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Abstract

Background:

Data on the use of ritonavir-boosted darunavir (DRV/r) and/or raltegravir (RAL) in resource-limited settings are rare and there is currently no published data regarding their use among African children. Botswana has recently made DRV/r and RAL available for patients failing second-line antiretroviral therapy (ART).

Methods:

Retrospective chart review of 4 multidrug-resistant pediatric patients on DRV/r- and/or RAL-based regimens. Viral load, CD4 count, adherence by pill count, and World Health Organization (WHO) clinical stage prior to and after switch to DRV/r- and/or RAL-based regimen were assessed. Antiretroviral therapy history, duration of virologic failure, and time to viral suppression were also noted. Genotypic resistance assays reviewed for mutations present prior to switch.

Results:

All patients achieved viral suppression, showed improved/stable CD4 counts, and obtained or maintained WHO clinical treatment stage I, even after long-standing virologic/immunologic failure.

Conclusions:

Well tolerated by and effective in our patients, DRV/r and RAL provide potentially lifesaving ART options for children and adolescents in resource-limited settings failing ART due to ritonavir-boosted lopinavir (LPV/r) resistance.

Keywords

HIV drug resistance pediatric antiretroviral therapy treatment failure HIV protease inhibitors integrase inhibitors Botswana

Introduction

With expansion of antiretroviral therapy (ART) programs, long-term survival for people living with HIV/AIDS (PLWA) in resource-limited settings has become the rule, including for children.¹ Yet, as cohorts’ experience with ART grows, treatment failure is becoming more common,² including failure on regimens containing ritonavir-boosted lopinavir (LPV/r), the mainstay for protease inhibitor (PI) therapy in Africa.³ LPV/r is part of standard second-line ART after failure on a non-nucleoside reverse transcriptase inhibitor (NNRTI)-containing first-line regimen in a growing number of African countries, including Botswana where standard second-line ART includes Abacavir (ABC) or Zidovudine (AZT) + Lamivudine (3TC) + LPV/r [4]. In addition, LPV/r-containing regimens are now recommended as first-line ART for infants and young children with previous nonnucleoside reverse transcriptase inhibitor (NNRTI) exposure via prevention of mother-to-child transmission (PMTCT) strategies.⁴ Indeed, recent data suggest that LPV/r-containing first-line regimens may be preferable for all infants and young children, regardless of NNRTI exposure.⁵ In Botswana, when these patients experience virological failure there is currently no standard second-line ART. A genotypic resistance assay is performed, and clinicians advised to consult a Ministry of Health-designated HIV Specialist in determining subsequent ART.⁴

Botswana has one of the world’s highest HIV prevalence rates, estimated at 25.0% among adults aged 15 to 49 years.⁶ Although there are a disproportionately large number of HIV-infected children (as many as 16 000 aged 0-14 years),⁶ the Botswana national ART program has achieved success in delivering ART to >90% of those in need, including over 10 000 children.⁷ Botswana national ART guidelines follow World Health Organization (WHO) recommendations of first-line ART with 2 nucleoside reverse transcriptase inhibitors (NRTIs) and 1 NNRTI for children without previous NNRTI exposure, and 2 NRTIs and 1 PI for those with previous NNRTI exposure.⁸ In addition, LPV/r is part of the standard second-line ART for children in Botswana.⁸ Genotypic resistance assays are available for those failing second-line ART and are used in developing subsequent treatment options; there is no standard national “third-line” or salvage ART regimen. Although uncommon, the number of children failing LPV/r-containing second-line regimens due to genotypically denoted LPV/r-resistance has increased in the recent years.

When children in Africa fail LPV/r-containing regimens, subsequent options are limited, and in many cases, inadequate. Recently, Botswana has made available raltegravir (RAL) and darunavir (DRV, boosted with mono-formulated ritonavir: DRV/r) for use by designated pediatric specialists in the treatment of children failing second-line ART with genotypically denoted LPV/r-resistance. Although DRV/r and RAL are both approved for use in children^9,10 and have shown clinical efficacy and good tolerability in children^11
–13, data from resource-limited settings are scarce, and there is no currently published data from the routine clinical use of DRV/r and/or RAL in African children. Our study provides the first African data on outcomes in HIV-infected children and adolescents treated in a routine clinical setting with DRV/r and/or RAL after failure of LPV/r-based ART due to LPV/r resistance.

