HIV-1 Drug Resistance and Third-Line Therapy Outcomes in Patients Failing Second-Line Therapy in Zimbabwe

HIV-1 Drug Resistance and Third-Line Therapy Outcomes in Patients Failing Second-Line Therapy in... Open Forum Infectious Diseases MAJOR ARTICLE HIV-1 Drug Resistance and Third-Line Therapy Outcomes in Patients Failing Second-Line Therapy in Zimbabwe 1,2 3 2 4 1,4,5 4 1,4 Cleophas Chimbetete, David Katzenstein, Tinei Shamu, Adrian Spoerri, Janne Estill, Matthias Egger, and Olivia Keiser 1 2 3 4 Institute of Global Health, University of Geneva, Geneva, Switzerland; Newlands Clinic, Harare, Zimbabwe; School of Medicine, University of Stanford, Stanford, California; Institute of Social and Preventive Medicine (ISPM), University of Bern, Bern, Switzerland; Institute of Mathematical Statistics and Actuarial Science, University of Bern, Bern, Switzerland Objectives. To analyze the patterns and risk factors of HIV drug resistance mutations among patients failing second-line treat- ment and to describe early treatment responses to recommended third-line antiretroviral therapy (ART) in a national referral HIV clinic in Zimbabwe. Methods. Patients on boosted protease inhibitor (PI) regimens for more than 6  months with treatment failure confirmed by 2 viral load (VL) tests >1000 copies/mL were genotyped, and susceptibility to available antiretroviral drugs was estimated by the Stanford HIVdb program. Risk factors for major PI resistance were assessed by logistic regression. Third-line treatment was provided as Darunavir/r, Raltegravir, or Dolutegravir and Zidovudine, Abacavir Lamivudine, or Tenofovir. Results. Genotypes were performed on 86 patients who had good adherence to treatment. The median duration of first- and second-line ART was 3.8 years (interquartile range [IQR], 2.3–5.1) and 2.6 years (IQR, 1.6–4.9), respectively. The median HIV viral 3 3 load and CD4 cell count were 65 210 copies/mL (IQR, 8728–208 920 copies/mL) and 201 cells/mm (IQR, 49–333 cells/mm ). Major     PI resistance-associated mutations (RAMs) were demonstrated in 44 (51%) non-nucleoside reverse transcriptase inhibitor RAMs in 72 patients (83%) and nucleoside reverse transcriptase inhibitors RAMs in 62 patients (72%). PI resistance was associated with age >24 years (P = .003) and CD4 cell count <200 cells/mm (P = .007). In multivariable analysis, only age >24 years was significantly associated (adjusted odds ratio, 4.75; 95% confidence interval, 1.69–13.38; P  = .003) with major PI mutations. Third-line DRV/r- and InSTI-based therapy achieved virologic suppression in 29/36 patients (81%) aer 6 m ft onths. Conclusions. e p Th revelance of PI mutations was high. Adolescents and young adults had a lower risk of acquiring major PI resistance mutations, possibly due to poor adherence to ART. Third-line treatment with a regimen of Darunavir/r, Raltegravir/ Dolutegravir, and optimized nucleoside reverse transcriptase inhibitors was effective. Keywords. HIV-1 drug resistance; second-line therapy; third-line ART outcomes; Zimbabwe. e Th many benefits of combination antiretroviral therapy (ART) of ART treatment failure [4]. In contrast to patients failing may be compromised by virologic failure and drug resistance firstline NNRTI- and nucleoside reverse transcriptase inhib- [1]. ART programs in countries hard hit by the HIV pandemic itor (NRTI)–based ART, the majority of patients failing with in Sub-Saharan Africa are facing increasing virologic failure a PI-based second-line ART regimen do not acquire major PI of firstline ART and high levels of drug resistance to non-nu- resistance-associated mutations [5, 6]. cleoside reverse transcriptase inhibitors (NNRTIs) [2]. The As more people with suboptimal adherence are on ART, the emergence of resistance to ART is a consequence of expanded number of patients failing first- and second-line ART regimens access to treatment and longer duration of ART exposure. To is increasing, and an increase in multiclass drug resistance is maintain the benefits of ART, international guidelines recom- expected [7]. Ongoing success of ART programs will require mend switching to second-line, boosted protease inhibitor an understanding of the emergence and patterns of HIV drug (PI)–based ART to maintain virologic suppression [3]. Routine resistance among individuals in whom treatment has failed. HIV viral load monitoring is essential for the early diagnosis Virologic failure occurs for multiple reasons, including subop- timal adherence and drug intolerance/toxicity leading to drug resistance. Aer s ft econd-line failure, evidence of multiclass Received 10 October 2017; editorial decision 22 December 2017; accepted 23 January 2018. resistance following exposure to boosted PI regimens requires Correspondence: C.  Chimbetete, MBChB, MPH,  56 Enterprise Road, Harare, Zimbabwe treatment with at least 2 fully active antiretroviral drugs to sup- (docchimbetete@gmail.com). press viremia, reduce the transmission of resistant virus, and Open Forum Infectious Diseases The Author(s) 2018. Published by Oxford University Press on behalf of Infectious Diseases optimize the effectiveness of third-line ART. Several factors, Society of America. This is an Open Access article distributed under the terms of the Creative such as the duration of PI use and viral load, have been iden- Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/ by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any tified as risk factors for developing PI resistance mutations [8]. medium, provided the original work is not altered or transformed in any way, and that the work Modeling provides evidence that genotyping to optimize third- is properly cited. For commercial re-use, please contact journals.permissions@oup.com line ART is more cost-effective than switching patients failing DOI: 10.1093/ofid/ofy005 HIV Drug Resistance and Third-Line ART in Zimbabwe • OFID • 1 Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy005/4835575 by Ed 'DeepDyve' Gillespie user on 16 March 2018 second-line to third-line based only on virologic failure [9]. adherence support program and who have acquired major PI In the Zimbabwe National ART program, boosted darunavir, resistance mutations are commenced on third-line ART. Third- recycling available nucleoside/nucleotide reverse transcriptase line regimens consist of Darunavir/ritonavir, Raltegravir or inhibitors, and an integrase strand transfer inhibitor (InSTI) are Dolutegravir, and an (optimized) NRTI based on GRT. provided, as recommended by the World Health Organization Enhanced Adherence Support Program (WHO) [3]. However, identification of multiclass drug resist- All patients suspected of second-line ART failure, that is, patients ance and eligibility for third-line therapy requires genotypic who had a VL >1000 copies/mL, were enrolled in a 6-week resistance testing (GRT). For patients who have developed mul- enhanced adherence support program before GRT between ticlass resistance to NNRTI, NRTIs, and boosted PIs, InSTIs August 1, 2013, and July 31, 2016. Patients who had elevated viral have been approved as a new class of ART with a high barrier to loads met in support groups of 8–10 participants once weekly for resistance and an exceptional safety profile [10]. While recently approximately 2.5 hours. Group cognitive behavioral counseling approved in Botswana for firstline treatment, InSTIs are cur - was aimed at discussion of HIV and ART, the identification of rently reserved for patients with evidence of multiclass resist- barriers and challenges to adherence, and the strengthening ance in most resource-limited countries, including Zimbabwe. of medication adherence. These meetings were facilitated by Darunavir, a second-generation protease inhibitor, has been trained counselors. There were separate groups for participants shown to be effective against HIV resistant to Atazanavir and age 24 years and younger and for those older than age 24 years. Lopinavir, and hence is a useful third-line option [11, 12]. All patients had the VL test repeated after the adherence support Third-line treatment should include new drugs with a mini- program. HIV-1 viral load was measured by the Roche COBAS mum risk of cross-resistance