1 Consensus HIV-1 Drug Resistance Mutations (DRMs) for Point of Care (POC) Testing Draft for comment: May 6, 2014 I. Rationale for POC Genotypic Resistance Testing More than ten million individuals in low- and middle-income countries (LMICs) are receiving antiretroviral (ARV) therapy (1). The global scale-up of ARV therapy has dramatically reduced HIVassociated mortality, mother-to-child HIV-1 transmission, and adult HIV-1 incidence (2-5). These public health accomplishments were made possible by the widespread administration of fixed-dose combinations of two nucleoside reverse transcriptase (RT) inhibitors (NRTIs) plus a non-nucleoside RT inhibitor (NNRTI) (6, 7). The margin of long-term ARV treatment success, however, is narrow because NNRTIbased regimens have a low genetic barrier to resistance. ARV treatment failure with a fixed-dose NRTI/NNRTI combination occurs in 10% to 30% of patients per year (8-10) and most patients with virological failure acquire NRTI and/or NNRTI resistance (10-12). As the number of patients with acquired ARV resistance has increased so has the proportion of newly infected patients with transmitted drug resistance (TDR) (11, 13, 14). In recent surveys in LMICs in SSA and SSEA, about three percent of ARV-naïve patients had genotypic evidence for TDR (11, 13, 14). In several recent large studies the proportion of ARV-naïve patients with genotypic resistance has been above five percent (15, 16). In many regions, the proportion of patients with transmitted NNRTI resistance has been increasing since ARV-scale-up (11, 13, 14). Additionally, a significant proportion of patients presenting for initial ARV therapy may have acquired drug-resistance stains as a result of previous unreported ARV-exposure (17). If the prevalence of drug resistance in patients presenting for initial ARV therapy becomes high enough to jeopardize ARV treatment success in a significant proportion of patients, patients and care providers would have reduced confidence in the treatability of HIV-1 infection. Fewer patients would present for care or seek to learn whether or not they were HIV-1-infected. Simply changing national recommendations for initial ARV therapy from an NNRTI-based regimen to a protease inhibitor (PI)based regimen would be suboptimal because PI-based regimens are more expensive and often less tolerable than NNRTI-based regimens. Pre-therapy genotypic resistance testing would therefore be useful to identify which patients should receive standard first-line therapy and which should receive a PIcontaining regimen. The use of genotypic resistance testing in tandem with virus load testing will improve the sensitivity and specificity of virological monitoring to identify patients whose ARV treatment regimens have reduced efficacy. Patients with elevated plasma HIV-1 RNA levels while receiving ARV therapy will not require a second confirmatory plasma HIV-1 RNA level if their initial elevated level was accompanied by genotypic evidence for drug resistance. Conversely, patients with elevated plasma HIV-1 RNA levels on ARV therapy but without evidence of genotypic resistance are candidates for adherence counseling rather than a change to more expensive and less tolerable second line regimens. Most LMICs have central laboratories capable of performing genotypic resistance testing using standard RT and protease sequencing. However, the use of these laboratories for genotypic resistance testing for individual patient management will not be practical in the near future as the infrastructure in many LMICs does not exist to support the expansion of these laboratories or the rapid transportation of samples to these laboratories. Moreover, the use of centralized rather than POC genotypic resistance testing requires additional potentially burdensome patient clinic visits. Most POC genotypic resistance tests are likely to be allele-specific point mutation assays. At least two such assays are undergoing field-testing in resource-limited settings for possible routine clinical use (18, 19). One commercial assay has been developed for detecting Mycobacterium tuberculosis rifampin- 2 resistance mutations (20). This document will review HIV-1 drug resistance knowledge to identify DRMs most useful for point mutation assays designed to detect HIV-1 resistance to the ARVs most likely to play a significant role in the next five years. The selection of a DRM for POC genotypic testing depends on the sensitivity and specificity of the DRM for identifying drug-resistant HIV-1 strains and on the implications of the DRM for the success of ARV therapy in the clinics likely to use POC tests. The remainder of this document describe (i) the characteristics of a DRM most relevant to its use in genotypic resistance testing; (ii) the clinical settings in which POC genotypic resistance testing is likely to be most useful; and (iii) how the