Monitoring and treatment in South Africa
Responding to antiretroviral failure and selecting an optimal regimen is very context specific. In South Africa, viral load monitoring is routinely available, but with only very limited access to antiretroviral resistance testing and with only two lines of therapy for children. In infants exposed to nevirapine, through a PMTCT intervention, there may effectively be only one regimen. In young sub-Saharan African children, who often start therapy in infancy, an NNRTI regimen seems less likely to suppress viral loads than a boosted PI regimen [5
], and in a recent multicentre study, P1060, an NVP regimen has been shown to be inferior to an LPV/r regimen both in children exposed to NVP through PMTCT [6
] and in those unexposed [7
Furthermore, children who were switched, when virologically suppressed from LPV/r to NVP were more likely to have viral loads of more than 1000 copies per ml and harbour resistance than those retained on LPV/r [8
]. This is in contrast to the multi-centre PENPACT-1 study where outcomes for PI and NNRTI regimens were similar; however, the median age was 6.5 years (much older than in P1060), and in 48%, the PI prescribed was nelfinavir, which has a lower genetic barrier than LPV/r.
Detected protease inhibitor resistance
We found that 14 out of 23 children with historic exposure to a regimen that included RTV sPI had major PI resistance, whereas none of 30 given LPV/r had major PI resistance. Nevertheless, it is not known if RTV sPI exposure per se
was aetiological in selecting for PI resistance, in all cases, as other factors, such as longer therapy duration[3
] and concomitant rifampicin use, could have contributed to PI resistance. The high prevalence of major PI resistance mutations (14 out of 82 or 17%) in this study cannot be extrapolated to the population as a whole as most of these specimens were referred from tertiary hospitals. However, this may represent a typical setting, which takes care of paediatric patients with long-term failure.
As we did not observe any PI resistance, despite having detectable viral loads, in nine of the 23 patients treated with LPV/r and prior RTV sPI treatment, and 30 out of 30 without prior RTV sPI, their virological failure was most likely due to poor adherence or inadequate dosage. This concurs with a French study that found a very low rate of PI resistance in children initiated on LPV/r despite a high prevalence of virologic failure [9
]. Three patients (patients 32, 334 and 344) harboured T74S, a common HIV-1 subtype C protease
polymorphism, which is found in higher frequencies in patients treated with PIs, especially nelfinavir. It has been reported to possibly restore fitness in patients with multiple PI resistance and to increase susceptibility to ritonavir and indinavir [10
Detected NRTI resistance mutations
Once major PI resistance was present, as expected, all children on lamivudine (3TC) had the M184V mutation. 3TC has a low genetic barrier and M184V occurs early during true drug failure [11
]. A high prevalence of M184V has been reported in other studies in children from sub-Saharan Africa [13
]. Nevertheless, 3TC is still preferred as a component of first-line therapy and often retained in second-line regimens for the following reasons: it has excellent tolerability and M184V increases susceptibility towards other NRTI components, such as AZT, D4T or tenofovir (TDF). Furthermore it reduces viral fitness, slows the accumulation of TAMs [12
] and may have clinical and immunological benefit [18
Outcomes after detecting PI resistance
Patients who continued on an LPV/r regimen had a better virological response than those switched to an NNRTI regimen. However, in two of three patients, who were switched to LPV/r after RTV sPI, additional mutations were observed in their second specimens, increasing PI resistance, thus questioning the durability of LPV/r therapy. However, despite significant PI resistance in 12, with additional TAMs in five children, there was no immunologic deterioration and viral loads remained relatively low in the majority.
This may be due to residual efficacy of the antiretroviral drugs (especially LPV/r), especially at increased plasma levels [19
] and the reduced fitness (crippling effect) of some resistance mutations, such as M184V
, and some PI resistance mutations. Nevertheless, children require ART for life. Inadequate response to therapy may have developmental and neurological consequences and could seriously compromise quality of life. Non-suppressive antiretroviral therapy may in the long run compromise future therapy options through the accumulation of resistance mutations, despite intermediate-term immunological and clinical benefits.
Criteria for genotypic resistance testing
The use of RTV sPI in children contributed to a cohort with an increased risk of PI resistance and therapy failure [20
]. Although children who never received an unboosted PI may also develop PI resistance, the current risk is probably too low to include this in criteria for genotypic resistance testing for resource-limited settings. A good adherence history, in combination with random LPV plasma concentration measurement (which cost only about US$40 in the South African state sector), may exclude patients with very poor adherence from unnecessary GRT, which is more expensive (about US$300 for in-house testing through the National Health Laboratory Service, the public laboratory service provider in South Africa).
An adequate plasma lopinavir concentration does not exclude periods of poor adherence as ingesting few doses before phlebotomy could result in adequate concentrations. However, a low level is indicative of poor adherence. Such random testing has been shown to be valuable in South African adults on second-line therapy [21
]. A further benefit of LPV plasma level monitoring is to facilitate dose adjustment and achieve virological suppression. An additional surrogate for adherence is macrocytosis in patients on either zidovudine or stavudine [22
Due to the reduced fitness of some resistant viruses, a high percentage of children with PI resistance may have relative low viral loads, as seen by the number with viral loads below 5000 copies/ml. Therefore, 1000 copies/ml may be an appropriate cut off for resistance testing in patients with a high pre-test probability of protease resistance (such as prior exposure to an RTV sPI ART regimen). Therefore, we recommend using 1000 copies/ml, rather than 5000 copies, as suggested by the World Health Organization, for those who might benefit from resistance testing.