to previously used regimens that Ampliprep/COBAS Taqman HIV-1 Test, version 2.0. are available in resource-limited settings [13]. ART Resistance Testing In this study, we analyzed the patterns of HIV drug resistance GRT was done for patients suspected of second-line failure mutations among patients failing second-line treatment and the with a confirmed viral load >1000 copies/mL after 6 weeks of risk factors for acquiring major PI resistance. We further describe enhanced adherence support, and had good adherence, as per early treatment responses to recommended third-line ART in an national guidelines. GRT was not done in patients who still had HIV clinic in Zimbabwe. Our broad aim was to inform planning confirmed poor adherence after adherence support. Patients for third-line ART programs in sub-Saharan Africa. with poor adherence continued to receive adherence sup- MATERIALS AND METHODS port, and repeated viral load tests were done every 3 months. Plasma viral RNA was extracted, reverse transcribed, and Study Setting and ART Treatment Guidelines 1.3 kb of the HIV-1 protease and reverse transcriptase genes Newlands Clinic is an HIV treatment center in Harare, was amplified as described by Manasa [15]. Amplicons were Zimbabwe, that is a national referral site for patients who are sequenced at MCLab Molecular Cloning Laboratories (http:// supposed to start third-line ART treatment after second-line www.mclab.com), San Francisco, California. The chromato- virologic failure. Firstline regimens comprise 2 nucleoside/ grams were assembled using Geneious software, version 8 NRTIs—among them tenofovir (TDF), zidovudine (ZDV), (http://www.geneious.com) [16], and consensus sequences abacavir (ABC), and lamivudine (3TC), and an NNRTI, either were analyzed using the Stanford University HIV Drug efavirenz (EFV) or nevirapine (NVP). Until 2013, stavudine Resistance Database’s HIVdb program, version 8.3 (https:// (D4T) was part of the national firstline ART regimen. Second- hivdb.stanford.edu/hivdb/by-sequences) [17]. The estimated line regimens include 2 NRTIs and a ritonavir-boosted PI, level of resistance to ART was determined by the genotypic either atazanavir or lopinavir. The NRTIs used in second-line susceptibility scores (GSS) associated with each of the drug are 3TC and AZT or TDF or ABC, depending on what the resistance mutations. The estimated level of resistance was patient received for firstline treatment. Protease inhibitor mon- calculated as follows: susceptible (total score 0–9), poten- otherapy is not part of national guidelines, and none of the tial low-level resistance (total score 10–14), low-level resist- patients studied received it. National guidelines recommend a ance (total score 15–29), intermediate resistance (total score change to a third-line regimen if virologic failure (2 consecutive 30–59), and high-level resistance (total score of 60 and above). HIV RNA tests >1000 copies/mL) occurs after at least 6 months of therapy and adherence is estimated to be greater than 95% Data Analysis by pill counts and/or pharmacy refill records [14]. National We analyzed the clinical data that were routinely collected for guidelines recommend that patients on second-line ART have each patient. We used univariable and multivariable logistic at least 1 viral load test done per year. Patients with an elevated regression to study the association between explanatory varia- VL (>1000 copies/mL) must have a repeat test done 3 months bles with the development of at least 1 major PI resistance-asso- after adherence support. At Newlands Clinic only, patients who ciated mutation (RAM). We included the following explanatory are deemed adherent (assessed by pill counts) after an intensive variables: age (≤24 and >24 years according to the WHO, which 2 • OFID • Chimbetete et al Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy005/4835575 by Ed 'DeepDyve' Gillespie user on 16 March 2018 defines adolescents and young adults as people aged ≤24 years 65 210 copies/mL (IQR, 8728–208 920 copies/mL) and 201     3 3 [18]), HIV RNA (≤100 000 and >100 000 copies/mL), sex, CD4 cells/mm (IQR, 49–333 cells/mm ), respectively. Participants cell count (≤200 and >200 cells/mm ), and duration of sec- had received firstline ART for a median of 3.8 years (IQR, 2.3– ond-line therapy (≤2 and >2 years). All variables were retained 5.1 years) and second-line ART for a median of 2.6 years (IQR, in multivariable logistic regression regardless of association in 1.6–4.9  years). Participants had received a median of 6 (IQR, univariable analysis. Statistical tests were 2-sided, with a signif- 6–7) antiretroviral medicines for first- and second-line ART. icance level of .05. There were no missing values. All statisti- Table 1 summarizes participant demographic and clinical char- cal analyses were performed in Stata, version 13.0 (StataCorp, acteristics at the time of GRT. There were differences in educa- College Station, TX). tion level (P = .006), CD4 cell counts (P = .032), HIV viral load (P = .039), and marital status (P = .001) between patients who Ethics had PI RAMs and those without. Only 2 participants received The study was approved by the Medical Research Council ART for the prevention of mother-to-child transmission; both of Zimbabwe (approval No. MRCZ/A/1336). Patients pro- had received single-dose nevirapine. vided written informed consent before being enrolled into the enhanced adherence support program. Drug Resistance–Associated Mutations Sanger sequencing was successful for all 86 patients. All patients RESULTS had subtype C virus. Wild-type virus was found in 12 (14%) A total of 186 participants received adherence support for sec- participants, and 74 (86%) had mutant virus. Most (n  =  72, ond-line failure, 61 achieved postadherence support viral loads 83%) had at least 1 NNRTI RAM, as summarized in Figure  1. of less than 1000 copies/mL, 3 were lost to follow-up, 1 was The most common NNRTI mutation was K103N (n = 30, 35%), transferred out, and 35 did not meet clinical criteria for geno- followed by Y181C (n = 26, 32%) and G190 (n = 24, 28%). typing due to confirmed poor adherence. Sixty-two participants (72%) had at least 1 NRTI RAM. The Of the 86 who were genotyped, 41 (48%) were female. Thirty- distribution of major NRTI mutations is summarized in Figures six (42%) had initiated firstline ART at Newlands Clinic and 1 and 2. Two-thirds had the NRTI mutation M184V (n  =  58, had been switched to a second-line regimen aer fa ft iling first- 67%), followed by the thymidine analogue mutations T215Y line ART, and 50 patients (58%) were referred to Newlands (n  =  31, 36%) and D67N (n  =  31, 36%). Overall, 13 (15%) Clinic, receiving second-line ART. The median age at geno- patients had the K65R mutation, which confers high-level typing was 27.7 years (IQR, 19.7–42.3 years). The median HIV resistance to Tenofovir. All 13 patients with the K65R mutation viral load and CD4 cell count at the time of genotyping were had been exposed to TDF for either first- or second-line ART. Table  1. Sociodemographic, Clinical, and Biological Characteristics of Study Population With HIV-1 Sequences (n  =  86) Comparing Those With and Without PI Mutations All Patients Parameter (n = 86) No PI Mutation (n = 42) Any PI Mutation (n = 44) P Value Median age (IQR), y 27.7 (19.7–42.3) 21.2 (18.0–38.3) 37.4 (25.9–46.9) .004 Gender, n (%) Female 41 (47.7) 22 (52.4) 19 (43.2) .729 Marital status, n (%) Single 47 (54.7) 32 (76.2) 15 (34.1) .001 Married 30 (34.9) 7 (11.7) 23 (52.3) Widowed 7 (8.1) 3 (7.1) 4 (9.1) Divorced 2 (2.3) 0 (0) 2 (4.6) Level of Education None 12 (14) 9 (21.4) 3 (6.8) .006 Primary 17 (19.7) 12 (28.6) 5 (11.4) Secondary 43 (50) 18 (42.9) 25 (56.8) Tertiary 14 (16.3) 3 (7.1) 11 (25) Clinical CD4 count, median (IQR), cell/mm 201 (49–333) 243 (132–379) 97 (22–277) .032 HIV RNA, median (IQR), copies/mL 65 210 (8728–208 920) 37 238 (4620–147 592) 79 362 (20 376–254 612) .039       Duration of ART (IQR), y 7.7 (5.3–9.4) 7.3 (5.0–9.4) 7.9 (6.1–9.5) .388 2nd-line ART duration (IQR), y 2.6 (1.6–4.9) 2.4 (1.6–4.7) 2.6 (1.7–5.2) .318 No. of ART drugs received, median (IQR) at GRT 6 (6–7) 6.5 (6–8) 6 (5–7) .081 Abbreviations: GRT, genotypic resistance testing; PI, protease inhibitor; RAM, resistance-associated mutation. HIV Drug Resistance and Third-Line ART in Zimbabwe • OFID • 3 Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy005/4835575 by Ed 'DeepDyve' Gillespie user on 16 March 2018 60 28 28 20 17 16 16 12 12 12 12 0123 45+ TAMs NRTI NNRTI PI Number of mutations Figure  1. Distribution of HIV drug resistance mutations (n  =  86). Abbreviations: NNRTI, non-nucleoside reverse transcriptase inhibitor; NRTI, nucleoside reverse tran- scriptase inhibitor; PI, protease inhibitor; TAM, thymidine analogue mutation. 