characteristics of different DRMs and clinical settings can be combined to identify the DRMs most useful for inclusion in a POC genotypic resistance test. Separate documents will model the potential health benefits and costs of POC genotypic resistance testing and will provide analyses to assist technical aspects of detecting HIV-1 DRMs. II. Characterization of HIV-1 Drug Resistance Mutations (DRMs) The NRTIs, NNRTIs, and PIs are the ARV classes used in most LMICs. The integrase inhibitors (INIs) are extraordinarily effective, safe, and well tolerated but have been used primarily in high-income countries. Should INIs become affordable, they will also play an important role in ARV treatment in LMICs. All NRTI and NNRTI DRMs are in the RT gene and there is practically no cross-resistance between these two drug classes. Each of the phenotypically and clinically established PI DRMs is in the protease gene and genotypic testing for PI resistance relies entirely upon the detection of protease mutations. It is not known, however, whether or not mutations outside of the protease gene can also reduce PI susceptibility (21, 22). Each of the INI-resistance mutations is in the integrase gene. A DRM can be characterized according to the following five criteria: (i) Its prevalence in virus isolates from ARV-naïve patients in regions with low-levels of TDR (polymorphism rate); (ii) Its prevalence in virus isolates from patients receiving ARV-therapy (treatment prevalence). (iii) The frequency with which the DRM occurs alone compared with the frequency with which it occurs with other DRMs (primary vs. secondary); (iv) Its contribution to reduced in vitro susceptibility either alone or in combination with other DRMs (in vitro phenotype); (v) Its association with a reduced virological response to an ARV in a new treatment regimen (clinical response). Virus subtype can influence the likelihood of acquiring a few DRMs but there is no evidence that DRMs have different effects in different subtypes. The Stanford HIV Drug Resistance Database (HIVDB) has an online genotypic resistance interpretation program to help clinicians and laboratories interpret HIV-1 genotypic resistance tests (http://hivdb.stanford.edu). The program’s output includes a list of penalty scores for each DRM in a submitted sequence and an estimate of reduced susceptibility for each ARV obtained by adding the penalty scores for each DRM in the submitted sequence. The mutation penalty scores can be found at http://hivdb.stanford.edu/DR/. Weighing the five characteristics described in the previous paragraph was instrumental to developing the scores. A score of 15 is associated with low-level resistance; a score of 30 indicates intermediate resistance; and a score of 60 indicates high-level resistance. In this document, mutations with a score of 30 or more are referred to as major DRMs. (i) Polymorphism rate. Most DRMs are nonpolymorphic in that they do not occur in the absence of selective drug pressure. Some DRMs are polymorphic and may occur naturally in ARV-naïve patients. Nonpolymorphic DRMs may directly reduce susceptibility or reduce susceptibility in combination with other DRMs. Polymorphic DRMs are uniformly accessory. The fourth column of Tables 1, 2, and 3 contains the polymorphism rate of the NRTI, NNRTI, and PI DRMs with an HIVDB mutation penalty score and a prevalence of ≥0.3% (about 1 in 300). Nonpolymorphic DRMs are useful for TDR surveillance because they are specific indicators of selective drug pressure and are also clinically more 3 significant than polymorphic DRMs (23). The nonpolymorphic DRMs used for TDR surveillance – surveillance DRMs (SDRMs) – are checked. (ii) Treatment prevalence. Nonpolymorphic DRMs that are selected during ARV therapy are a high priority for inclusion in a list of consensus DRMs for POC testing. Assays that include a sufficiently large set of commonly occurring DRMs will have a high sensitivity for detecting both transmitted and acquired drug resistance. The fifth column of Tables 1, 2, and 3 contains the prevalence of NRTI, NNRTI, and PI DRMs in pooled sequences from NRTI-, NNRTI-, and PI-experienced patients. The evolution of a mutation during ARV therapy provides Darwinian evidence that the mutation is associated with resistance to the ARV that selected the mutation. The next draft of this document will indicate which DRMs are most relevant to each ARV by having separate lists of DRMs selected by each ARV (rather than ARV class). (iii) Primary vs. secondary. HIV-1 strains from patients with virological failure often contain several DRMs associated with resistance to an ARV they are receiving. Usually, the first or primary DRM reduces ARV susceptibility by itself, whereas the mutations that follow lead to further reductions in susceptibility or compensate for the reduced fitness associated with a primary DRM (24). The sixth