a rate of 14% for the K65R mutationamong65subtypeCsamples [Pillayetal., 2008].Anotherrecent study from South Africa showed a rate of 10% forK65R among patients who failed a first regimenthat included stavudinie (d4T) [Orrell et al., 2008]. AThai study showed that 7% of patients possessedK65R after failing an initial d4T/3TC/NVP regimen[Sungkanuparph et al., 2008].

K65R may also be less prevalent in subtype A thansubtype C infections [Gupta et al., 2005]. One studyshowed that the combination of TDF with either efavir-enz or nevirapine may also favor the emergence of K65Rin certain subtypes [Von Wyl et al., 2008], although thiswas not proved in the current study. This study alsoshowed the diversity of HIV-1 subtypes in Israel andtheir origin. Patients harboring subtype C viruses emi-grated from Ethiopia, those with CRF01_AE originatedin the Ukraine, while those with subtype B were bornmainly in Israel.

TABLE I. Characteristics of Patients Harboring Viruses (a) Without and (b) With the K65R Mutation

Patientno. Gender Age VLa CD4b Last regimenc Previous drugsd

Country oforigin

CDCstaging

Hepatitisvirus

co-infection

(a)1 m 55 3.11 176 ABC, 3TC, EFV AZT, DDC Israel A32 f 29 3.58 744 ABC, 3TC, NVP D4T Israel A13 f 53 3.34 309 ABC, AZT, 3TC, NFV Ethiopia A34 m 31 4.85 90 ABC, DDI, DDC AZT, 3TC, IDV, RTV, NFV Israel A25 m 31 3.49 362 ABC, EFV, r/LPV AZT, 3TC, D4T, IDV Argentina A2 HCV6 m 42 5.30 353 ABC, NVP, NFV AZT, 3TC, D4T, DDI, RTV, IDV Israel C37 m 42 4.23 410 ABC, SQV, NFV AZT, 3TC, IDV Israel C38 f 34 4.06 35 ABC, TDF, EFV AZT, D4T, 3TC, DDC, r/LPV, NFV Ethiopia A39 f 36 3.84 616 AZT, 3TC, ABC NFV Ethiopia A1

10 m 59 4.27 327 AZT, 3TC, ABC Ethiopia A211 f 46 3.19 495 AZT, ABC, r/LPV D4T, 3TC, IDV Israel A2 HCV12 m 45 4.00 558 AZT, DDI Argentina A313 m 29 4.66 233 AZT, DDI, r/LPV ABC, EFV Ukraine C2 HCV14 m 31 2.95 305 D4T, ABC, NFV AZT, 3TC, IDV Argentina A2 HCV15 m 35 3.47 539 D4T, DDI, EFV Israel A116 m 41 4.15 198 DDI, 3TC AZT, IDV Israel C317 f 33 2.82 315 DDI, 3TC, EFV ABC Israel C2 HCV18 m 47 3.06 165 DDI, 3TC, EFV AZT, NFV Nigeria A319 f 37 3.32 255 DDI, 3TC, NVP AZT, D4T, EFV, IDV Ethiopia A220 f 33 4.26 174 DDI, ABC, 3TC AZT Ethiopia A321 m 22 5.14 82 DDI, D4T, EFV Ukraine A3 HCV22 m 33 4.10 186 DDI, D4T, RTV AZT, 3TC Israel A323 m 33 4.39 45 DDI, EFV, Hu ABC, 3TC, SQV, NFV Israel C324 f 29 4.69 513 DDI, EFV, r/SQV D4T, 3TC, ABC, IDV Israel C325 m 47 3.38 54 TDF, 3TC, AZT, r/LPV DDI, DDC, DLV, r/APV, IDV Brazil A3 HCV26 m 45 3.57 42 TDF, 3TC, EFV Israel A327 m 41 >5 171 TDF, 3TC, EFV Israel A328 f 29 3.56 27 TDF, 3TC, EFV Ethiopia B329 m 35 >5 304 TDF, 3TC, NVP D4T, AZT, r/LPV Israel B230 f 60 3.31 336 TDF, 3TC, NVP r/APV Israel A231 m 42 4.11 392 TDF, 3TC, r/LPV D4T, DDI, 3TC, EFV, IDV, NFV Israel C232 m 39 3.72 312 TDF, 3TC, r/LPV AZT, D4T, ABC, DDI, NVP, SQV Israel A233 m 47 6.68 1 TDF, 3TC, r/LPV, SQV AZT, D4T, DDI, NFV, IDV, r/APV Ethiopia C334 m 52 3.38 456 TDF, 3TC, EFV D4T, DDI, 3TC Ukraine A2 HCV35 m 38 5.87 24 TDF, FTC, r/LPV AZT, 3TC, EFV Tajikistan C336 f 52 4.55 415 TDF, FTC, r/LPV UK B2