25 (30%) had 3 or more TAMs. Figure 1 highlights the observed At the time of GRT, of the patients with the K65R mutation, 4  were receiving TDF/3TC, 2 AZT/3TC, and 7 ABC/3TC, as frequency of TAMs among the participants; 66 (76.7%) partici- the  NRTI backbone for second-line treatment. The multidrug pants had pathway 2 TAMs [19]. NRTI drug resistance mutations T69Ins and Q151M were pres- PI RAMs were present in 50 (58%) participants, and major ent in 7 (8%) and 4 (5%) participants, respectively. Overall, 46 PI RAMs were present in 44 (51%) participants. Figures 1 and (54%) had at least 1 thymidine analogue mutation (TAM), and 2 summarize the frequency of major PI mutations and the Most frequent mutations (n = 86) L76V I47V,A L90M I84V,A I50V,L M46I,L A98G G190S,E Y181C K103N,S T69DIns K65R K219Q M41L K70R T215Y D67N M184V/I 0% 10% 20% 30% 40% 50% 60% 70% 80% Proportion of patients Figure 2. Distribution of specific HIV drug resistance mutations. Abbreviation: HIVDR, HIV drug resistance. 4 • OFID • Chimbetete et al Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy005/4835575 by Ed 'DeepDyve' Gillespie user on 16 March 2018 HIVDR mutations % of participants NRTI NNRTI PI number of major PI mutations per patient. The most common of darunavir, raltegravir, and optimized NRTI. Two patients PI mutation was M46I (n = 28, 33%), followed by I50V (n = 18, with PI mutations continued on second-line (1 had very poor 21%) and V82A (n = 18, 21%). A total of 24 participants (28%) adherence due to psychiatric illness, and the other was receiv- had at least 3 major PI RAMs. ing palliative care for disseminated cancer of the cervix), and Figure  3 highlights the results of estimated resistance for 6 patients died before commencing third-line ART. Figure  4 potential third-line ART drugs. e Th HIV drug resistance in- highlights the outcomes of the patients who received third- terpretation for PI showed that 40 (48%) participants had a line therapy. The median age of patients at commencement of virus susceptible to atazanavir, 62 (74%) were fully susceptible third-line therapy was 41 years (IQR, 30–47.5 years). Patients to darunavir, and 44 (52%) were fully susceptible to lopinavir. were severely immunosuppressed with a median CD4 cell 3 3 For NNRTI, full susceptibility was predicted for 18 (21%) to count of 147.5 cells/mm (IQR, 28–252.5 cells/mm ) and a nevirapine, 19 (23%) to efavirenz, 21 (25%) to etravirine, and median HIV viral load of 57 774 copies/mL (IQR, 18 809– 19 (22%) to rilpivirine. For the NRTIs, full susceptibility to 215 624 copies/mL) at commencement of third-line therapy. Lamivudine/Emtricitabine was noted in 26 (31%), to Abacavir At the time of analysis among participants commenced on or Didanosine was noted in 25 (30%), to Zidovudine was noted third-line ART, none had been lost to follow-up and 2 had in 44 (51%), and to Tenofovir was noted in 35 (42%). died, 1 due to chronic renal failure (diagnosed while the participant was on firstline therapy) and 1 due to acute alco- Risk Factors for PI Drug Resistance Mutations hol-induced pancreatitis. Table  2 summarizes the risk factors for developing PI muta- At week 24 on third-line therapy, the median CD4 cell count tions. In univariable analysis, participants who had a CD4 cell increased from 147.5 to 251.5 cells/mm (IQR, 187.5–381 cells/ 3 3 count of <200 cells/mm (odds ratio, 3.67 cells/mm ; 95% con- mm ). At week 24 on third-line therapy, 29/36 (81%) partici- fidence interval [CI], 1.43–9.43 cells/mm ) were more likely pants achieved viral suppression of <50 copies/mL, 5/36 (14%) to have major PI mutations. Age >24 years was independently patients had VL between 50 and 1000 copies/mL and 1/36 (3%) associated with the risk of having major PI mutations in mul- had died. One, a 17-year-old adolescent, had a week 24 VL of tivariable analysis (adjusted odds ratio,  4.75  years; 95% CI, 2244 copies/mL and has been receiving adherence support, and 1.69–13.38  years). HIV viral load and CD4 cell count were to date he has not managed to achieve virological suppression. not independently associated with the risk of having major er Th e were no reported discontinuations due to toxicity of any PI mutations; neither was the duration of receiving PI-based of the third-line medicines. second-line ART. Among the 39 patients who had no PI mutations and con- tinued on second-line ART with ongoing adherence sup- Early Third-Line Outcomes port, only 8 achieved virological suppression 24 weeks after The decision to switch to third-line ART was based on HIV drug resistance testing. Two participants were recom- the presence of major PI RAMs conferring resistance to menced on firstline (because they had wild-type virus), Atazanavir and Lopinavir. A  total of 36 patients (19 females and both achieved virological suppression after 24 weeks of and 17 males) were commenced on a third-line ART regimen firstline ART. Rilpivirine Etravirine TDF AZT ABC Atazanavir Lopinavir Darunavir ART Medicine S PLR LR IR HLR Figure 3. HIV drug resistance interpretation. Abbreviations: ABC, Abacavir; AZT, Zidovudine; HLR, high-level resistance; IR, intermediate resistance; LR, low-level resist- ance; PLR, potential low-level resistance; S, susceptible; TDF, Tenofovir. HIV Drug Resistance and Third-Line ART in Zimbabwe • OFID • 5 Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy005/4835575 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Proportion of patients Table 2. Risk Factors for Major Protease Inhibitor Resistance Mutations Univariable Multivariable Risk Factor OR (95% CI) P Value OR (95% CI) P Value VL > 100 000 copies/mL 2.04 (0.84–4.92) .114 2.14 (0.75–6.12) .155 2nd-line duration > 2 y 1.31 (0.5–3.14) .541 1.65 (0.61–4.50) .327 Age > 24 y 4.11 (1.62–10.43) .003 4.75 (1.69–13.34) .003 CD4 < 200 cells/mm 3.67 (1.43–9.43) .007 2.53 (0.90–7.15) .079 Abbreviations: CI, confidence interval; OR, odds ratio; VL, viral load. DISCUSSION (<24  years), were less likely to have acquired major PI drug resistance mutations upon failing PI-based second-line treat- Among patients referred for second-line failure and geno- ment. Viral load and immunological status at resistance typed after 6 weeks of aggressive adherence support, 14% had testing were not independently associated with major PI wild-type virus, suggesting very low adherence, and 86% had RAMs. Early third-line treatment outcomes were excellent, mutant virus. Among those with drug resistance mutations, with 30/36 patients achieving viral loads <50 copies/mL at all had 1 or more NNRTI and/or NRTI mutations and 44/86 24 weeks. (51%) had major PI resistance mutations. Younger patients 186 participants received EAC 61 resuppressed 35 confirmed poor adherence 3 LTFU 1 transferred out 86 participants had GRT 42 had no major 44 had PI RAMs PI RAMs 6 deceased 1 deceased 39 continued 2nd-line 36 received 3rd-line 2 switched to 1st 2 continued 2nd 24 Weeks outcome VL at 24 Weeks 1 had VL >1000 30 had VL >1000 5 had VL 50–1000 1 had VL of 183 29 had VL <50 8 had VL <50 1 died Figure 4. Outcomes of patients failing second-line antiretroviral therapy who received genotypic resistance testing. Abbreviations: EAC, enhanced adherence counseling; GRT, genotypic resistance testing; LTFU, loss to follow-up; PI, protease inhibitor; RAM, resistance-associated mutation; VL, viral load. 6 • OFID • Chimbetete et al Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy005/4835575 by Ed 'DeepDyve' Gillespie user on 16 March 2018 This study has some limitations. Our sample size was small. either viral loads were not done prior to that time or the data However, as resistance data