Methods

Opened in 2003, the Botswana-Baylor Children’s Clinical Centre of Excellence (COE) is Botswana’s largest pediatric ART clinic with >2000 HIV-infected children in care, of whom more than 1800 receive ART.¹⁴ Of these, approximately 400 patients are on LPV/r-containing second-line or salvage regimens, of which roughly 75% have achieved viral suppression (< 400 copies/mL at last viral load assay). Complicated patients, including those failing second-line and salvage ART, are managed by a multidisciplinary team that has developed a framework for the use of advanced ART regimens in multidrug-resistant patients. In Botswana’s national ART program, use of DRV/r and/or RAL in children and adolescents is a decision made by a pediatric HIV expert on a case-by-case basis, as mandated by the Botswana Ministry of Health.⁸ Criteria for approval include persistent virologic failure in the presence of good adherence and documented substantial genotypic LPV/r resistance.⁸

In this study, a retrospective chart review of the 4 multidrug-resistant patients currently on DRV/r- and/or RAL-based regimens at the COE was performed. Viral load (VL), CD4 count, adherence by pill count, and WHO clinical stage at the clinic visit prior to and at the most current visit (9-24 months) after switch to DRV/r- and/or RAL-based regimen were assessed. In addition, ART history, duration of virologic failure—defined in Botswana National Treatment Guidelines as 2 or more sequential VL >400 copies/mL⁸—and time to viral suppression after switch were noted. Genotypic resistance testing was performed by HIV-1 ViroSeq Genotypic System (Abbott Molecular, Abbott Park, Illinois, USA) at the Botswana-Harvard HIV Reference Laboratory; assays were reviewed for NRTI-, NNRTI-, and PI-associated mutations prior to switch. Assays were reviewed for NRTI-, NNRTI-, and PI-associated mutations prior to switch. Drug resistance mutations were defined using the Stanford University HIV Drug Resistance Database.

Results

Results are illustrated in Tables 1 and 2.

Table 1.

Patient Demographics, ART Regimen, Outcome Variables, ART Adherence, and Genotype Results for 4 Patients on DRV/r- ± RAL-Based ART at the Botswana-Baylor Children’s Clinical Centre of Excellence.

Patient (sex: M-male; F-female)	Age, y	ART Regimen Prior to Switch and Current	Duration of Virologic Failure	Time to Viral Suppression	Months Since Switch	VL	CD4	WHO Clinical Stage	Pill Count	NRTI Mutations/Pattern of Resistance^a	NNRTI Mutations/Pattern of Resistance^a	PI Mutations/Pattern of Resistance^a
1 (M) pre	12	TDF-FTC-LPV/r	5 years			190 000	344/20%	TIV	100%	D67N, L74V, L100I, M184V, T215E, K219Q/ Intermediate-level resistance to AZT, D4T. High-level resistance to ABC, DDI, 3TC and FTC. **Susceptible to TDF.	K103N/ Low-level resistance to ETR. High-level resistance to NVP and EFV.	L10I, K20M, E35D, M36I, R41K, I54V, H69K, V77I, V82A, L88M, I93L/ Intermediate-level resistance to LPV/r, SQV/r, NFV, ATV/r. **Susceptible to DRV/r.
1 post	13	TDF-FTC-DRV/r-RAL		6 weeks	24	<400	729/32%	TI	100%
2 (F) pre	10	AZT-3TC-LPV/r	Intermittent x 5 years; persistent x 14 months			2600	414/14%	TIII	100%	M41L, D67N, V118I, M184V, L210W, T215Y/ Intermediate-level resistance to DDI and TDF. High-level resistance to AZT, D4T, ABC, 3TC, and FTC.	K101E, Y181C, G190A/ NNRTI: High-level resistance to NVP, EFV, and ETR.	L10F, L33F, M36I, M46I, I54V, L63P, A71V, V82A, L90M/ High-level resistance to LPV/r, SQV/r, NFV, ATV/r. **Susceptible to DRV/r.
2 post	11	TDF-FTC-DRV/r-RAL		6 weeks	17	<400	874/22%	TI	101%
3 (M) pre	9	AZT-3TC-LPV/r-SQV	21 months			2300	783/30%	TI	100%	M41L, V75A, A98G, V108I, M184V/ Low-level resistance to D4T, DDI, and ABC. High-level resistance to 3TC and FTC. **Susceptible to AZT and TDF.	Y181C/ Intermediate-level resistance to EFV and ETR. High-level resistance to NVP.	I54V, V82A/ Intermediate-level resistance to LPV/r, SQV/r, NFV, ATV/r. **Susceptible to DRV/r.
3 post	10	AZT-3TC-DRV/r-RAL		10 weeks	15	<400	730/36%	TI	97%
4 (F) pre	20	AZT-3TC-LPV/r	Off/on for 3 years; persistently × 13 months			4500	504/20%	TI	99%	M184V/ Potential low-level resistance to ABC. High-level resistance to 3TC and FTC. **Susceptible to AZT, D4T, DDI, and TDF.	None/ **Susceptible to NVP, EFV, and ETR.	L10F, L24I, M46I, I54V, A71V, L76V, V82A/ Low-level resistance to DRV/r. Intermediate-level resistance to SQV/r. High-level resistance to LPV/r, NFV, and ATV/r.
4 post	21	TDF-FTC-DRV/r		4 weeks	9	<400	484/21%	TI	98%