Defining criteria for third-line therapy
Children who are switched to an NNRTI regimen at the time of PI failure are likely to have an increased risk of failure and resistance, due to the low genetic barrier of the regimen, previous exposure to NVP for PMTCT, and probable sub-optimal adherence. Furthermore, those failing a PI regimen, but with NRTI resistance (such as TAMs) are unlikely to achieve full virological suppression on a second-line NNRTI regimen, and thus rapidly acquire NNRTI resistance. There is therefore a need for a durable third-line combination. However, third-line therapy for children not responding to the currently available regimens is more costly than standard first- or second-line therapy. Therefore, defining indications for third-line therapy in a cost-effective manner is essential.
Two candidate PIs for salvage are TPV/r and DRV/r. Proposed criteria for GRT and third-line ART regimens in children in a resource-limited setting are provided here:
Criteria for paediatric genotype resistance testing (GRT)
Criteria A and B must be met
A) Failure of LPV/r regimen (two sequential viral loads >1000 copies/ml, despite confirmed adherence of >90%) while LPV drug levels are within the therapeutic range.
B) Prior exposure to an unboosted sPI regimen, such as RTV.
Criteria for third-line ART regimens following GRT
Criteria A AND (B OR C) must be met
A) Susceptible or low-level resistance to the proposed high barrier salvage PI (DRV/r or TPV/r) by the Stanford HIVDB RIS.
B) Intermediate resistance to LPV/r (including either Rega, HIVDB, ANRS or Geno2Pheno) with three or more TAMs.
C) High-level LPV/r resistance (including either Rega, HIVDB, ANRS or Geno2Pheno).
Selecting a third-line regimen
Although continuing therapy without virological suppression is likely to result in additional resistance accumulation, stopping antiretroviral therapy causes rapid clinical and immunological deterioration [24
]. In selecting an optimal third-line regimen, a balance between tolerability, residual activity, fitness benefit and the resistance threshold of a regimen should be sought. Where possible, there should be at least good susceptibility (no more than low-level resistance) to two of a three-drug regimen. The most essential component is a PI with a high barrier to resistance, such as DRV/r or TPV/r. The choice is guided by the resistance pattern and the age of the patient. TPV/r is available in a liquid formulation for children as young as two years, whereas DRV/r is only available in tablets for children older than six years. DRV has a better side-effect profile than TPV.
Therapy history and genotypic resistance testing should guide the choice of the best NRTI backbone. When the selective pressure of a particular drug is removed, resistance may become undetectable, but remains clinically relevant. Almost all who previously failed a regimen including 3TC have the M184V mutation irrespective of the current genotypic result. TAMs and other NRTI resistance mutations, such as K65R, L74V and multiple NRTI resistance mutations, are especially valuable in determining the best NRTI backbone. Quite often, in the presence of TAMs, tenofovir (TDF) is the only NRTI with full susceptibility. However, there is no formulation for patients who weigh less than 30kg. Didanosine (DDI) often shows susceptibility. However, its poor tolerability could contribute to a high failure rate.
Even when the genotype suggests combining DDI and abacavir (ABC), one should consider that resistance to both drugs are conferred by the same mutations (L74V and K65R), thus potentiating rapid failure. Rarely, susceptibility to all NRTIs is lost, necessitating the use of other drugs classes in combination with DRV/r or TPV/r. 3TC could be retained despite resistance, as we have discussed. Other valuable salvage drugs, such as the integrase inhibitor, raltegravir, the second-generation NNRTI, etravirine, and the CCR5 inhibitor, maraviroc, are not yet licensed for children.
Recent data has shown that raltegravir is valuable in paediatric treatment [25
]. When raltegravir is used in treatment-experienced children, due to its low genetic barrier, the proposed regimen must be able to achieve full virological suppression. It should therefore combine potent and high genetic barrier drugs, such as DRV/r or TPV/r. The same applies to etravirine, especially with prior exposure to NNRTIs. However, the addition of raltegravir or etravirine may double the cost of the regimen. A practical approach is to combine a high-barrier PI (DRV/r or TPV/r) with at least one other drug with full sensitivity and a third with some beneficial effect (such as 3TC). Expedited viral load testing should occur within two to three months.
Successful third-line therapy of paediatric patients is hindered by the lack of paediatric formulations and high costs, with dosing especially problematic for children younger than six years, largely a result of the low priority that is given globally to the development of paediatric formulations and regimens [26
]. 3TC monotherapy and other sub-optimal interim measures, although being used in some resource-limited settings, are not evidence based, whereas continued PI therapy, even when it does not achieve virological success, could nevertheless render immunological and clinical benefit in children [27
], but at the potential cost of resistance accumulation.