aer s ft econd-line failure are scarce, were not provided. It is important for HIV treatment programs we believe that these results are important to clinicians look- to oer r ff outine viral load testing to enable early diagnosis of ing aer p ft atients failing second-line ART. Population-based treatment failure and hence prevent accumulation of TAMs. sequencing, used in this analysis, is not able to detect minority TAMs have the potential to confer resistance to all drugs in the resistant viral strains, thus potentially underestimating resist- NRTI class. ance [20]. Moreover, the durability of suppression on third-line Despite the absence of NNRTI exposure during second-line therapy was only established for 6 months, and longer follow-up ART, the virus from almost 84% of patients had at least 1 major is essential. Lack of resistance patterns aer fir ft stline failure was NNRTI resistance mutation, and the virus from 40% had a also a limitation. mutation at the K103N codon, strongly associated with resist- Patterns of DRM demonstrate that despite interventions to ance to nevirapine and efavirenz. The persistence of NNRTI improve adherence, almost half of the patients who had GRT resistance is consistent with genotypic analyses of 2 South done did not acquire major PI resistance-associated mutations. African cohorts that failed PI-based ART [5, 25] and precludes A total of 61 (33%) patients out of the original 186 resuppressed recycling of firstline NNRTI drugs in third-line therapy. The aer ad ft herence support, highlighting that they had a virus sus- high levels of NNRTI resistance mutations may indicate exten- ceptible to second-line ART. This provides evidence that viro- sive drug resistance including NRTI mutations prior to the logic failure is likely due to poor adherence, leading to reduced onset of second-line therapy. drug exposure. A  number of studies from resource-limited e n Th eed for evidence regarding the implementation of third- settings have reported low rates of PI resistance aer fa ft ilure of line ART in resource-limited settings has been recognized by the second-line ART [5, 6, 21], oen a ft ttributing this finding to poor WHO [3]. Our data demonstrate the effectiveness of third-line medication adherence. The presence of major protease inhibitor ART in a cohort of patients who are infected with HIV subtype mutations at the time of second-line failure ranges from 0% to C.  e Th majority of patients achieved virologic suppression on 50% [22]. A  recent national survey in Kenya reported a 25% regimens including darunavir/ritonavir, raltegravir, and NRTIs, prevalence of PI mutations among patients failing second-line suggesting that this can be used as a standardized third-line reg- ART [23]. The high prevalence of PI resistance in our cohort imen in Zimbabwe. Data on the treatment outcomes of third- can be attributed to possible selection bias. Only patients with line ART are still very scarce in sub-Saharan Africa; however, 2 reported good adherence (aer a ft t least 6 weeks of enhanced other reports have provided evidence of effectiveness [34, 35]. adherence counseling) had a GRT done, that is, only 86 out of In a small Indian cohort, early treatment outcomes showed the original 186. The association of younger age and PI resist- excellent effectiveness of third-line ART [36]. Although the ance is consistent with findings from similar studies in South small cohort size limits wider assumptions of efficacy, the pre- Africa and the United Kingdom [5, 24, 25]. These studies show liminary outcomes suggest that third-line therapy can be effect- that second-line failure in young people is oen ft due to poor ively implemented in a resource-limited setting with excellent adherence rather than development of PI RAMs. Age may pro- rates of virologic suppression. Furthermore, our results support vide an explanation for some of the patient-level, regimen-spe- the use of darunavir/ritonavir and an InSTI backbone for third- cific, and structural factors associated with the absence of PI line ART, as recommended by the WHO [3]. mutations and reduced adherence to second-line ART [26–29]. CONCLUSIONS Social and structural obstacles to adherence can include inac- cessible clinic location or lack of access to transportation, work/ Prevalence of RAMs was high among participants failing sec- child care responsibilities, and low health care provider to ond-line ART. However, only half of these participants had patient ratio as a consequence of the rapid growth in ART roll- major PI RAMs, which necessitate the switch to third-line out programs [30, 31]. Optimizing treatment adherence and treatment. The presence of major PI RAMs was significantly retention at all stages in the cascade of HIV care is critical to the associated with an increase in age. Younger participants were prevention of resistance. more likely to fail second-line treatment due to poor adherence As expected in Africa, the predominant NRTI mutation rather than development of PI resistance. GRT is essential to observed in our cohort was MI84V, which confers high-level identify those with triple class resistance, and those who require resistance to Lamivudine and Emtricitabine [32]. The observed third-line therapy to regain and sustain virologic suppression. prevalence of TAMs was high, the commonest being T215Y and A  Darunavir/r, Integrase strand transfer inhibitor and opti- D67N. TAMs are known to accumulate in patients who remain mized NRTI (based on GRT) regimen was effective in achieving on a failing ART regimen due to delays in detecting treatment virologic suppression in early follow-up. Our results show that failure [33]. Patients may have been failing on second-line ART third-line regimens for patients with multidrug-resistant HIV for a long time before enrolling in the adherence program, but in Africa are likely to be effective. HIV Drug Resistance and Third-Line ART in Zimbabwe • OFID • 7 Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy005/4835575 by Ed 'DeepDyve' Gillespie user on 16 March 2018 16. Kearse M, Moir R, Wilson A, et  al. Geneious Basic: an integrated and extend- Acknowledgments able desktop software platform for the organization and analysis of sequence data. Financial support. e a Th uthors acknowledge Ruedi Luethy for his Bioinformatics 2012; 28:1647–9. support of the study. Research reported in this publication was supported 17. Liu TF, Shafer RW. Web resources for HIV type 1 genotypic-resistance test inter- by the Ruedi Luethy Foundation and the National Institute of Allergy and pretation. Clin Infect Dis 2006; 42:1608–18. Infectious Diseases of the National Institutes of Health under award num- 18. World Health Organization. HIV and Adolescents: Guidance for HIV Testing and ber U01AI069924 (PIs: Egger and Davies). The content is solely the respon- Counselling and Care for Adolescents Living With HIV. Geneva: WHO; 2015. sibility of the authors and does not necessarily represent the official views 19. Marcelin AG, Delaugerre C, Wirden M, et al. Thymidine analogue reverse tran- of the National Institutes of Health. O. Keiser and J. Estill were supported scriptase inhibitors resistance mutations profiles and association to other nucleo- side reverse transcriptase inhibitors resistance mutations observed in the context by a professorship grant from the Swiss National Science Foundation (grant of virological failure. J Med Virol 2004; 72:162–5. No. 163878). 20. Kozal MJ, Chiarella J, St John EP, et al. Prevalence of low-level HIV-1 variants with Potential conifl cts of interest. All authors: no reported conflicts of reverse transcriptase mutation K65R and the effect of antiretroviral drug expos- interest. All authors have submitted the ICMJE Form for Disclosure of ure on variant levels. Antivir Ther 2011; 16:925–9. Potential Conflicts of Interest. Conflicts that the editors consider relevant to 21. El-Khatib Z, Ekstrom AM, Ledwaba J, et al. Viremia and drug resistance among the content of the manuscript have been disclosed. 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HIV-1 Drug Resistance and Third-Line Therapy Outcomes in Patients Failing Second-Line Therapy in Zimbabwe