Abbreviations: ART, antiretroviral therapy; NRTI, nucleoside reverse transcriptase inhibitor; NNRTI, nonnucleoside reverse transcriptase inhibitor; PI, protease inhibitor; WHO, World Health Organization; VL, viral load; AZT, zidovudine; D4T, stavudine; ABC, abacavir; DDI, didanosine; 3TC, lamivudine; FTC, emtricitabine; TDF, tenofovir; ETR, etravirine; NVP, nevirapine; EFV, efavirenz; LPV/r, lopinavir/ritonavir; SQV/r, saquinavir/ritonavir; NFV, nelfinavir; DRV/r, darunavir/ritonavir; RAL, raltegravir.

^aResistance predictions per Stanford University HIV Drug Resistance Database (http: hivdb.stanford.edu/).

Table 2.

Antiretroviral Therapy History of Patient.

Patient	ART history (month/year)
1	D4T-3TC-EFV (6/05-3/06); AZT-DDI-NFV (3/06-5/07); AZT-DDI-LPV/r (5/07-1/08); DDI-3TC-LPV/r (1/08-11/08); ABC-AZT-3TC-LPV/r (11/08-5/09); TDF-FTC-LPV/r (5/09-5/10) with suspension (8/09-9/09); TDF-FTC-DRV/r-RAL (5/10-present)
2	AZT-3TC-NVP (5/02-11/03); DDI-D4T-NFV (11/03-2005); Defaulted (2005-1/06); 3TC-SQV/r-LPV/r (1/06-4/06); ABC-AZT-LPV/r (4/06-12/06); ABC-AZT-3TC-LPV/r (12/06-1/10); Suspended (1/10-6/10); AZT-3TC-LPV/r (6/10-12/10); TDF-FTC-DRV/r-RAL (12/10-present)
3	DDI-D4T-NVP (1/02-2/05); AZT-3TC-LPV/r (2/05-2/06); Suspended (2/06-5/07); AZT-3TC-LPV/r (5/07-3/08); AZT-3TC-ABC-LPV/r (3/08-7/08); AZT-3TC-SQV/r-LPV/r (7/08-2/11); AZT-3TC-DRV/r-RAL (2/11-present)
4	AZT-3TC-NVP (10/05-5/06); DDI-D4T-NFV (5/06-5/07); DDI-D4T-LPV/r (5/07-2008); AZT-3TC-LPV/r (2008-8/11); TDF-FTC-DRV/r (8/11-present)

Abbreviations: ART, antiretroviral therapy; AZT, zidovudine; D4T, stavudine; ABC, abacavir; DDI, didanosine; 3TC, lamivudine; FTC, emtricitabine; TDF, tenofovir; ETR, etravirine; NVP, nevirapine; EFV, efavirenz; LPV/r, lopinavir/ritonavir; SQV/r, saquinavir/ritonavir; NFV, nelfinavir; DRV/r, darunavir/ritonavir; RAL, raltegravir.

Discussion

Although most LPV/r resistance being noted at present in African settings is occurring in patients with a history of prior unboosted PI use,¹⁵ such histories are not uncommon in our setting. Cases of LPV/r resistance emerging after LPV/r as the only PI exposure, too, have been noted in multiple reports.^3,16,17 Also, LPV/r resistance may develop more easily in infants and young children,³ making management of LPV/r-resistant HIV a growing concern in our setting and similar settings where pediatric cohorts are accumulating experience on ART, and particularly where early adherence to ART may be challenging. Options for management of ART failure with LPV/r resistance are few—second-line regimens containing an NNRTI are often not durably potent, given most HIV-infected children, especially in southern Africa, have been previously exposed to an NNRTI, and continued use of LPV/r has not shown long-term durability when LPV/r resistance is present, as further resistance mutations accumulate.¹⁵ Without any other options, adding ritonavir-boosted saquinavir (SQV/r) or another PI to LPV/r has been tried in our and other similar settings in the past; yet this approach carries a high pill burden, is poorly tolerable to many patients, and increases the risk of PI toxicity and side effects.

