Lipid and phase specificity of α-toxin from S. aureus.

The pore forming toxin Hla (α-toxin) from Staphylococcus aureus is an important pathogenic factor of the bacterium S. aureus and also a model system for the process of membrane-induced protein oligomerisation and pore formation. It has been shown that binding to lipid membranes at neutral or basic pH requires the presence of a phosphocholine-headgroup. Thus, sphingomyelin and phosphatidylcholine may serve as interaction partners in cellular membranes. Based on earlier studies it has been suggested that rafts of sphingomyelin are particularly efficient in toxin binding. In this study we compared the oligomerisation of Hla on liposomes of various lipid compositions in order to identify the preferred interaction partners and conditions. Hla seems to have an intrinsic preference for sphingomyelin compared to phosphatidylcholine due to a higher probability of oligomerisation of membrane bound monomer. We also can show that increasing the surface density of Hla-binding sites enhances the oligomerisation efficiency. Thus, preferential binding to lipid rafts can be expected in the cellular context. On the other hand, sphingomyelin in the liquid disordered phase is a more favourable binding partner for Hla than sphingomyelin in the liquid ordered phase, which makes the membrane outside of lipid rafts the more preferred region of interaction. Thus, the partitioning of Hla is expected to strongly depend on the exact composition of raft and non-raft domains in the membrane.

Control of kinetics by cooperative interactions.

Cooperative effects in ligand binding and dissociation kinetics are much less investigated than steady state kinetics or equilibrium binding. Nevertheless, cooperativity in ligand binding leads necessarily to characteristic properties with respect to kinetic properties of the system. In case of positive cooperativity as found in oxygen binding proteins, a typical property is an autocatalytic ligand dissociation behavior leading to a time dependent, apparent ligand dissociation rate. To follow systematically the influence of the various potentially involved parameters on this characteristic property, simulations based on the simple MWC model were performed which should be relevant for all types of models based on the concept of an allosteric unit. In cases where the initial conformational distribution is very much dominated by the R-state, the intrinsic kinetic properties of the T-state are of minor influence for the observed ligand dissociation rate. Even for fast conformational transition rates, the R-state properties together with the size of the allosteric unit and the allosteric equilibrium constant define the shape of the curve. In such a case, a simplified model of the MWC-scheme (the irreversible n-chain model) is a good approximation of the full scheme. However, if in the starting conformational distribution some liganded T-molecules are present (a few percent is enough), the average off-rates can be significantly altered. Thus, the assignment of the initial rates to R-state properties has to be done with great care. However, if the R-state strongly dominates initially it is even possible to get an estimation of the lower limit for the number of interacting subunits from kinetic data: similar to the Hill-coefficient for equilibrium conditions, a measure for "kinetic cooperativity" can be derived by comparing initial and final ligand dissociation rates.

Unusual oxygen binding behavior of a 24-meric crustacean hemocyanin.

Hemocyanins from Crustacea usually are found as 1x6 or 2x6-meric assemblies. An exception is the hemocyanin isolated from thalassinidean shrimps where the main component is a 24-meric structure. Our analysis of oxygen binding data of the thalassinidean shrimp Upogebia pusilla based on a three-state MWC-model revealed that despite the 24-meric structure the functional properties can be described very well based on the hexamer as allosteric unit. In contrast to the hemocyanins from other thalassinidean shrimps the oxygen affinity of hemocyanin from U. pusilla is increased upon addition of l-lactate. A particular feature of this hemocyanin seems to be that l-lactate already enhances oxygen affinity under resting conditions which possibly compensates the rather low intrinsic affinity observed in absence of l-lactate. The fast rate of oxygen dissociation might indicate that in this hemocyanin a higher cooperativity is less important than a fast response of saturation level to changes in oxygen concentration.

The molecular heterogeneity of hemocyanin: Structural and functional properties of the 4x6-meric protein of Upogebia pusilla (Crustacea).