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Open Forum Infectious Diseases MAJOR ARTICLE HIV-1 Drug Resistance and Third-Line Therapy Outcomes in Patients Failing Second-Line Therapy in Zimbabwe 1,2 3 2 4 1,4,5 4 1,4 Cleophas Chimbetete, David Katzenstein, Tinei Shamu, Adrian Spoerri, Janne Estill, Matthias Egger, and Olivia Keiser 1 2 3 4 Institute of Global Health, University of Geneva, Geneva, Switzerland; Newlands Clinic, Harare, Zimbabwe; School of Medicine, University of Stanford, Stanford, California; Institute of Social and Preventive Medicine (ISPM), University of Bern, Bern, Switzerland; Institute of Mathematical Statistics and Actuarial Science, University of Bern, Bern, Switzerland Objectives. To analyze the patterns and risk factors of HIV drug resistance mutations among patients failing second-line treat- ment and to describe early treatment responses to recommended third-line antiretroviral therapy (ART) in a national referral HIV clinic in Zimbabwe. Methods. Patients on boosted protease inhibitor (PI) regimens for more than 6  months with treatment failure confirmed by 2 viral load (VL) tests >1000 copies/mL were genotyped, and susceptibility to available antiretroviral drugs was estimated by the Stanford HIVdb program. Risk factors for major PI resistance were assessed by logistic regression. Third-line treatment was provided as Darunavir/r, Raltegravir, or Dolutegravir and Zidovudine, Abacavir Lamivudine, or Tenofovir. Results. Genotypes were performed on 86 patients who had good adherence to treatment. The median duration of first- and second-line ART was 3.8 years (interquartile range [IQR], 2.3–5.1) and 2.6 years (IQR, 1.6–4.9), respectively. The median HIV viral 3 3 load and CD4 cell count were 65 210 copies/mL (IQR, 8728–208 920 copies/mL) and 201 cells/mm (IQR, 49–333 cells/mm ). Major     PI resistance-associated mutations (RAMs) were demonstrated in 44 (51%) non-nucleoside reverse transcriptase inhibitor RAMs in 72 patients (83%) and nucleoside reverse transcriptase inhibitors RAMs in 62 patients (72%). PI resistance was associated with age >24 years (P = .003) and CD4 cell count <200 cells/mm (P = .007). In multivariable analysis, only age >24 years was significantly associated (adjusted odds ratio, 4.75; 95% confidence interval, 1.69–13.38; P  = .003) with major PI mutations. Third-line DRV/r- and InSTI-based therapy achieved virologic suppression in 29/36 patients (81%) aer 6 m ft onths. Conclusions. e p Th revelance of PI mutations was high. Adolescents and young adults had a lower risk of acquiring major PI resistance mutations, possibly due to poor adherence to ART. Third-line treatment with a regimen of Darunavir/r, Raltegravir/ Dolutegravir, and optimized nucleoside reverse transcriptase inhibitors was effective. Keywords. HIV-1 drug resistance; second-line therapy; third-line ART outcomes; Zimbabwe. e Th many benefits of combination antiretroviral therapy (ART) of ART treatment failure [4]. In contrast to patients failing may be compromised by virologic failure and drug resistance firstline NNRTI- and nucleoside reverse transcriptase inhib- [1]. ART programs in countries hard hit by the HIV pandemic itor (NRTI)–based ART, the majority of patients failing with in Sub-Saharan Africa are facing increasing virologic failure a PI-based second-line ART regimen do not acquire major PI of firstline ART and high levels of drug resistance to non-nu- resistance-associated mutations [5, 6]. cleoside reverse transcriptase inhibitors (NNRTIs) [2]. The As more people with suboptimal adherence are on ART, the emergence of resistance to ART is a consequence of expanded number of patients failing first- and second-line ART regimens access to treatment and longer duration of ART exposure. To is increasing, and an increase in multiclass drug resistance is maintain the benefits of ART, international guidelines recom- expected [7]. Ongoing success of ART programs will require mend switching to second-line, boosted protease inhibitor an understanding of the emergence and patterns of HIV drug (PI)–based ART to maintain virologic suppression [3]. Routine resistance among individuals in whom treatment has failed. HIV viral load monitoring is essential for the early diagnosis Virologic failure occurs for multiple reasons, including subop- timal adherence and drug intolerance/toxicity leading to drug resistance. Aer s ft econd-line failure, evidence of multiclass Received 10 October 2017; editorial decision 22 December 2017; accepted 23 January 2018. resistance following exposure to boosted PI regimens requires Correspondence: C.  Chimbetete, MBChB, MPH,  56 Enterprise Road, Harare, Zimbabwe treatment with at least 2 fully active antiretroviral drugs to sup- (docchimbetete@gmail.com). press viremia, reduce the transmission of resistant virus, and Open Forum Infectious Diseases The Author(s) 2018. Published by Oxford University Press on behalf of Infectious Diseases optimize the effectiveness of third-line ART. Several factors, Society of America. This is an Open Access article distributed under the terms of the Creative such as the duration of PI use and viral load, have been iden- Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/ by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any tified as risk factors for developing PI resistance mutations [8]. medium, provided the original work is not altered or transformed in any way, and that the work Modeling provides evidence that genotyping to optimize third- is properly cited. For commercial re-use, please contact journals.permissions@oup.com line ART is more cost-effective than switching patients failing DOI: 10.1093/ofid/ofy005 HIV Drug Resistance and Third-Line ART in Zimbabwe • OFID • 1 Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy005/4835575 by Ed 'DeepDyve' Gillespie user on 16 March 2018 second-line to third-line based only on virologic failure [9]. adherence support program and who have acquired major PI In the Zimbabwe National ART program, boosted darunavir, resistance mutations are commenced on third-line ART. Third- recycling available nucleoside/nucleotide reverse transcriptase line regimens consist of Darunavir/ritonavir, Raltegravir or inhibitors, and an integrase strand transfer inhibitor (InSTI) are Dolutegravir, and an (optimized) NRTI based on GRT. provided, as recommended by the World Health Organization Enhanced Adherence Support Program (WHO) [3]. However, identification of multiclass drug resist- All patients suspected of second-line ART failure, that is, patients ance and eligibility for third-line therapy requires genotypic who had a VL >1000 copies/mL, were enrolled in a 6-week resistance testing (GRT). For patients who have developed mul- enhanced adherence support program before GRT between ticlass resistance to NNRTI, NRTIs, and boosted PIs, InSTIs August 1, 2013, and July 31, 2016. Patients who had elevated viral have been approved as a new class of ART with a high barrier to loads met in support groups of 8–10 participants once weekly for resistance and an exceptional safety profile [10]. While recently approximately 2.5 hours. Group cognitive behavioral counseling approved in Botswana for firstline treatment, InSTIs are cur - was aimed at discussion of HIV and ART, the identification of rently reserved for patients with evidence of multiclass resist- barriers and challenges to adherence, and the strengthening ance in most resource-limited countries, including Zimbabwe. of medication adherence. These meetings were facilitated by Darunavir, a second-generation protease inhibitor, has been trained counselors. There were separate groups for participants shown to be effective against HIV resistant to Atazanavir and age 24 years and younger and for those older than age 24 years. Lopinavir, and hence is a useful third-line option [11, 12]. All patients had the VL test repeated after the adherence support Third-line treatment should include new drugs with a mini- program. HIV-1 viral load was measured by the Roche COBAS mum risk of cross-resistance to previously used regimens that Ampliprep/COBAS Taqman HIV-1 Test, version 2.0. are available in resource-limited settings [13]. ART Resistance Testing In this study, we analyzed