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Resistance testing methodologies and mechanisms of resistance.



This review summarises recent data on resistance testing methodologies and mechanisms of antiretroviral resistance, focusing on emerging data and including preliminary observations that, while requiring confirmation, point to new exciting developments in the field.

RESISTANCE TESTING TECHNOLOGIES

Antiretroviral drug resistance testing is a valuable tool in the management of HIV-infected persons. However, there are limitations to current routine resistance assays. In particular it has become increasingly evident that standard tests cannot reliably detect resistant variants present within the viral quasispecies at prevalence below 20-25%. Limited data from persons receiving NNRTI-based regiments indicate that low-frequency resistant mutants can affect responses to antiretroviral treatment. It can be proposed, however, that the impact of low-frequency mutants depends on multiple factors, including the frequency within the quasispecies, the effects of the mutation on drug susceptibility and virus fitness, the specific drugs and drug-classes affected within a given regimen, and the patient's level of adherence.

Several new technologies allow the detection of low-frequency mutants with high sensitivity (Table 1).

It is important to keep in mind that the high degree of variability of the HIV HIV (Human Immunodeficiency Virus), either of two closely related retroviruses that invade T-helper lymphocytes and are responsible for AIDS. There are two types of HIV: HIV-1 and HIV-2. HIV-1 is responsible for the vast majority of AIDS in the United States.  genome dictates that point mutations conferring drug resistance emerge spontaneously during virus replication, leading to a natural background of drug resistant mutants. Thus, ultrasensitive resistance assays must be able to reliably and reproducibly detect resistance that matters, while avoiding the detection of the natural background. The current consensus is that the optimal sensitivity is around 0.1-1%, a threshold above which resistant mutants may negatively affect treatment responses, at least in the context of NNRTI-based therapy.

As an example of the limitations of routine genotypic testing, Table 2 illustrates the results of clonal sequencing of plasma viral RNA RNA: see nucleic acid.
RNA
 in full ribonucleic acid

One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic
 from a patient who underwent the unstructured discontinuation of efavirenz-based therapy after achieving sustained virological virological

pertaining to viruses.
 suppression <50 copies/ml. At the time of virological rebound, while population sequences obtained by routine genotypic resistance testing showed a wild-type virus, analysis of 200 clones derived from five separate PCR PCR polymerase chain reaction.

PCR
abbr.
polymerase chain reaction


Polymerase chain reaction (PCR) 
 reactions showed NNRTI- as well as NRTI-resistant mutants at frequencies between 0.5% and 2%.

It is currently debated how easily the more sensitive assays can be introduced into routine diagnostic practice. Available methodologies are either extremely labour intensive (e.g. clonal analysis/single genome sequencing), complex to set up for the variety of possible resistance mutations and subtypes (e.g. allele-specific PCR), or very expensive (e.g. ultra-deep sequencing).

Among some exciting new methodologies being developed, pyrosequencing combined with DNA barcoding is an elegant approach, likely to have broad applications in the future [1]. This method combines DNA barcoding and massively parallel pyrosequencing to achieve full sequencing of the genomic regions of interest and a sensitivity of detection of 5%. Multiple samples (seven or more) are tested grouped together in a single reaction and later identified through unique sequence tags. This approach allows multiplex analysis of many barcoded samples in a single sequencing experiment, thereby offering the potential to drive down the cost of each genotype determination.

RESISTANCE TO SMCCR5 INHIBITORS

Recent studies have identified two different mechanisms of resistance to small molecule CCR 1. CCR - condition code register.
2. CCR - (Database) concurrency control and recovery.
5 (smCCR5) inhibitors such as maraviroc.

In the first mechanism, changes in the virus envelope allow the virus to bind to to contract; as, to bind one's self to a wife s>.

See also: Bind
 the co-receptor despite the alteration in shape caused by the presence of the inhibitor [2]. The phenotypic effect is shown by a plateau in the maximal percentage inhibition (MPI MPI - Message Passing Interface ) with increasing drug concentrations. As a result of mutations in the stems of the V3 loop, resistant virus binds with higher affinity to the drug-bound co-receptor than wild-type virus. Mutations at codons 13 and 26 appear to be especially common in resistant strains, but changes outside the V3 loop may also be important. To date the mutation pattern remains to be clearly defined, making the genotypic identification of resistance problematic.

