As treatment options for HIV-infected patients have improved over time in terms of greater effectiveness, tolerability, and sheer number of available antiretrovirals, we found that a decreasing proportion of ARV-treated patients in our diverse US-based cohort had viremia and thus were candidates for GT to optimize cART (). Among ARV-experienced patients in our cohort who underwent GT in the context of routine outpatient HIV care during 1999–2008, viruses harboring resistance mutations to the main ARV classes were detected with either a constant or possibly decreasing frequency over time; resistance mutations to protease inhibitors decreased significantly in viral strains of patients who had ever been exposed to PIs.
The magnitude and temporal trends in the frequency of resistance mutations among HIV strains isolated from patients who undergo GT can be influenced by a myriad of factors, including but not necessarily limited to (i) changes in the sociodemographic and clinical characteristics of the patient population over time, particularly in a dynamic cohort such as the HOPS in which patients can enter or leave care; (ii) changes in the nature and extent of ARV exposure over time (e.g., decreasing exposure to mono- or dual-NRTI regimens, increasing exposure to newer more potent and tolerable cART regimens as first-line or salvage therapy); (iii) more widespread utilization of GT and changes in the characteristics of patients considered appropriate candidates for such testing; (iv) archiving of mutations in the absence of selective antiretroviral drug pressures; (v) cohort-level changes in the degree of patient adherence to prescribed ARVs and the related likelihood of experiencing viremia while prescribed cART.
It is important to note that our findings regarding the extent of and trends in the frequency of ARV resistance mutations apply only to the decreasing annual proportion of HOPS patients eligible for resistance testing [16
], that is, persons who experienced viremia while receiving cART. Our aim was to describe what clinicians may see in practice when testing ARV-experienced patients. Modeling of population-wide prevalence of resistance in the HOPS cohort (including both viremic and virologically suppressed patients on cART), which was not undertaken here but has been attempted in other populations [25
], would presumably reveal lower prevalence of resistance cohort-wide and potentially different trends over time. Such modeling approaches typically require strong assumptions about the likelihood of archived resistance mutations among patients who are currently virologically suppressed and imputations of missing data for many ARV-experienced patients who did not undergo GT (the bulk of ARV-experienced patients in the HOPS, per ) but can provide useful comparative data in large samples under different assumed scenarios.
Although we did not detect statistically significant declines in the frequency of any ARV resistance among HIV strains from all HOPS patients who underwent GT in clinical practice and among the subset who were solely cART-experienced, recent population-based studies provide supporting evidence for such declines. These studies suggest that the incidence of ARV resistance is decreasing [19
] due to increasing use of cART regimens that are more tolerable, potent, easier to adhere to, and consequently less likely to result in resistance [18
] and due to smaller proportions of patients failing antiretroviral therapy [7
] or being sufficiently viremic while receiving cART to allow for the performance of GT.
We also found that HOPS patients who underwent GT were increasingly less likely to have virus with PI resistance, whereas potential declines in NNRTI resistance were more modest and nonsignificant over time. These trends might reflect shorter persistence of PI mutations than NNRTI mutations in the absence of drug pressure or greater use of ritonavir-boosted PIs, which are more potent and confer greater barriers to the emergence of resistance. Among ARV-naïve patients initiating cART who were followed in British Columbia, Canada, the development of ARV drug resistance was associated with the use of non-boosted PI-based or NNRTI-based regimens [18
], suboptimal levels of ARV adherence (particularly in the 80%–95% range), low pre-cART CD4+ cell counts, and high baseline HIV RNA levels [18
], and was reduced for patients who initiated cART in 2002–2004 versus earlier [18
]. In the Swiss Cohort study, the estimated prevalence of resistance in a population of over 8,000 ARV-exposed patients also decreased by approximately 12% during 1999 to 2007 and was driven by loss to follow up or death of patients with mono-dual NRTI exposure countered by continued enrollment of patients starting cART with NNRTI-based or boosted PI-based regimens [17
We excluded from our primary analyses genotypic tests performed on plasma HIV RNA samples with ≤1,000
copies/mL because such assays may provide unreliable results (i.e., appear falsely negative for resistance mutations) due to insufficient HIV RNA copy and such patients may have low level self-limited viremia (viral “blips”) rather than true virologic failure. Indeed, when comparing results from genotypic tests performed when HIV RNA >1,000 copies/mL versus ≤1,000 copies/mL in the latest years of the study, we found that the frequency of “insufficient specimens” was higher, while the frequency of detected resistance was lower in the latter group, especially when HIV RNA <500 copies/mL. Aside from concern for bias due to nonevaluable samples, including the relatively few results from genotypic tests performed with HIV RNA between 500 and 1,000
copies/mL would have likely accentuated the observed decreases in the frequencies of resistance at GT through 2008. Our analytic approach ensured more comparable data over time, and the declines in resistance we observed are likely conservative.