The structural properties of the hemocyanin isolated from the Mediterranean mud shrimp, Upogebia pusilla (Decapoda: Thalassinidea), were investigated. Our intent was to make use of the U. pusilla case to perform a structural comparison between crustacean and chelicerate 4x6-meric hemocyanins. The thalassinidean hemocyanin appears similar in size but different in structural organization compared to the chelicerate 4x6-mer. Ultracentrifuge analyses on the purified protein revealed a sedimentation coefficient of 39S, typical of 4x6 hemocyanins. Electron micrographs are in agreement with a model in which four 2x6-meric building blocks are arranged in a tetrahedron-like quaternary structure and not in the quasi-square-planar orientation characteristic of the chelicerate protein. Size-exclusion chromatography-fast protein chromatography analysis showed elevated instability of the protein in absence of divalent ions or at pH values higher than 8.0. This analysis also shows that the dissociation of the U. pusilla 4x6-meric hemocyanin into hexamers occurs without any intermediate 2x6-meric state, in contrast with the dissociation profile of the chelicerate protein exhibiting several dissociation intermediates. The oxygen-binding properties of U. pusilla hemocyanin were studied to disclose possible effects by the typical allosteric effectors that modulate the functional properties of crustacean hemocyanin. A marked Bohr and lactate effect, but no significant influence of urate, on the oxygen affinity of U. pusilla hemocyanin were found.

Antiviral activity and cross-resistance profile of P-1946, a novel human immunodeficiency virus type 1 protease inhibitor.

The HIV protease inhibitor P-1946 is a member of a novel family of l-Lysine derivatives. The compound is a specific HIV-1 protease inhibitor that has potent and selective in vitro antiviral activity (EC50 152 nM) against a range of isolates resistant to commercially available protease inhibitors. The presence of at least four primary and four secondary drug resistance mutations is required to achieve greater than four-fold resistance to P-1946. P-1946's favorable resistance profile makes it a good lead for the development of new agents active against existing PI-resistant virus in treatment-experienced patient.

Structural properties, conformational stability and oxygen binding properties of Penaeus monodon hemocyanin.

Hemocyanin sequences allineament shows the presence of highly invariant regions especially in the active site and in the tight intersubunits interaction sites. Comparing the aminoacids in contact regions between monomers is possible to interpret the stability of hexamers.

Natural variation of drug susceptibility in wild-type human immunodeficiency virus type 1.

Wild-type viruses from the ViroLogic phenotype-genotype database were evaluated to determine the upper confidence limit of the drug susceptibility distributions, or "biological cutoffs," for the PhenoSense HIV phenotypic drug susceptibility assay. Definition of the natural variation in drug susceptibility in wild-type human immunodeficiency virus (HIV) type 1 isolates is necessary to determine the prevalence of innate drug resistance and to assess the capability of the PhenoSense assay to reliably measure subtle reductions in drug susceptibility. The biological cutoffs for each drug, defined by the 99th percentile of the fold change in the 50% inhibitory concentration distributions or the mean fold change plus 2 standard deviations, were lower than those previously reported for other phenotypic assays and lower than the clinically relevant cutoffs previously defined for the PhenoSense assay. The 99th percentile fold change values ranged from 1.2 (tenofovir) to 1.8 (zidovudine) for nucleoside reverse transcriptase RT inhibitors (RTIs), from 3.0 (efavirenz) to 6.2 (delavirdine) for nonnucleoside RTIs, and from 1.6 (lopinavir) to 3.6 (nelfinavir) for protease inhibitors. To evaluate the potential role of intrinsic assay variability in the observed variations in the drug susceptibilities of wild-type isolates, 10 reference viruses with different drug susceptibility patterns were tested 8 to 30 times each. The median coefficients of variation in fold change for the reference viruses ranged from 12 to 18% for all drugs except zidovudine (32%), strongly suggesting that the observed differences in wild-type virus susceptibility to the different drugs is related to intrinsic virus variability rather than assay variability. The low biological cutoffs and assay variability suggest that the PhenoSense HIV assay may assist in defining clinically relevant susceptibility cutoffs for resistance to antiretroviral drugs.