A second mechanism of resistance is characterised by a switch in viral tropism from R5 to X4. In most cases the switch is thought to occur within the viral population rather than at the individual virus level. One study examined 10 patients who experienced virological failure in clinical trials of maraviroc [3]. The patients had R5 virus at study entry and X4 or dual tropic/mixed (DM) virus at failure, as determined by Monogram Bioscience's Trofile assay. The assay has a reported sensitivity of 10% for the detection of X4 virus. Using clonal analyses of the envelope gene, the investigators demonstrated that in seven patients the X4/DM virus detected at failure was phylogenetically phy·lo·ge·net·ic  
adj.
1. Of or relating to phylogeny or phylogenetics.

2. Relating to or based on evolutionary development or history: a phylogenetic classification of species.
 related to X4/DM virus present at low frequency (up to 7%) in the baseline sample (Figure 1). These data provide support to the concept that tropism tropism (trōp`ĭzəm), involuntary response of an organism, or part of an organism, involving orientation toward (positive tropism) or away from (negative tropism) one or more external stimuli.  shift occurs at the virus population level, when selective suppression of R5 virus allows outgrowth of the X4/dual tropic population.

[FIGURE 1 OMITTED]

One issue of significant current interest is related to the ideal sensitivity of tropism assays for the detection of X4 virus. Reflecting these concerns, the Trofile assay has been recently modified to achieve increased sensitivity. However, in analogy with the issues discussed in the context of low-frequency drug-resistance species, the threshold prevalence of X4 or dual tropic virus that impacts on responses to smCCR5 inhibitors remains to be clearly established. Virus strains using the CXCR CXCR Chemokine, CXC Motif, Receptor
CXCR Alpha Chemokine Receptor
4 co-receptor are likely to be present in most patients with established HIV infection even if at low or very low frequency and an assay with too great a sensitivity may, paradoxically, lead to unnecessary avoidance of smCCR5 inhibitors. The threshold above which responses are affected is likely to depend on multiple determinants, including the activity of other drugs in the regimen. Although data are available to demonstrate the pre-existence of low-frequency X4/DM virus in patients subsequently failing maraviroc-based therapy, there are no published data on the prevalence of such low-frequency variants among patients who achieved virological suppression. While in many circumstances it may be sufficient to use a tropism test with good rather than 'perfect' sensitivity to determine whether the dominant quasi-species is R5, it will always remain an important principle of antiretroviral therapy to ensure that the smCCR5 inhibitor is well supported by other active drugs in the regimen, and that prolonged virological failure is avoided.

Reassuringly, limited data indicate that after smCCR5 discontinuation, resistant virus reverts to minority species and in cases of tropism switch, R5 virus again becomes the dominant quasispecies. These observations suggest that the viral population selected during therapy appears to have no inherent advantage over the pre-treatment population in the absence of drug pressure. The long-term impact of the possible enrichment of resistant variants or X4 and dual tropic virus remains unknown at present.

PATHWAYS OF TENOFOVIR RESISTANCE

The K65R mutation in reverse transcriptase confers resistance to tenofovir and intermediate levels of cross-resistance to abacavir, didanosine didanosine /di·dan·o·sine/ (-dan´o-sen) 2, an analogue of dideoxyadenosine; an antiretroviral agent used for the treatment of advanced HIV-1 infection and acquired immunodeficiency syndrome, administered orally. , and lamivudine, and also has resistance effects for stavudine (not evident in phenotypic testing), while conferring hypersusceptibility to zidovudine zidovudine /zi·do·vu·dine/ (zi-do´vu-den) a synthetic nucleoside (thymidine) analogue that inhibits replication of some retroviruses, including the human immunodeficiency virus; used in the treatment of HIV infection and AIDS. . There has been some recent evidence showing a greater propensity for HIV-1 subtype C to develop the K65R mutation under in vitro selection with tenofovir compared with subtype B or other non-B subtypes [4]. Whether this is a true subtype effect remains to be clearly shown. A single nucleotide change is required for a K [right arrow] R substitution in both subtype B (AAA to AGA) and subtype C (AAG to AGG AGG Aggregate
AGG Allgemeines Gleichbehandlungsgesetz
AGG African Gold Group, Inc.
AGG Arnall Golden Gregory LLP (Atlanta, GA)
AGG Aggravated
AGG Asociación de Gerentes de Guatemala
).