the patterns of HIV drug resistance GRT was done for patients suspected of second-line failure mutations among patients failing second-line treatment and the with a confirmed viral load >1000 copies/mL after 6 weeks of risk factors for acquiring major PI resistance. We further describe enhanced adherence support, and had good adherence, as per early treatment responses to recommended third-line ART in an national guidelines. GRT was not done in patients who still had HIV clinic in Zimbabwe. Our broad aim was to inform planning confirmed poor adherence after adherence support. Patients for third-line ART programs in sub-Saharan Africa. with poor adherence continued to receive adherence sup- MATERIALS AND METHODS port, and repeated viral load tests were done every 3 months. Plasma viral RNA was extracted, reverse transcribed, and Study Setting and ART Treatment Guidelines 1.3 kb of the HIV-1 protease and reverse transcriptase genes Newlands Clinic is an HIV treatment center in Harare, was amplified as described by Manasa [15]. Amplicons were Zimbabwe, that is a national referral site for patients who are sequenced at MCLab Molecular Cloning Laboratories (http:// supposed to start third-line ART treatment after second-line www.mclab.com), San Francisco, California. The chromato- virologic failure. Firstline regimens comprise 2 nucleoside/ grams were assembled using Geneious software, version 8 NRTIs—among them tenofovir (TDF), zidovudine (ZDV), (http://www.geneious.com) [16], and consensus sequences abacavir (ABC), and lamivudine (3TC), and an NNRTI, either were analyzed using the Stanford University HIV Drug efavirenz (EFV) or nevirapine (NVP). Until 2013, stavudine Resistance Database’s HIVdb program, version 8.3 (https:// (D4T) was part of the national firstline ART regimen. Second- hivdb.stanford.edu/hivdb/by-sequences) [17]. The estimated line regimens include 2 NRTIs and a ritonavir-boosted PI, level of resistance to ART was determined by the genotypic either atazanavir or lopinavir. The NRTIs used in second-line susceptibility scores (GSS) associated with each of the drug are 3TC and AZT or TDF or ABC, depending on what the resistance mutations. The estimated level of resistance was patient received for firstline treatment. Protease inhibitor mon- calculated as follows: susceptible (total score 0–9), poten- otherapy is not part of national guidelines, and none of the tial low-level resistance (total score 10–14), low-level resist- patients studied received it. National guidelines recommend a ance (total score 15–29), intermediate resistance (total score change to a third-line regimen if virologic failure (2 consecutive 30–59), and high-level resistance (total score of 60 and above). HIV RNA tests >1000 copies/mL) occurs after at least 6 months of therapy and adherence is estimated to be greater than 95% Data Analysis by pill counts and/or pharmacy refill records [14]. National We analyzed the clinical data that were routinely collected for guidelines recommend that patients on second-line ART have each patient. We used univariable and multivariable logistic at least 1 viral load test done per year. Patients with an elevated regression to study the association between explanatory varia- VL (>1000 copies/mL) must have a repeat test done 3 months bles with the development of at least 1 major PI resistance-asso- after adherence support. At Newlands Clinic only, patients who ciated mutation (RAM). We included the following explanatory are deemed adherent (assessed by pill counts) after an intensive variables: age (≤24 and >24 years according to the WHO, which 2 • OFID • Chimbetete et al Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy005/4835575 by Ed 'DeepDyve' Gillespie user on 16 March 2018 defines adolescents and young adults as people aged ≤24 years 65 210 copies/mL (IQR, 8728–208 920 copies/mL) and 201     3 3 [18]), HIV RNA (≤100 000 and >100 000 copies/mL), sex, CD4 cells/mm (IQR, 49–333 cells/mm ), respectively. Participants cell count (≤200 and >200 cells/mm ), and duration of sec- had received firstline ART for a median of 3.8 years (IQR, 2.3– ond-line therapy (≤2 and >2 years). All variables were retained 5.1 years) and second-line ART for a median of 2.6 years (IQR, in multivariable logistic regression regardless of association in 1.6–4.9  years). Participants had received a median of 6 (IQR, univariable analysis. Statistical tests were 2-sided, with a signif- 6–7) antiretroviral medicines for first- and second-line ART. icance level of .05. There were no missing values. All statisti- Table 1 summarizes participant demographic and clinical char- cal analyses were performed in Stata, version 13.0 (StataCorp, acteristics at the time of GRT. There were differences in educa- College Station, TX). tion level (P = .006), CD4 cell counts (P = .032), HIV viral load (P = .039), and marital status (P = .001) between patients who Ethics had PI RAMs and those without. Only 2 participants received The study was approved by the Medical Research Council ART for the prevention of mother-to-child transmission; both of Zimbabwe (approval No. MRCZ/A/1336). Patients pro- had received single-dose nevirapine. vided written informed consent before being enrolled into the enhanced adherence support program. Drug Resistance–Associated Mutations Sanger sequencing was successful for all 86 patients. All patients RESULTS had subtype C virus. Wild-type virus was found in 12 (14%) A total of 186 participants received adherence support for sec- participants, and 74 (86%) had mutant virus. Most (n  =  72, ond-line failure, 61 achieved postadherence support viral loads 83%) had at least 1 NNRTI RAM, as summarized in Figure  1. of less than 1000 copies/mL, 3 were lost to follow-up, 1 was The most common NNRTI mutation was K103N (n = 30, 35%), transferred out, and 35 did not meet clinical criteria for geno- followed by Y181C (n = 26, 32%) and G190 (n = 24, 28%). typing due to confirmed poor adherence. Sixty-two participants (72%) had at least 1 NRTI RAM. The Of the 86 who were genotyped, 41 (48%) were female. Thirty- distribution of major NRTI mutations is summarized in Figures six (42%) had initiated firstline ART at Newlands Clinic and 1 and 2. Two-thirds had the NRTI mutation M184V (n  =  58, had been switched to a second-line regimen aer fa ft iling first- 67%), followed by the thymidine analogue mutations T215Y line ART, and 50 patients (58%) were referred to Newlands (n  =  31, 36%) and D67N (n  =  31, 36%). Overall, 13 (15%) Clinic, receiving second-line ART. The median age at geno- patients had the K65R mutation, which confers high-level typing was 27.7 years (IQR, 19.7–42.3 years). The median HIV resistance to Tenofovir. All 13 patients with the K65R mutation viral load and CD4 cell count at the time of genotyping were had been exposed to TDF for either first- or second-line ART. Table  1. Sociodemographic, Clinical, and Biological Characteristics of Study Population With HIV-1 Sequences (n  =  86) Comparing Those With and Without PI Mutations All Patients Parameter (n = 86) No PI Mutation (n = 42) Any PI Mutation (n = 44) P Value Median age (IQR), y 27.7 (19.7–42.3) 21.2 (18.0–38.3) 37.4 (25.9–46.9) .004 Gender, n (%) Female 41 (47.7) 22 (52.4) 19 (43.2) .729 Marital status, n (%) Single 47 (54.7) 32 (76.2) 15 (34.1) .001 Married 30 (34.9) 7 (11.7) 23 (52.3) Widowed 7 (8.1) 3 (7.1) 4 (9.1) Divorced 2 (2.3) 0 (0) 2 (4.6) Level of Education None 12 (14) 9 (21.4) 3 (6.8) .006 Primary 17 (19.7) 12 (28.6) 5 (11.4) Secondary 43 (50) 18 (42.9) 25 (56.8) Tertiary 14 (16.3) 3 (7.1) 11 (25) Clinical CD4 count, median (IQR), cell/mm 201 (49–333) 243 (132–379) 97 (22–277) .032 HIV RNA, median (IQR), copies/mL 65 210 (8728–208 920) 37 238 (4620–147 592) 79 362 (20 376–254 612) .039       Duration of ART (IQR), y 7.7 (5.3–9.4) 7.3 (5.0–9.4) 7.9 (6.1–9.5) .388 2nd-line ART duration (IQR), y 2.6 (1.6–4.9) 2.4 (1.6–4.7) 2.6 (1.7–5.2) .318 No. of ART drugs received, median (IQR) at GRT 6 (6–7) 6.5 (6–8) 6 (5–7) .081 Abbreviations: GRT, genotypic resistance testing; PI, protease inhibitor; RAM, resistance-associated mutation. HIV Drug Resistance and Third-Line ART in Zimbabwe • OFID • 3 Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy005/4835575 by Ed 'DeepDyve' Gillespie user on 16 March 2018 60 28 28 20 17 16 16 12 12 12 12 0123 45+ TAMs NRTI NNRTI PI Number of mutations Figure  1. Distribution of HIV drug resistance mutations (n  =  86). Abbreviations: NNRTI, non-nucleoside reverse transcriptase inhibitor; NRTI, nucleoside reverse tran- scriptase inhibitor; PI, protease inhibitor; TAM, thymidine analogue mutation. 