Updated European recommendations for the clinical use of HIV drug resistance testing.

In most European countries, HIV drug resistance testing has become a routine clinical tool. However, its practical implementation in a clinical context is demanding. The European HIV Drug Resistance Panel was established to make recommendations to clinicians and virologists on this topic and to propose quality control measures. The panel recommends resistance testing for the following indications: i) drug-naive patients with acute or recent infection; ii) therapy failure, including suboptimal treatment response, when treatment change is considered; iii) pregnant HIV-1-infected women and paediatric patients with detectable viral load when treatment initiation or change is considered; and iv) genotype source patient when post-exposure prophylaxis is considered. In addition, for drug-naive patients with chronic infection in whom treatment is to be started, the panel suggests that resistance testing should be strongly considered and recommends testing the earliest sample for drug resistance if suspicion of resistance is high or prevalence of resistance in this population exceeds 10%. The panel does not favour genotyping over phenotype, however it is anticipated that genotyping will be used more often because of its greater accessibility, lower cost and faster turnaround time. For the interpretation of resistance data, clinically validated systems should be used to the greatest extent possible. It is mandatory that laboratories performing HIV resistance tests take regular part in quality assurance programs. Similarly, it is necessary that HIV clinicians and virologists take part in continuous education and meet regularly to discuss problematic clinical cases. Indeed, resistance test results should be used in the context of all other clinically relevant information for predicting therapy response. The panel also encourages the timely collection of epidemiological information to estimate the impact of transmission of resistant HIV and the prevalence of HIV-1 non-B subtypes in the different European countries.

A potential role for water in the modulation of oxygen-binding by tarantula hemocyanin.

Hemocyanin from the tarantula Eurypelma californicum is a large respiratory protein with an exceptional high cooperativity. In contrast to hemocyanins from other species, no physiological allosteric effectors other than protons have been identified so far for this 24-meric oligomer. Here we report for the first time the mediating effects of water activity on the oxygen binding properties of a hemocyanin. Oxygen binding curves were measured in presence of several concentrations of glycine and sucrose since both substances reduce water activity. A pronounced shift of the p(50) was observed in both cases but in different directions: adding sucrose shifts the p(50) towards lower values whereas presence of glycine shows the same tendency as for human hemoglobin. Furthermore, prolonged incubation in sucrose slightly distorts the oxygen binding characteristics of spider hemocyanin. Therefore, only the influence of glycine was further analysed. An analysis based on the nested MWC model indicates, that presence of glycine leads to a preferential population of the two states with lower oxygen affinity (tR and tT) compared to the high affinity states rT and rR. The results corroborate the presence of hierarchically organized interactions in this hemocyanin.

Postexposure prophylaxis for human immunodeficiency virus infection after sexual or injection drug use exposure: identification and characterization of the source of exposure.

In a nonrandomized study of nonoccupational postexposure prophylaxis (PEP), a cross-sectional evaluation of subjects who were the source of human immunodeficiency (HIV) exposure was performed to characterize partners of index subjects seeking nonoccupational PEP against HIV. Among 401 index subjects, 64 (16%) recruited a source subject. Those in a steady relationship and those who knew that the source subject was HIV antibody positive were more likely to recruit their source subject. Source subjects reported high rates of past (78%) and current (69%) antiretroviral use; 46% of those using antiretroviral drugs had detectable plasma HIV-1 RNA levels. Antiretroviral resistance was detected in many source subjects who reported any use of antiretrovirals and was rare among source subjects who reported no history of antiretroviral use. Clinicians often make treatment decisions on the basis of incomplete knowledge of the source subject's HIV status or antiretroviral treatment history. The treatment history, particularly nonuse of a class of antiretroviral drugs, can be used to predict drug resistance.