Our findings suggestive of declines over time in rates of ARV resistance are notable for at least two reasons. First, because of the method we employed to analyze cumulative frequency of ARV resistance to date, by “carrying forward” previously identified mutations that may have become archived, the frequency of resistance in later years should tend to be higher (because patients have had the opportunity to accumulate more mutations over time as revealed by successive genotypic tests performed) than would be suggested if results were based on a single genotypic test that year. Further, one might expect to find higher overall frequency of mutations over time, as newer ARVs are added to the three drug classes, with new, often ARV-specific, resistance mutations being identified and detected using contemporary genotypic tests.
We believe that three factors most likely explain the lower frequency of resistance among HOPS patients who underwent GT over time: (i) the increasing use of more tolerable and potent cART regimens associated with higher barriers to resistance or more complex mutational pathways necessary for resistance to develop; (ii) the decreasing proportion of HOPS patients under observation having mono-dual NRTI exposure; (iii) the increasing and more widespread use of GT in the HOPS with possible testing of patients at lower risk of virologic failure due to resistance. The decreasing frequency of resistance to PIs in our population was associated with increased use of more potent PIs (i.e., ritonavir boosting). This trend could have also occurred if exposures to PIs were increasingly distant, which would have allowed greater opportunity for PI resistance mutations to become archived before patients underwent GT. However, we found no evidence that the time between PI exposure among exposed patients and time of genotypic test differed over the study observation period.
In a cohort study of 1,587 HIV-infected patients at the University of North Carolina (2000–2006), of whom 607 had at least one genotype, there were 27.2% of patients with TCR among 437 patients who had GT and any exposure to each of the three drug classes. This was lower than the 40.6% estimated for patients with TCE in our study, possibly because we required a minimum of three months of exposure on each of the three drug classes to define TCE [25
]. Our findings of high frequency of any resistance mutations in the genotyped population are consistent with the findings from an early large genotypic substudy of patients with HIV viral load >500 copies/mL in the population-based HIV Cost and Service Utilization Study (HCSUS), which documented that 76% of tested patients had evidence of genotypic resistance to one or more antiretroviral drugs in 1996–1998 [23
The findings from our ecological analyses should be interpreted in light of several additional caveats and limitations. First, most patients receiving antiretrovirals in the HOPS were virologically suppressed and, over time, fewer were eligible for GT during the study period, resulting in a progressively smaller and more select sample of patients with GT results, raising the concern for potential bias and limiting statistical power. Second, we relied on data collected in the course of regular HIV care since our purpose was to reflect what a treating clinician would see. Not all eligible patients had GT performed, and trends in and magnitude of resistance frequencies detected may have been different if all ARV-experienced, viremic patients had undergone scheduled GT at regular intervals [16
]. Third, some mutations that arose during prior courses of ARV therapy (before the advent of GT or before HOPS entry) may have no longer been apparent because of a lack of continuing ARV drug pressure; however, we do not believe this effect would have systematically biased our findings over calendar years away from the null hypothesis. Fourth, patients who harbored the greatest number of mutations were also more likely to have had advanced HIV disease and an extensive ARV treatment history that involved mono-dual NRTI exposure (data not shown). These patients may have died or been lost to follow up at rates higher than other HOPS patients during the study period. Moreover, the demographics of patients who underwent GT shifted toward a higher percentage of women, persons of color, and those with heterosexual risk for HIV infection, all factors which are associated with more recent HIV diagnoses and less exposure to ART in the HOPS (data not shown). These dynamics may explain in part the decreasing trends in resistance in our open cohort. Finally, we have no systematic data on pre-ARV resistance profiles for the majority of patients and therefore could not evaluate the role of primary transmitted drug resistance or incidence rates of HIV resistance.
In conclusion, the frequency of ARV resistance mutations detected for HIV among patients in the HOPS who were tested in the course of routine clinical practice was high and tended to decrease during 1999–2008. The decreasing frequency of genotypic resistance corresponded with at least two trends: an increasing use of more tolerable and potent cART, including boosted PI regimens, and a decreasing proportion of HOPS patients with histories of exposure to mono- and dual-NRTI ARV regimens. Our findings support the continuing need for routine HIV resistance testing and monitoring of evolving ARV resistance patterns among ARV-treated patients [15
] who experience virologic nonsuppression while on therapy. Available evidence suggests that adoption of such testing as routine would facilitate tailoring of more effective cART [29
], reduce the likelihood of further resistance evolution, and ultimately augment cART-related reductions in HIV-related mortality [7