25 (30%) had 3 or more TAMs. Figure 1 highlights the observed At the time of GRT, of the patients with the K65R mutation, 4  were receiving TDF/3TC, 2 AZT/3TC, and 7 ABC/3TC, as frequency of TAMs among the participants; 66 (76.7%) partici- the  NRTI backbone for second-line treatment. The multidrug pants had pathway 2 TAMs [19]. NRTI drug resistance mutations T69Ins and Q151M were pres- PI RAMs were present in 50 (58%) participants, and major ent in 7 (8%) and 4 (5%) participants, respectively. Overall, 46 PI RAMs were present in 44 (51%) participants. Figures 1 and (54%) had at least 1 thymidine analogue mutation (TAM), and 2 summarize the frequency of major PI mutations and the Most frequent mutations (n = 86) L76V I47V,A L90M I84V,A I50V,L M46I,L A98G G190S,E Y181C K103N,S T69DIns K65R K219Q M41L K70R T215Y D67N M184V/I 0% 10% 20% 30% 40% 50% 60% 70% 80% Proportion of patients Figure 2. Distribution of specific HIV drug resistance mutations. Abbreviation: HIVDR, HIV drug resistance. 4 • OFID • Chimbetete et al Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy005/4835575 by Ed 'DeepDyve' Gillespie user on 16 March 2018 HIVDR mutations % of participants NRTI NNRTI PI number of major PI mutations per patient. The most common of darunavir, raltegravir, and optimized NRTI. Two patients PI mutation was M46I (n = 28, 33%), followed by I50V (n = 18, with PI mutations continued on second-line (1 had very poor 21%) and V82A (n = 18, 21%). A total of 24 participants (28%) adherence due to psychiatric illness, and the other was receiv- had at least 3 major PI RAMs. ing palliative care for disseminated cancer of the cervix), and Figure  3 highlights the results of estimated resistance for 6 patients died before commencing third-line ART. Figure  4 potential third-line ART drugs. e Th HIV drug resistance in- highlights the outcomes of the patients who received third- terpretation for PI showed that 40 (48%) participants had a line therapy. The median age of patients at commencement of virus susceptible to atazanavir, 62 (74%) were fully susceptible third-line therapy was 41 years (IQR, 30–47.5 years). Patients to darunavir, and 44 (52%) were fully susceptible to lopinavir. were severely immunosuppressed with a median CD4 cell 3 3 For NNRTI, full susceptibility was predicted for 18 (21%) to count of 147.5 cells/mm (IQR, 28–252.5 cells/mm ) and a nevirapine, 19 (23%) to efavirenz, 21 (25%) to etravirine, and median HIV viral load of 57 774 copies/mL (IQR, 18 809– 19 (22%) to rilpivirine. For the NRTIs, full susceptibility to 215 624 copies/mL) at commencement of third-line therapy. Lamivudine/Emtricitabine was noted in 26 (31%), to Abacavir At the time of analysis among participants commenced on or Didanosine was noted in 25 (30%), to Zidovudine was noted third-line ART, none had been lost to follow-up and 2 had in 44 (51%), and to Tenofovir was noted in 35 (42%). died, 1 due to chronic renal failure (diagnosed while the participant was on firstline therapy) and 1 due to acute alco- Risk Factors for PI Drug Resistance Mutations hol-induced pancreatitis. Table  2 summarizes the risk factors for developing PI muta- At week 24 on third-line therapy, the median CD4 cell count tions. In univariable analysis, participants who had a CD4 cell increased from 147.5 to 251.5 cells/mm (IQR, 187.5–381 cells/ 3 3 count of <200 cells/mm (odds ratio, 3.67 cells/mm ; 95% con- mm ). At week 24 on third-line therapy, 29/36 (81%) partici- fidence interval [CI], 1.43–9.43 cells/mm ) were more likely pants achieved viral suppression of <50 copies/mL, 5/36 (14%) to have major PI mutations. Age >24 years was independently patients had VL between 50 and 1000 copies/mL and 1/36 (3%) associated with the risk of having major PI mutations in mul- had died. One, a 17-year-old adolescent, had a week 24 VL of tivariable analysis (adjusted odds ratio,  4.75  years; 95% CI, 2244 copies/mL and has been receiving adherence support, and 1.69–13.38  years). HIV viral load and CD4 cell count were to date he has not managed to achieve virological suppression. not independently associated with the risk of having major er Th e were no reported discontinuations due to toxicity of any PI mutations; neither was the duration of receiving PI-based of the third-line medicines. second-line ART. Among the 39 patients who had no PI mutations and con- tinued on second-line ART with ongoing adherence sup- Early Third-Line Outcomes port, only 8 achieved virological suppression 24 weeks after The decision to switch to third-line ART was based on HIV drug resistance testing. Two participants were recom- the presence of major PI RAMs conferring resistance to menced on firstline (because they had wild-type virus), Atazanavir and Lopinavir. A  total of 36 patients (19 females and both achieved virological suppression after 24 weeks of and 17 males) were commenced on a third-line ART regimen firstline ART. Rilpivirine Etravirine TDF AZT ABC Atazanavir Lopinavir Darunavir ART Medicine S PLR LR IR HLR Figure 3. HIV drug resistance interpretation. Abbreviations: ABC, Abacavir; AZT, Zidovudine; HLR, high-level resistance; IR, intermediate resistance; LR, low-level resist- ance; PLR, potential low-level resistance; S, susceptible; TDF, Tenofovir. HIV Drug Resistance and Third-Line ART in Zimbabwe • OFID • 5 Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy005/4835575 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Proportion of patients Table 2. Risk Factors for Major Protease Inhibitor Resistance Mutations Univariable Multivariable Risk Factor OR (95% CI) P Value OR (95% CI) P Value VL > 100 000 copies/mL 2.04 (0.84–4.92) .114 2.14 (0.75–6.12) .155 2nd-line duration > 2 y 1.31 (0.5–3.14) .541 1.65 (0.61–4.50) .327 Age > 24 y 4.11 (1.62–10.43) .003 4.75 (1.69–13.34) .003 CD4 < 200 cells/mm 3.67 (1.43–9.43) .007 2.53 (0.90–7.15) .079 Abbreviations: CI, confidence interval; OR, odds ratio; VL, viral load. DISCUSSION (<24  years), were less likely to have acquired major PI drug resistance mutations upon failing PI-based second-line treat- Among patients referred for second-line failure and geno- ment. Viral load and immunological status at resistance typed after 6 weeks of aggressive adherence support, 14% had testing were not independently associated with major PI wild-type virus, suggesting very low adherence, and 86% had RAMs. Early third-line treatment outcomes were excellent, mutant virus. Among those with drug resistance mutations, with 30/36 patients achieving viral loads <50 copies/mL at all had 1 or more NNRTI and/or NRTI mutations and 44/86 24 weeks. (51%) had major PI resistance mutations. Younger patients 186 participants received EAC 61 resuppressed 35 confirmed poor adherence 3 LTFU 1 transferred out 86 participants had GRT 42 had no major 44 had PI RAMs PI RAMs 6 deceased 1 deceased 39 continued 2nd-line 36 received 3rd-line 2 switched to 1st 2 continued 2nd 24 Weeks outcome VL at 24 Weeks 1 had VL >1000 30 had VL >1000 5 had VL 50–1000 1 had VL of 183 29 had VL <50 8 had VL <50 1 died Figure 4. Outcomes of patients failing second-line antiretroviral therapy who received genotypic resistance testing. Abbreviations: EAC, enhanced adherence counseling; GRT, genotypic resistance testing; LTFU, loss to follow-up; PI, protease inhibitor; RAM, resistance-associated mutation; VL, viral load. 6 • OFID • Chimbetete et al Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy005/4835575 by Ed 'DeepDyve' Gillespie user on 16 March 2018 This study has some limitations. Our sample size was small. either viral loads were not done prior to that time or the data However, as resistance data aer s ft econd-line failure are scarce, were not provided. It is important for HIV treatment programs we believe that these results are important to clinicians look- to oer r ff outine