A mechanistic explanation has been recently proposed, and is related to the way the sequence of the virus affects the points at which the reverse transcriptase stops or pauses before continuing its activity [5,6]. When HIV replicates, the viral RNA template is used by the reverse transcriptase to produce a DNA DNA: see nucleic acid.
DNA
 or deoxyribonucleic acid

One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes.
 copy. This process is characterised by disruptions or dissociations of the enzyme and there are indications that the places at which these occur are the points at which mutations can also occur. It has been proposed that the sequence of the template influences the likelihood of the reverse transcriptase stopping, pausing or dissociating from the template. In particular, it has been suggested that subtype C sequences favour pausing at or near position 65, which will then favour the evolution of K65R. This is interesting because it reveals how the sequence variability associated with subtypes may influence the development of resistance. The clinical significance of these findings, however, remains to be determined. Currently available, albeit limited, clinical data from tenofovir-treated patients do not suggest a higher rate of K65R development among patients with subtype C [7]. Prospective studies of patients infected with different HIV-1 subtypes are required to determine whether they show a different propensity for the development of K65R.

A less common pathway of tenofovir resistance is characterised by the emergence of the K70E mutation, which shows resistance effects overall similar to those observed with K65R. It had been previously proposed that the K70E mutation may appear transiently prior to the emergence of K65R. Recent data, however, indicate that under continuous virological pressure with tenofovir the K70E mutation evolves into the rarer mutation K70G [8]. The change from K70E to K70G is a two-step process in which the wild-type lysine lysine (lī`sēn), organic compound, one of the 20 amino acids commonly found in animal proteins. Only the l-stereoisomer appears in mammalian protein.  at position 70 changes to glutamic acid and then to glycine glycine (glī`sēn), organic compound, one of the 20 amino acids commonly found in animal proteins. Glycine is the only one of these amino acids that is not optically active, i.e.  (K[AAA] [right arrow E[GAA] [right arrow] G[GGA GGA Generalized Gradient Approximation
GGA Good Game All
ggA Geschützte Geographische Angabe (German: Protected Geographical Indication)
GGA Global Gecko Association
GGA Georgia Geocachers Association
]). This unusual resistance pattern emerged in an HIV-infected individual receiving dual antiretroviral therapy with tenofovir/emtricitabine due to poor compliance with triple therapy. Ongoing virus replication led to the initial emergence of K70E with the emtricitabine resistance mutation M184V, followed by the loss of K70E and emergence of K70G. Clonal analysis showed that K70G and M184V co-existed on the same viral genome. The phenotypic effects of the K70G mutation in isolation or with M184V had not been previously described. Using the Antivirogram assay (Virco, Belgium) phenotypic resistance to lamivudine, emtricitabine, abacavir, didanosine and tenofovir was observed, with preserved susceptibility to zidovudine and stavudine (Table 3).

RESISTANCE TO ETRAVIRINE (TMC TMC Technology Marketing Corporation (Norwalk, Connecticut)
TMC Texas Medical Center (Houston, TX)
TMC Traffic Message Channel
TMC The Movie Channel
TMC Traffic Management Center
125)