column of Tables 2, 3, and 4 indicate the frequency with which each DRM occurs in the absence of other major DRMs (defined as other DRMs with a high HIVDB mutation penalty score). It is important to note, however, that some DRMs that usually occur in the presence of another DRM may have a primary role in conferring resistance to additional DRMs. For example, many primary NNRTI DRMs usually occur in the presence of M184V because this mutation develops rapidly during 3TC or FTC selective pressure. K65R, L74V, and T215Y are primary mutations that often follow M184V and confer resistance to ARVs other than 3TC and FTC. In addition, several of the rare mutations that are found alone may reflect transmitted DRMs (e.g., the T215 revertants: T215S/C/D/I/V) (iv) In vitro susceptibility: Phenotypic in vitro susceptibility assays measure the susceptibility of a virus to an antiviral agent in cell culture. Antiviral susceptibility is usually reported as the concentration of antiviral agent that inhibits viral replication by 50% (IC50). The IC50 is compared to that of a drugsensitive reference virus, and expressed as a ratio (often referred to as fold change) of the experimental virus versus the control. In vitro susceptibility data must be interpreted in the context of the susceptibility assay used and the ARV tested. For the same viruses, different assays may produce different levels of fold change in susceptibility (25). The clinical significance of reductions in susceptibility may vary markedly between ARVs belonging to the same or different ARV classes. The NRTIs must be triphosphorylated to their active form, a process that occurs at different rates in different cell lines. The dynamic susceptibility range between wild type and the most NRTI-resistant isolates is as low as 5-fold for tenofovir and more than 200-fold for AZT, 3TC, and FTC (26, 27). Similar but less pronounced differences in the dynamic susceptibility range exist within the NNRTI, PI, and INI classes (28-30). The contribution to reduced susceptibility of many NRTI, NNRTI, and INI DRMs has been studied using laboratory clones containing site-directed mutations and using drug-resistant clinical virus isolates from patients. The effect of a DRM or combination of DRMs when placed in a laboratory clone on ARV susceptibility yields an unbiased assessment of a mutation’s phenotypic effect. However, the number of DRMs and DRM combinations studied in this manner particularly in the same phenotypic assay is limited. This is particularly the case for DRMs with highly context-dependent phenotypic effects, particularly PI DRMs. The contribution of a DRM to reduced ARV susceptibility in clinical isolates can be studied using regression analyses in which the presence or absence of a DRM is an explanatory variable and the fold reduction in susceptibility is the outcome variable. Tables 4, 5, and 6 contain the regression coefficients 4 indicating the contribution of NRTI, NNRTI, and PI DRMs to reductions in susceptibility for the most relevant NRTIs, NNRTIs, and PIs used in LMICs. The regression coefficients indicate the relative contribution of a DRM to reduced ARV susceptibility while controlling for the other DRMs in a virus sequence. The regression models were described in three publications (27-29). (v) Clinical or virological response to ARV therapy. Two types of studies have evaluated the effect of a DRM on the virological response to an ARV treatment regimen: 1. Studies of the effect of a pre-therapy DRM on the virological response to an initial ARV treatment regimen; and 2. Studies of the effect of a pre-therapy DRM in an ARV-experienced patient on the virological response to a change in ARV treatment. In regions where genotypic resistance testing is routinely performed before therapy, genotypic results are used to guide therapy. Therefore, there are few studies in which it has been possible to examine the effect of a DRM on the response to an initial ARV regimen. These studies have involved the retrospective testing of cryopreserved blood samples obtained from patients in whom genotype resistance testing was not performed prior to the start of therapy (31-35). Most of these studies concluded that pre-therapy DRMs are associated with an increased risk of virological failure (16, 34). However, because most patients in these studies have wild type viruses prior to therapy, these studies have had insufficient power to assess the effects of most individual DRMs. Nonetheless, in aggregate these studies suggest that NNRTI DRMs pose a higher risk to the success of first-line therapy than NRTI DRMs. This conclusion is supported by additional studies in which ARV therapy was selected on the basis of genotypic resistance testing using standard direct PCR Sanger sequencing but was followed by assays for low-abundant variants not detectable by direct PCR Sanger sequencing. In