Comparison of genotyping and phenotyping methods for determining susceptibility of HIV-1 to antiretroviral drugs.

OBJECTIVE(S): To compare antiretroviral resistance susceptibility testing of patient HIV-1 strains using genotype and phenotype methods. DESIGN: Eighteen plasma samples with viral load > 2000 HIV-1 RNA copies/ml were randomly selected for testing by both methods. Disease and treatment data were available for all patients. METHODS: Samples were analysed genotypically using a kit assay (HIV-1 Genotyping Systems, Applied Biosystems), performed by the Clinical Research Laboratory at Macfarlane Burnet Centre for Medical Research. Samples were analysed phenotypically using a rapid phenotypic assay (PhenoSenseTM HIV, ViroLogic), performed by the manufacturer. Results from both methods were interpreted using a defined protocol. Each susceptibility assay was performed and interpreted by individuals unaware of either the clinical data or the results of the other susceptibility assay. Concordance was defined categorically as either the presence of reduced susceptibility (> 2.5-fold change) in the phenotypic assay and resistance associated mutations in the genotypic assay, or the absence of these findings in both assays. RESULTS: Concordance between phenotypic and genotypic susceptibility testing was 81% for nucleoside reverse transcriptase inhibitors, 91% for non-nucleoside reverse transcriptase inhibitors and 90% for protease inhibitors. Complete concordance between phenotype and genotype for all 14 drugs evaluated was observed in three (17%) patient samples. CONCLUSIONS: Phenotypic and genotypic susceptibility appear to provide similar results. However, interpretation of genotypic results can be complicated, and both methods still require clinical validation.

Two types of urate binding sites on hemocyanin from the crayfish Astacus leptodactylus: an ITC study.

The oxygen binding behaviour of hemocyanins from Crustacea is regulated by small organic compounds such as urate and L-lactate. We investigated the binding characteristics of urate and the related compound caffeine to the 2 x 6-meric hemocyanin of A. leptodactylus under fully oxygenated conditions employing isothermal titration calorimetry (ITC). An analysis of urate and caffeine binding based on a model of n identical binding sites resulted in approximately four binding sites for caffeine and eight for urate. This result suggests that the binding process for these effectors is more complex than this most simple model. Therefore, we introduced a number of alternative models. Displacement experiments helped to select the appropriate model. Based on these experiments, at least two different types of binding sites for urate and caffeine exist on the 2 x 6-meric hemocyanin of A. leptodactylus. The two binding sites differ strongly in their specificity towards the two analogues. It can be hypothesized that two different subunit types (beta and gamma) are responsible for the two types of binding sites.

Phenotypic hypersusceptibility to non-nucleoside reverse transcriptase inhibitors in treatment-experienced HIV-infected patients: impact on virological response to efavirenz-based therapy.

BACKGROUND: Enhanced susceptibility to non-nucleoside reverse transcriptase inhibitors (NNRTI) was recently described in association with increased resistance to nucleoside analogs (nucleoside reverse transcriptase inhibitors; NRTI). OBJECTIVES: To determine the prevalence of NNRTI hypersusceptibility, the genotypic correlates, and its impact on virologic response to efavirenz-based salvage therapy. METHODS: Genotype and phenotype testing was performed retrospectively on baseline isolates from 30 patients who received salvage therapy containing efavirenz. NNRTI hypersusceptibility was defined as a 50% inhibitory concentration (IC(50)) of < 0.5 that of the wild-type control. RESULTS: Eight isolates had major NNRTI mutations. Among the 22 isolates with no major NNRTI mutations, 11 (50%) were hypersusceptible to efavirenz, 10 (45%) to delavirdine, and eight (36%) to nevirapine. Among eight isolates with NNRTI mutations, NNRTI resistance was present, but at lower than expected levels. The number of NRTI mutations was correlated inversely with the fold decrease in susceptibility to efavirenz (Spearman's rho, -0.57; P = 0.005), delavirdine (rho, -0.43; P = 0.04), and nevirapine (rho, -0.69; P < 0.001). Excluding subjects with NNRTI mutations, subjects with efavirenz hypersusceptibility at baseline had significantly better virologic suppression over 24 weeks than those without efavirenz hypersusceptibility (P < 0.001). CONCLUSION: NNRTI hypersusceptibility is common in heavily treated but NNRTI naive patients and is related directly to NRTI resistance mutations. Among patients receiving efavirenz-containing regimens, NNRTI hypersusceptibility was associated with an improved virologic outcome after 24 weeks of therapy. A reversal of phenotypic resistance was seen in patients with NNRTI mutations in the presence of multiple NRTI mutations, but no obvious virologic benefit of this phenomenon was seen in this study.