The DUET 1 and 2 studies randomised Adj. 1. randomised - set up or distributed in a deliberately random way
randomized

irregular - contrary to rule or accepted order or general practice; "irregular hiring practices"
 treatment-experienced patients (n = 600 per trial) with viral load >5000 copies/ml while on stable antiretroviral therapy for at least 8 weeks to receive TMC125 or placebo, each combined with ritonavir-boosted darunavir (DRV/r) and an optimised NRTI Noun 1. NRTI - an antiviral drug used against HIV; is incorporated into the DNA of the virus and stops the building process; results in incomplete DNA that cannot create a new virus; often used in combination with other drugs  backbone, with optional use of enfuvirtide (T20). Patients had [greater than or equal to] 1 NNRTI NNRTI Non-nucleoside reverse transcriptase inhibitor, see there  resistance mutation (either at screening or in documented historical genotype) and [greater than or equal to] 3 primary protease mutations. Pooled data from the two studies were analysed to identify NNRTI mutations significantly associated with reduced responses to the TMC125 arm [9].

The genotypic score included both mutations with well-known effects on NNRTI susceptibility such as Y181C and others with unknown effects such as V90I. Overall, 13 mutations showed a highly statistically significant correlation with responses in the multivariate analysis, after adjusting for baseline parameters including phenotypic susceptibility to DRV DRV Driver
DRV Drive
DRV Device Driver (file name extension)
DRV Democratic Republic of Vietnam
DRV Darunavir (pharmaceutical drug-HIV treatment)
DRV Daily Reference Value
DRV Deutscher Raiffeisen Verband
 (Table 4).

In general, as the number of mutations increased, the response to TMC125 was reduced, with the greatest loss of responses seen in the presence of three or more mutations. Given the prominent role of mutations at codon codon: see nucleic acid.  181 in conferring resistance to TMC125, it can be concluded that a history of failure on nevirapine nevirapine /ne·vir·a·pine/ (ne-vir´ah-pen) a nonnucleoside inhibitor of HIV-1reverse transcriptase, used in combination with other antiretroviral agents in the treatment of HIV infection. , particularly if protracted, may be considered predictive of a reduced response to TMC125.

These data are extremely interesting as they indicate that NNRTI sequencing may now be proposed as a valid treatment strategy. To test the hypothesis fully and evaluate the impact of low-frequency NNRTI resistant mutants, clinical data are eagerly awaited on the use of second generation NNRTIs (TMC125 or TMC278) in combination with NRTIs (without ritonavir-boosted PIs or other drug classes) in patients who failed standard first-line efavirenz efavirenz /ef·a·vi·renz/ (ef´ah-vi?renz) an antiretroviral, inhibiting reverse transcriptase; used in the treatment of HIV infection.

e·fa·vir·enz
n.
 or nevirapine therapy.

PATHWAYS OF LOPINAVIR RESISTANCE

Studies have reported that the L76V mutation in protease is selected by lopinavir (LPV LPV lymphotropic papovavirus. ) use in first-line therapy. The MONARK study randomised treatment-naive patients to receive lopinavir/ritonavir (LPV/r) either alone or with a backbone of zidovudine/lamivudine. Low-level viraemia Noun 1. viraemia - the presence of a virus in the blood stream; "viremia spread the smallpox virus to the internal organs"
viremia

pathology - any deviation from a healthy or normal condition
 (>50 but < 400 copies/ml) occurred more commonly in the monotherapy arm than the triple therapy arm over 48 weeks. During the study period, 32 (38.5%) and 7 (13.2%) patients, respectively, underwent genotypic resistance testing, which was initiated at the clinician's discretion when viral load was >500 copies/ml [10]. Overall five patients in the monotherapy arm and none in the triple therapy arm developed major PI resistance mutations. The resistance analysis in the five patients is summarised in Table 5. Three of the five patients showed the emergence of L76V, either alone or with M46I.

The phenotypic effects of the L76V + M46I mutation pattern on PI susceptibility have also been investigated by site-directed mutagenesis [11]. Overall intermediate levels of resistance to LPV and amprenavir were observed (Table 6).

While its phenotypic effect appears to be modest, the L76V mutation is part of the darunavir score and may therefore reduce the genetic barrier to the drug. Although the mutation has been observed in patients infected with subtype CRFAG, it remains to be demonstrated whether there exists a subtype-related effect on its emergence.