these studies, the presence of low-abundance NNRTI DRMs, particularly K103N, was associated with an increased risk of ARV treatment failure (36-38). Many studies have attempted to determine the effect of individual DRMs on the virological response to an ARV in a salvage therapy regimen. Most of these studies have had too few patients relative to the large number of covariates associated with response to salvage therapy. First, patients usually had complicated past ARV treatment histories. Second, multiple DRMs were usually present in the pre-salvage therapy virus sequences making it difficult to determine which mutations were most likely to have influenced the virological response to therapy. Third, the pre-therapy virus levels were heterogeneous. Fourth, the salvage regimens were usually heterogeneous making it difficult to assess the effect of DRMs on individual ARVs. Fifth, in nearly all of these studies, the pre-salvage therapy genotype influenced the ARVs selected for treatment. Nonetheless, several large studies of patients in ARV registration trials – in which the variability in the patient characteristics and in the ARVs used for salvage therapy was minimized – led to an improved understanding of the clinical significance of several of the most common DRMs. Table 10 summarizes the most informative of these studies. Effect of subtype on DRM prevalence: There are differences in the proportions of nonpolymorphic DRMs in different subtypes. Many of these differences reflect differences in ARV usage patterns in different regions. A few differences, however, are directly related to inter-subtype sequence differences. For example, V106M occurs more commonly in subtype C viruses from patients treated with nevirapine or efavirenz than in viruses belonging to any other subtype because this mutation requires a single basepair change in subtype C viruses – GTG (V) => ATG (M) – but a two base-pair change in each of the other subtypes – GTA (V) => ATG (M) (39, 40). By virtue of a different mechanism, subtype C viruses may be predisposed to develop the NRTI-resistance mutation K65R (41). 5 III. Clinical Scenarios for POC Testing Patients with virological failure on an initial NRTI/NNRTI treatment regimen: The use of POC genotypic resistance testing in tandem with virus load testing will improve the sensitivity and specificity of virologic monitoring to identify patients in whom ARV treatment regimens may have reduced efficacy. In patients with detectable levels of plasma viremia, the presence of a major HIV-1 DRM confirms that virological failure occurred and suggests that the next appropriate management step would be to change therapy. The absence of a DRM in patients with detectable viremia suggests that counseling to improve adherence and follow-up plasma HIV-1 RNA testing are necessary (42). To identify DRMs that would be the most sensitive and specific indicators of virological failure in patients receiving a first-line NRTI/NNRTI-containing regimen, we analyzed publicly available RT sequences from 4,019 patients with virological failure on the regimens most commonly used or currently recommended for initial therapy in LMICs. Table 11 summarizes the number of patients according to first-line treatment and HIV-1 subtype. Sixty-two percent (n=2,494), 26% (n=1,363), 10% (n=406), and 2% (n=75) received a d4T, AZT, TDF, or ABC-containing regimen, respectively. About one-half of the patients received EFV and one-half received NVP. About one-half of the patients were infected with subtype C viruses and about 18% with CRF01_AE viruses. Subtypes A, B, G, and CRF02_AG each accounted for 5% to 7% of viruses. Subtypes D, F, and miscellaneous subtypes each accounted for 1% to 3% of viruses. Of the 4,019 sequences, 73% (n=2,966) had ≥1 major NRTI DRM and ≥1 major NNRTI DRM. Nine percent (n=349) had a major NNRTI DRM but no major NRTI DRM. Two percent (n=81) had a major NRTI DRM but no major NNRTI DRM. Sixteen percent (n=636) had no major NRTI- or NNRTI DRM. Table 12 shows the most common major NRTI DRMs: M184V (91%), K65R (10%), and the thymidine analog mutations (TAMs) K70R (15%), T215Y (12%), and T215F (9%). Table 13 shows the most common patterns of NNRTI DRMs. The most common patterns in sequences without M184V were K65R alone (2.3%), M184I alone (1.1%), K65R+M184V (0.5%), K65R+Y115F (0.4%), K65R+Q151M (0.2%), T215Y (0.2%), and Q151M+M184V (0.2%). M184V and/or K65R occurred in 98% of sequences with one or more NRTI-DRM. The importance of K65R as the second most common major NRTI-DRM is more evident in the 406 patients with virological failure on a first-line TDF-containing regimen (Table 14). Although TDF-regimens comprise about 10% of this dataset, they are being increasingly used as a result of their generally favorable safety and efficacy profile. Table 12 also shows the most common NNRTI DRMs: K103N (46%), Y181C (28%), G190A (21%), V106M (17%), and K101E (13%). K103N, V106M, Y181C, and G190A were present in 90% of sequences with ≥1 major NNRTI DRM. Table 13 shows that the most common DRM patterns without one of these four mutations included: Y188L (3.2%), G190S/E/Q (1.5%), Y181I/V (1.4%), V106A + F227L (0.6%), and K101E + G190S (0.6%). Drug-resistance before starting an initial ARV treatment regimen: Should the levels of TDR continue to increase, pre-therapy POC genotypic resistance testing will be useful to identify patients in whom a first-line PI-containing regimen would be preferable to a first-line NNRTI-containing regimen. Pretherapy POC testing will also be useful in regions where the patients frequently change clinics and may present with unreliable treatment histories. NNRTI and NRTI resistance are the most common forms of genotypic resistance in previously untreated patients (11, 13, 14). Table 15 contains data from a completed analysis of RT sequences from 51,200 patients in 294 published studies (manuscript available upon request) that examined drug-resistance in ARV-naïve populations using the WHO-list for surveillance DRMs (SDRMs). The NNRTI SDRMs are 6 all major DRMs in that they have an HIVDB mutation penalty score ≥30. Nearly all NRTI SDRMs have a mutation penalty score ≥15. The next draft of this document will also include a table listing only the major NRTI DRMs. The NNRTI DRMs K103N, Y181C, G190A, and K101E accounted for 80% of viruses with one or more major NNRTI DRM and were the four most common mutations in all regions and subtypes (Table 15). V106M was the fifth most common NNRTI DRM in subtype C viruses consistent with previous reports that this mutation results from a single base-pair change only in subtype C viruses (39, 40). M184V and the TAMs were the most common NRTI DRMs in all regions. Although M184V is the most commonly occurring NRTI DRM in patients with virological failure on a first- or second-line regimen, it was present in fewer than 20% of viruses with an NRTI SDRM in untreated patients, consistent with its reduced replication fitness and frequent reversion to wild type in the absence of ARV-selection pressure (43, 44). As noted above in the section “Virological response to ARV therapy”, most available data suggest that NNRTI DRMs are more likely than NRTI DRMs to reduce the virological response to initial ARV therapy. In addition, the clinical significance of pre-therapy TAMs will be further reduced in patients receiving TDF-containing initial ARV regimens. Virological failure on a second-line PI-containing regimen: The most common second-line ARV regimens in LMICs include ritonavir-boosted lopinavir (LPV/r) usually in combination with AZT/3TC or TDF/3TC. Despite the high-level of NRTI resistance in many viruses from patients starting second-line therapy, the virological response to a second-line LPV/r-containing regimen is usually about 80% during the first year of therapy in adults and somewhat lower in children (45-52). When virological failure occurs in LMIC patients receiving a second-line LPV/r-containing regimen, the proportion of patients with PI DRMs is low (53, 54). These data are consistent with the observation from studies in high-income countries that the viruses from most patients with detectable viremia on an initial boosted PI-containing regimen do not have phenotypic PI resistance or viruses with PI DRMs (34, 55-57). Nonetheless, the spectrum of PI DRMs associated with virological failure on initial LPV/r and ATV/rcontaining regimens is becoming evident. Among protease sequences from 1,049 patients receiving LPV/r, 16.6% had one or more major PI DRMs including V82A (8.5%), L76V (4.9%), L90M (2.7%), I84V (2.6%), V82S/T/M/F (2.6%), I47V/A (2.2%), I50V (0.8%), and V32I (0.8%). Among protease sequences from 124 patients receiving ATV/r, 31.5% had one or more major PI DRMs including I50L (26%), N88S (4.0%), and L90M (4.0%). IV. Selection of DRMs for POC Testing Tier 1: NNRTI DRMs: K103N, V106M, Y181C, G190A NRTI DRMs: K65R, M184V Sensitivity of Tier 1 DRMS for Detecting Transmitted and Acquired Drug Resistance* ARV Class ARV-Naïve Patients Patients on Standard 1st-Line Regimens Sensitivity for detecting NNRTI 75% 90% resistance Sensitivity for detecting NRTI 20% 98% resistance Sensitivity for detecting either NRTI or 95% 99% NNRTI resistance *Transmitted and acquired drug resistance are defined as an HIVDB score of ≥30 for one or more of the following NRTIs: 3TC, FTC, TDF, AZT, ABC and for NVP or EFV 7 Tier 2: NNRTI DRMs: K101E, V106A, Y181I/V, Y188L, G190S/E/Q NRTI DRMs: M41L, K65N, K70R/E, L74V/I, Y115F, Q151M, M184I, T215Y/F a. Improved sensitivity for detecting transmitted NRTI and NNRTI resistance b. Improved sensitivity for detecting acquired NRTI and NNRTI resistance including resistance to NRTIs other than 3TC and FTC. c. 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