Download Consensus HIV-1 Drug Resistance Mutations (DRMs) for Point of

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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-
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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
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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
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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).
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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
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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
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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. Sensitive for detecting acquired NRTI and NNRTI resistance on non-standard regimens
Tier 3: PI DRMs
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