(b)1 m 35 2.96 392 TDF, FTC, EFV AZT, 3TC, r/LPV Ukraine A2 HCV2 m 34 4.18 99 TDF, 3TC, EFV Ethiopia A33 f 39 4.46 132 TDF, DDI, NFV Ethiopia C34 f 3.33 554 TDF, 3TC, EFV Ethiopia C15 m 73 3.38 266 DDI, AZT, IDV Ethiopia C36 m 35 3.84 575 AZT, DDI, NVP AZT, 3TC, NFV Nigeria A17 m 45 >5 182 TDF, FTC, APV Israel B38 m 3.5 267 TDF, 3TC, EFV Israel B2

aViral load (VL) log10.bCD4 counts (no. of cells/ml).cHAART regimen at the time of resistance testing.dDrugs in previous HAART regimen except for drugs also included in the last regimen.

K65R Resistance Reverse Transcriptase Mutation 1511

J. Med. Virol. DOI 10.1002/jmv

Some limitations of this study include the small num-ber of patients followed up, although statistical analyseswere performed. Some resistance mutations might havebeen missed since a population-based sequencing meth-od was used that only detects viral species which arerepresented above 20% of the total.

More data are required on different patterns of drugresistance mutations which can be expected to occur inpatients infected by different viral subtypes [Katabiraand Oelrichs, 2007].

ACKNOWLEDGMENTS

We thank Esther Eshkol for editorial assistance.

REFERENCES

Brenner BG, Oliveira M, Doualla-Bell F, Moisi DD, Ntemgwa M,Frankel F, Essex M, Wainberg MA. 2006. HIV-1 subtype C virusesrapidly develop K65R resistance to tenofovir in cell culture. AIDS20:F9–F13.

Doualla-Bell F, Avalos A, Brenner B, Gaolathe T, Mine M, Gaseitsiwe S,Oliveira M, Moisi D, Ndwapi N, Moffat H, Essex M, Wainberg MA.2006. High prevalence of the K65R mutation in human immunode-ficiency virus type 1 subtype C isolates from infected patients inBotswana treated with didanosine-based regimens. AntimicrobAgents Chemother 50:4182–4185.

Gallant JE, DeJesus E, Arribas JR, Pozniak AL, Gazzard B, Campo RE,Lu B, McColl D, Chuck S, Enejosa J, Toole JJ, Cheng AK, Study 934Group. 2006. Tenofovir DF, emtricitabine, and efavirenz vs. zido-vudine, lamivudine, and efavirenz for HIV. N Engl J Med354:251–260.

Gianotti N, Seminari E, Fusetti G, Salpietro S, Boeri E, Galli A,Lazzarin A, Clementi M, Castagna A. 2004. Impact of a treatmentincluding tenofovir plus didanosine on the selection of the 65Rmutation in highly drug-experienced HIV-infected patients. AIDS18:2205–2208.

Grossman Z, Lorber M, Maayan S, Bar-Yacov N, Levy I, Averbuch D,Istomin V, Chowers M, Sthoeger Z, Ram D, Rudich H, Mileguir F,Pavel R, Almaliach R, Schlaeffer F, Kra-Oz Z, Mendelson E, Scha-piro JM, Riesenberg K. 2005. Drug-resistant HIV infection amongdrug-naive patients in Israel. Clin Infect Dis 40:294–302.

GuZ,Fletcher RS,ArtsEJ,WainbergMA,Parniak MA.1994.TheK65Rmutant reverse transcriptase of HIV-1 cross-resistant to 20,30-di-deoxycytidine, 20,30-dideoxy-30-thiacytidine, and 20,30-dideoxyino-sine shows reduced sensitivity to specific dideoxynucleosidetriphosphate inhibitors in vitro. J Biol Chem 269:28118–28122.

Gu Z, Arts EJ, Parniak MA, Wainberg MA. 1995. Mutated K65Rrecombinant reverse transcriptase of human immunodeficiencyvirus type 1 shows diminished chain termination in the presenceof 20,30-dideoxycytidine 50-triphosphate and other drugs. Proc NatlAcad Sci USA 92:2760–2764.