Previously reported protease genotype data obtained over several years from our treatment cohort who have failed PI-containing regimens indicate DRV/r remains an option even after failure of sequential unboosted PIs with a variety of major PI mutations,¹⁷ and the same has been observed in other settings.^15,16 Hence, DRV/r and RAL represent very potential choices for salvage ART in children and adolescents who have failed LPV/r and have high-grade PI resistance. Studies of DRV/r in treatment-experienced children and adolescents have yielded evidence of safety, tolerability, and potency in younger populations in resource-rich settings,^11,12 where DRV/r has been widely recommended as salvage therapy for children and adolescents older than 3 years with high degrees of treatment experience and antiretroviral (ARV) resistance.¹² Likewise, RAL has been evaluated in treatment-experienced pediatric populations in clinical trials in both resource-rich and resource-limited settings, showing potency, durability, and safety and is US Food and Drug Administration (FDA) approved for children aged 2 years and older.^10,13 Both DRV/r and RAL in combination have been studied (along with etravirine [ETR]) in treatment-experienced adolescents to good success.¹⁸

Although our experience reported here involves a small number of patients, sustained virologic suppression has been achieved in patients previously experiencing persistent viremia with complete adherence to current ART line; immunologic indices improved or were stable at nonimmunosuppressed levels; and no intolerance or toxicity was noted, in all patients to date, including when high levels of resistance to PIs are present, including LPV/r.

Like most patients in southern Africa with LPV/r resistance, patients 1, 2, and 4 have a history of unboosted PI use prior to receiving LPV/r—in each of their cases nelfinavir (NFV) had been previously used as part of the second-line ART under older Botswana national guidelines. Patient 3’s LPV resistance developed without prior unboosted PI use, after approximately 3 years of documented persistent poor adherence to LPV/r and saquinavir/ritonovir (SQV/r).

Most patients in our setting who have failed first- and second-line ART have considerable NRTI mutations,¹⁴ making the construction of durable NRTI backbones challenging; patients 1, 2, and 3 fit this profile, and we opt to use RAL along with DRV/r in such cases. Patient 4’s genotype while on zidovudine–lamivudine-LPV/r (AZT-3TC-LPV/r), however, demonstrated no NRTI mutations other than M184V, allowing us to hold RAL, with its low genetic barrier to resistance, for future treatment options, and swap DRV/r for LPV/r with a tenofovir–emtricitabine (TDF-FTC) backbone standard in Botswana for the patient’s age group.

The success seen in our Botswana pediatric cohort is congruent with the adult cohorts in resource-rich settings, where there is considerable experience with DRV/r and RAL, including patients who failed LPV/r, and considerable evidence of efficacy of these ARV drugs toward clinical, immunologic, and virologic end points in such cases.¹⁹

Pediatric data such as ours from routine clinical use of DRV/r and/or RAL in any setting, resource-rich or resource-limited, are scanty, and we are not aware of previously published reports from sub-Saharan Africa. Yet the success seen in our cohort is compared with non-trial data from resource-rich settings. In a French cohort 11 of 12 heavily treatment-experienced adolescents achieved viral suppression (<400 copies/mL) with a regimen of DRV/r and RAL and ETR.¹⁸ Similarly, in a recently reported Spanish cohort of 19 children and adolescents (mean age 16 years) failing conventional ART of a variety of backbones, the addition of RAL resulted in viral suppression in 17 children.²⁰ As in our population, in both of these cohorts viral suppression was sustained, substantial immunologic improvement was noted in most patients, and no substantial DRV or RAL intolerance or toxicity was noted.²⁰

Given the mounting evidence of the utility and safety of DRV/r, RAL, and other newer ARV options in children, appropriately there have been calls for expanded ART options for children in resource-limited settings failing LPV/r-based second-line ART,⁵ for whom only few if any options currently exist. Specifically, DRV/r and RAL have been recommended for evaluation in such populations.⁵ Our data provide evidence that, when made available, DRV/r and RAL present viable options for salvage therapy in most African children and adolescents with LPV/r and other multidrug resistance.

Although further monitoring of our population and prospective evaluation of larger numbers of patients are necessary for more robust conclusions to be drawn, broader access to these and other salvage therapies for HIV-infected youth in the developing world merits exploration. Despite best efforts in assuring adherence and other psychosocial supports which reduce the likelihood of ART failure, the number of children and adolescents requiring approaches beyond standard second-line ART will only grow with time, heightening the urgency of an emerging area of concern in the global pediatric HIV response. Investments will be required, as most settings in Africa currently lack the readily available VL monitoring and resistance genotyping available in Botswana and South Africa, upon which more sophisticated approaches to salvage ART such as described here depend.

Footnotes

Authors’ Note

This study was approved by the Health Research and Development Committee (HRDC), Ministry of Health, Botswana, and the Institutional Review Board, Baylor College of Medicine, USA. All authors contributed significantly to the programmatic conception, data interpretation, and construction of this manuscript. We thank Mr. Vincent Mabikwa, COE M&E Officer, and Dr. Olukemi Ogundipe, Texas Children's Hospital Global Health Fellow, for assistance in data collection, and thank the Government of Botswana for their support of our program and their advocacy for universal access to care and treatment for HIV-infected children in Botswana.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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