REFERENCES

[1.] Hoffmann C, Minkah N, Leipzig J et al. DNA bar coding and pyro-sequencing to identify rare HIV drug resistance HIV drug resistance Antiretroviral drug resistance AIDS The resistance of a strain of HIV to an agent–eg, a reverse transcriptase inhibitor, which occurs in 5%-20% of those newly infected with HIV  mutations. Nucleic Acids Res, 2007, 35, 91.

[2.] Mori J, Mosley M, Lewis M et al. Characterization of maraviroc resistance in patients failing treatment with CCR5-tropic virus in MOTIVATE 1 and MOTIVATE 2. XVI International HIV Drug Resistance Workshop, June 12-16, 2007, Barbados. Abstract 10.

[3.] Lewis M, Simpson P, Fransen S et al. CXCR4-using virus detected in patients receiving maraviroc in the phase III studies MOTIVATE 1 and 2 originates from a pre-existing minority of CXCR4-using virus. XVI International HIV Drug Resistance Workshop, 12-16 June, 2007, Barbados. Abstract 56.

[4.] Brenner BG, Oliveira M, Doualla-Bell F et al. HIV-1 subtype C viruses rapidly develop K65R resistance to tenofovir in cell culture. AIDS, 2006, 20, F9-F13.

[5.] Harrigan PR, Chihwei Sheen T, Wynhoven B et al. Silent mutations at RT codons 65 and 66 are very strongly associated with treatment and drug resistance and are implicated in reverse transcription efficiency. XVI International HIV Drug Resistance Workshop, 12-16 June, 2007, Barbados. Abstract 113.

[6.] Coutsinos D, Invernizzi CF, Xu H et al. In vitro molecular characterization of the development of the K65R and M184V mutations in subtype C HIV-1. XVI International HIV Drug Resistance Workshop, 12-16 June, 2007, Barbados. Abstract 114.

[7.] Miller MD, Margot N, McColl D, Cheng AK. K65R development among subtype C HIV-1-infected patients in tenofovir DF clinical trials. AIDS, 2007, 21, 265-266.

[8.] Bradshaw D, Malik S, Booth C et al. Novel drug resistance pattern associated with the mutations K70G and M184V in Human Immunodeficiency Virus human immunodeficiency virus
n.
HIV.


Human immunodeficiency virus (HIV)
A transmissible retrovirus that causes AIDS in humans.
 type 1 reverse transcriptase. Antimicrob Agents Chemother, 2007, 51, 4489-4491.

[9.] Vingerhoets J, Buelens A, Peeters M et al. Impact of baseline NNRTI mutations on the virological response to TMC125 in the phase III clinical trials DUET-1 and DUET-2. XVI International HIV Drug Resistance Workshop, 12-16 June, 2007, Barbados. Abstract 32.

[10.] Delaugerre C, Flandre P, Chaix ML et al. Protease gene mutations in a trial comparing first-line lopinavir/ritonavir monotherapy to lopinavir/ritonavir + zidovudine/lamivudine (MONARK Trial). XVI International HIV Drug Resistance Workshop, 12-16 June, 2007, Barbados. Abstract 75.

[11.] Nijhuis M, Wensing AMJ AMJ Academy of Management Journal
AMJ American Muslims for Jerusalem
AMJ Advisory Material Joint
AMJ Ahmadiyya Muslim Jamaat
AMJ Ahmadiyya Muslim Jama'at
, Bierman W et al. A novel genetic pathway involving L76V and M46I leading to lopinavir/ritonavir. XVI International HIV Drug Resistance Workshop; 12-16 June, 2007, Barbados. Abstract 127.

Correspondence to:

Dr Anna Maria Geretti, MD, PhD, FRCPath, Department of Virology virology, study of viruses and their role in disease. Many viruses, such as animal RNA viruses and viruses that infect bacteria, or bacteriophages, have become useful laboratory tools in genetic studies and in work on the cellular metabolic control of gene expression , Royal Free Hospital and Royal Free & University College Medical School, Pond Street, London NE3 2QG, UK. Email: a.geretti@medsch.ucl.ac.uk
Table 1: Ultrasensitive methods for the detection of
drug-resistant mutants.