viral load testing to enable early diagnosis of ing aer p ft atients failing second-line ART. Population-based treatment failure and hence prevent accumulation of TAMs. sequencing, used in this analysis, is not able to detect minority TAMs have the potential to confer resistance to all drugs in the resistant viral strains, thus potentially underestimating resist- NRTI class. ance [20]. Moreover, the durability of suppression on third-line Despite the absence of NNRTI exposure during second-line therapy was only established for 6 months, and longer follow-up ART, the virus from almost 84% of patients had at least 1 major is essential. Lack of resistance patterns aer fir ft stline failure was NNRTI resistance mutation, and the virus from 40% had a also a limitation. mutation at the K103N codon, strongly associated with resist- Patterns of DRM demonstrate that despite interventions to ance to nevirapine and efavirenz. The persistence of NNRTI improve adherence, almost half of the patients who had GRT resistance is consistent with genotypic analyses of 2 South done did not acquire major PI resistance-associated mutations. African cohorts that failed PI-based ART [5, 25] and precludes A total of 61 (33%) patients out of the original 186 resuppressed recycling of firstline NNRTI drugs in third-line therapy. The aer ad ft herence support, highlighting that they had a virus sus- high levels of NNRTI resistance mutations may indicate exten- ceptible to second-line ART. This provides evidence that viro- sive drug resistance including NRTI mutations prior to the logic failure is likely due to poor adherence, leading to reduced onset of second-line therapy. drug exposure. A  number of studies from resource-limited e n Th eed for evidence regarding the implementation of third- settings have reported low rates of PI resistance aer fa ft ilure of line ART in resource-limited settings has been recognized by the second-line ART [5, 6, 21], oen a ft ttributing this finding to poor WHO [3]. Our data demonstrate the effectiveness of third-line medication adherence. The presence of major protease inhibitor ART in a cohort of patients who are infected with HIV subtype mutations at the time of second-line failure ranges from 0% to C.  e Th majority of patients achieved virologic suppression on 50% [22]. A  recent national survey in Kenya reported a 25% regimens including darunavir/ritonavir, raltegravir, and NRTIs, prevalence of PI mutations among patients failing second-line suggesting that this can be used as a standardized third-line reg- ART [23]. The high prevalence of PI resistance in our cohort imen in Zimbabwe. Data on the treatment outcomes of third- can be attributed to possible selection bias. Only patients with line ART are still very scarce in sub-Saharan Africa; however, 2 reported good adherence (aer a ft t least 6 weeks of enhanced other reports have provided evidence of effectiveness [34, 35]. adherence counseling) had a GRT done, that is, only 86 out of In a small Indian cohort, early treatment outcomes showed the original 186. The association of younger age and PI resist- excellent effectiveness of third-line ART [36]. Although the ance is consistent with findings from similar studies in South small cohort size limits wider assumptions of efficacy, the pre- Africa and the United Kingdom [5, 24, 25]. These studies show liminary outcomes suggest that third-line therapy can be effect- that second-line failure in young people is oen ft due to poor ively implemented in a resource-limited setting with excellent adherence rather than development of PI RAMs. Age may pro- rates of virologic suppression. Furthermore, our results support vide an explanation for some of the patient-level, regimen-spe- the use of darunavir/ritonavir and an InSTI backbone for third- cific, and structural factors associated with the absence of PI line ART, as recommended by the WHO [3]. mutations and reduced adherence to second-line ART [26–29]. CONCLUSIONS Social and structural obstacles to adherence can include inac- cessible clinic location or lack of access to transportation, work/ Prevalence of RAMs was high among participants failing sec- child care responsibilities, and low health care provider to ond-line ART. However, only half of these participants had patient ratio as a consequence of the rapid growth in ART roll- major PI RAMs, which necessitate the switch to third-line out programs [30, 31]. Optimizing treatment adherence and treatment. The presence of major PI RAMs was significantly retention at all stages in the cascade of HIV care is critical to the associated with an increase in age. Younger participants were prevention of resistance. more likely to fail second-line treatment due to poor adherence As expected in Africa, the predominant NRTI mutation rather than development of PI resistance. GRT is essential to observed in our cohort was MI84V, which confers high-level identify those with triple class resistance, and those who require resistance to Lamivudine and Emtricitabine [32]. The observed third-line therapy to regain and sustain virologic suppression. prevalence of TAMs was high, the commonest being T215Y and A  Darunavir/r, Integrase strand transfer inhibitor and opti- D67N. TAMs are known to accumulate in patients who remain mized NRTI (based on GRT) regimen was effective in achieving on a failing ART regimen due to delays in detecting treatment virologic suppression in early follow-up. Our results show that failure [33]. Patients may have been failing on second-line ART third-line regimens for patients with multidrug-resistant HIV for a long time before enrolling in the adherence program, but in Africa are likely to be effective. HIV Drug Resistance and Third-Line ART in Zimbabwe • OFID • 7 Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy005/4835575 by Ed 'DeepDyve' Gillespie user on 16 March 2018 16. Kearse M, Moir R, Wilson A, et  al. Geneious Basic: an integrated and extend- Acknowledgments able desktop software platform for the organization and analysis of sequence data. Financial support. e a Th uthors acknowledge Ruedi Luethy for his Bioinformatics 2012; 28:1647–9. support of the study. Research reported in this publication was supported 17. Liu TF, Shafer RW. Web resources for HIV type 1 genotypic-resistance test inter- by the Ruedi Luethy Foundation and the National Institute of Allergy and pretation. Clin Infect Dis 2006; 42:1608–18. Infectious Diseases of the National Institutes of Health under award num- 18. World Health Organization. HIV and Adolescents: Guidance for HIV Testing and ber U01AI069924 (PIs: Egger and Davies). The content is solely the respon- Counselling and Care for Adolescents Living With HIV. Geneva: WHO; 2015. sibility of the authors and does not necessarily represent the official views 19. Marcelin AG, Delaugerre C, Wirden M, et al. Thymidine analogue reverse tran- of the National Institutes of Health. O. Keiser and J. Estill were supported scriptase inhibitors resistance mutations profiles and association to other nucleo- side reverse transcriptase inhibitors resistance mutations observed in the context by a professorship grant from the Swiss National Science Foundation (grant of virological failure. J Med Virol 2004; 72:162–5. No. 163878). 20. Kozal MJ, Chiarella J, St John EP, et al. Prevalence of low-level HIV-1 variants with Potential conifl cts of interest. All authors: no reported conflicts of reverse transcriptase mutation K65R and the effect of antiretroviral drug expos- interest. All authors have submitted the ICMJE Form for Disclosure of ure on variant levels. Antivir Ther 2011; 16:925–9. Potential Conflicts of Interest. Conflicts that the editors consider relevant to 21. El-Khatib Z, Ekstrom AM, Ledwaba J, et al. Viremia and drug resistance among the content of the manuscript have been disclosed. 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Glob Health Action 2014; 7. 8 • OFID • Chimbetete et al Downloaded from https://academic.oup.com/ofid/article-abstract/5/2/ofy005/4835575 by Ed 'DeepDyve' Gillespie user on 16 March 2018

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Published: Feb 1, 2018

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