Gupta RK, Chrystie IL, O’Shea S, Mullen JE, Kulasegaram R, Tong CY.2005. K65R and Y181C are less prevalent in HAART-experiencedHIV-1 subtype A. AIDS 19:1916–1919.

Katabira ET, Oelrichs RB. 2007. Scaling up antiretroviral treatmentin resource-limited settings: Successes and challenges. AIDS21:S5–S10.

Orrell C, Walensky RP, Losina E, Freedberg KA, Wood R. 2008. HIV-1clade C resistance genotypes after first virological failure in a largecommunity ART programme. Ninth International Congress onDrug Therapy in HIV Infection Glasgow, UK. 9–13 November2008. Abstract 113. J Int AIDS Soc 11:O3.

Parikh UM, Zelina S, Sluis-Cremer N, Mellors JW. 2007. Molecularmechanisms of bidirectional antagonism between K65R and thymi-dine analog mutations in HIV-1 reverse transcriptase. AIDS21:1405–1414.

Pillay V, Pillay C, Kantor R, Venter F, Levin L, Morris L. 2008. HIV type1 subtype C drug resistance among pediatric and adult SouthAfrican patients failing antiretroviral therapy. AIDS Res HumRetroviruses 24:1449–1454.

Stone C, Ait-Khaled M, Craig C, Griffin P, Tisdale M. 2004. Humanimmunodeficiency virus type 1 reverse transcriptase mutationselection during in vitro exposure to tenofovir alone or combinedwith abacavir or lamivudine. Antimicrob Agents Chemother48:1413–1415.

Sungkanuparph S, Manosuthi W, Kiertiburanakul S, Saekang N,Pairoj W, Chantratita W. 2008. Prevalence and risk factors fordeveloping K65R mutations among HIV-1 infected patients whofail an initial regimen of fixed-dose combination of stavudine, lam-ivudine, and nevirapine. J Clin Virol 41:310–313.

Valer L, Martin-Carbonero L, de Mendoza C, Corral A, Soriano V. 2004.Predictors of selection of K65R: Tenofovir use and lack of thymidineanalogue mutations. AIDS 18:2094–2096.

Von Wyl V, Yerly S, B€oni J, B€urgisser P, Klimkait T, Battegay M,Bernasconi E, Cavassini M, Furrer H, Hirschel B, Vernazza PL,Rickenbach M, Ledergerber B, G€unthard HF, Swiss HIV CohortStudy. 2008. Factors associated with the emergence of K65R inpatients with HIV-1 infection treated with combination antiretro-viral therapy containing tenofovir. 1. Clin Infect Dis 46:1299–1309.

Wainberg MA. 2005. Generic HIV drugs-enlightened policy for globalhealth. N Engl J Med 352:747–750.

Wainberg MA, Miller MD, Quan Y, Salomon H, Mulato AS, Lamy PD,Margot NA, Anton KE, Cherrington JM. 1999. In vitro selection andcharacterization of HIV-1 with reduced susceptibility to PMPA.Antivir Ther 4:87–94.

Waters L, Nelson M, Mandalia S, Bower M, Powles T, Gazzard B,Stebbing J. 2008. The risks and incidence of K65R and L74Vmutations and subsequent virologic responses. Clin Infect Dis46:96–100.

Winston A, Pozniak A, Mandalia S, Gazzard B, Pillay D, Nelson M.2004. Which nucleoside and nucleotide backbone combinationsselect for the K65R mutation in HIV-1 reverse transcriptase. AIDS18:949–951.

TABLE II. Subtype Distribution Among Viruses HarboringK65R

SubtypeK65R

presentK65Rabsent P-valuea TAMsb

A 1 1CRF01_AE 1 3 NSCRF02_AG 2 NSC 4 7 <0.05 3D 1 1F 3 1G 1Non-B 7 16 <0.05 6c

B 1 20 8cd

Total 8 36 14

aThe P-value represents comparison of subtype B and other subtypes withand without the K65R mutation.bThymidine analogue-associated mutations (TAMs) include: M41L, D67N,K70R, L210W, T215YF, K219QE.cNo statistical differences were observed for the presence of TAMs between Band non-B subtypes.dOne virus possessed the multi-resistance Q151M mutation.

1512 Turner et al.

J. Med. Virol. DOI 10.1002/jmv