Allele-specific real-time PCR

Parallel allele-specific sequencing (PASS)

Pyrosequencing

Ultra-deep sequencing

Clonal analysis

Single genome sequencing

Table 2: Clonal analysis (n=200 clones) of plasma
HIV RNA (reverse transcriptase codons 1-250) from
a treatment-experienced patient lacking resistance
by routine genotyping.

Mutation detected    No of clones (%)    Drugs affected

A62V                      1 (0.5)        Possibly NRTIs
V75A                      1 (0.5)        Stavudine
K103N                     4 (2.0)        Efavirenz, nevirapine
V106A                     1 (0.5)        Efavirenz, nevirapine
Y115C                     2 (1.0)        Unknown
Q151R                     1 (0.5)        Unknown
V179G                     1 (0.5)        Possibly NNRTIs
M184T                     1 (0.5)        Lamivudine
Y188D                     1 (0.5)        NNRTIs

Table 3: Phenotypic resistance (fold-changes in [IC.sub.50])
of HIV-1 clinical isolates with the reverse transcriptase
mutations K70E+M184V, K70G+M184V or K70G alone.

(Adapted from reference 8.)

Drug            K70E+M184V   K70G+M184V   K70    BCO (a)
                                           G

Lamivudine        >29.3        >84.5      4.0      2.4
Emtricitabine     >40.8        >63.4      NA       3.5
Zidovudine         <0.3         <0.8      1.0      2.7
Stavudine           0.4          0.9      1.3      2.3
Didanosine          1.6          5.3      2.1      2.2
Abacavir            1.3          3.2      2.1      2.2
Tenofovir           1.7          1.5      1.9      2.1

Drug                   VircoType
                        CCO (b)

                  Lower        Upper

Lamivudine         1.0          3.4
Emtricitabine      NA           NA
Zidovudine         1.2          9.6
Stavudine          0.9          2.0
Didanosine         0.9          2.6
Abacavir           0.8          1.9
Tenofovir          0.9          2.1

(a) Antivirogram biological cut-off; (b) VircoTYPE HIV-1
lower and upper clinical cut-off; NA, not available.

Table 4: Mutations associated with reduced responses to
etravirine in the DUET 1 and 2 studies.

V90I    A98G      L100I        K101E/P
V106I   V179D/F   Y181C/I/V    G190A/S

Table 5: Resistance analysis of monotherapy arm
of the MONARK trial. (Adapted from reference 10.)

                               Viral
                               load
                               ([log.
                    Study      sub.10]
Patient   Subtype   visit      copies/ml)

1         B         Baseline   4.8
                    week 40    2.9

2         CRFAG     Baseline   4.8
                    week 44    2.8

3         CRFAG     Baseline   5.0
                    week 62    2.6

4         B         Baseline   4.4
                    week 76    3.1

5         CRFAG     Baseline   4.6
                    week 90    2.5

                                            Lopinavir
                                            fold
Patient   Protease genotype (a)             change

1         L63P/S V771                       1.2
          M46I L63P V77I                    1.4

2         I13V K20I M36I H69K               0.6
          I13V K20I M36I H69K L76V          1.3

3         I13V K20I M36I H69K               0.6
          I13V K20I M36I M46I H69K L76V     2.7

4         L10I/L L63P A71A/T                1.5
          L10F V82A/V L63P                  1.1

5         I13V K20I M36I H69K L89M          0.9
          I13V K20I M36I H69K L76V L89M     NA

(a) Emerging mutations in bold; NA, not available.

Table 6: Phenotypic susceptibility to protease
inhibitors of M46I+L76V site-directed mutants.

Fold-change
Mutations      Lopinavir    Atazanavir   Saquinavir

M46I + L76V        12           0            0

Fold-change
Mutations      Tipranavir   Amprenavir   Darunavir

M46I + L76V        1            5            1
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Title Annotation:PRESENTATION
Author:Geretti, Anna Maria
Publication:Journal of HIV Therapy
Geographic Code:4EUUK
Date:Dec 1, 2007
Words:3057
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