Laboratory guidelines for the practical use of HIV drug resistance tests in patient follow-up.

HIV drug resistance is one of the major limitations in the successful treatment of HIV-infected patients using currently available antiretroviral combination therapies. When appropriate, drug susceptibility profiles should be taken into consideration in the choice of a specific combination therapy. Guidelines recommending resistance testing in certain circumstances have been issued. Many clinicians have access to resistance testing and will increasingly use these results in their treatment decisions. In this document, we comment on the different methods available, and the relevant issues relating to the clinical application of these tests. Specifically, the following recommendations can be made: (i) genotypic and phenotypic HIV-1 drug resistance analyses can yield complementary information for the clinician. However, insufficient information currently exists as to which approach is preferable in any particular clinical setting; (ii) when HIV-1 drug resistance testing is required, it is recommended that testing be performed on plasma samples obtained before starting, stopping or changing therapy, on samples that have a viral load above the detection limit of the resistance test; (iii) the panel recommends that genotypic and phenotypic HIV-1 drug resistance testing for clinical purposes be performed in a certified laboratory under strict quality control and quality assurance standards; and (iv) the panel recommends that resistance testing laboratories provide clinicians with resistance reports that include a list of drug-related resistance mutations (genotype) and/or a list of drug-related fold resistance values (phenotype), with interpretations of each by an experienced virologist. The interpretation of genotypic and phenotypic analysis is a complex and developing science, and in order to understand HIV-1 drug resistance reports, communication between the requesting clinician and the expert that interpreted the resistance report is recommended.

Cerebrospinal fluid response to structured treatment interruption after virological failure.

OBJECTIVE: Structured antiretroviral treatment interruption (STI) has been advocated as a therapeutic strategy for HIV-1 infection. We report initial observations of cerebrospinal fluid (CSF) HIV-1 infection in five patients undergoing serial lumbar punctures (LPs) during STI undertaken following virological failure. DESIGN AND METHODS: In this prospective observational study we quantified HIV-1 RNA concentrations and assessed both phenotypic drug susceptibility profiles and genotypic antiviral drug resistance mutations in CSF and plasma during the period of treatment interruption. CSF white blood cells were also counted, and patients' neurological status monitored. RESULTS: In four of the patients, CSF HIV-1 concentration increased more rapidly than that of the plasma, with consequent reduction in the ratio between plasma and CSF viral loads (pVL : cVL). Three individuals developed robust, though asymptomatic CSF lymphocytic pleocytosis. In all patients the predominant HIV-1 quasispecies shifted simultaneously in CSF and plasma from a drug-resistant to a more drug-susceptible phenotype with identical and simultaneous changes in genotypes associated with drug resistance. CONCLUSIONS: STI may be accompanied by previously unrecognized changes in tissue viral exposures and lymphocyte traffic. Hence, despite 'virological failure' as evidenced by persistent plasma viremia, ongoing antiretroviral treatment prior to its interruption appeared to suppress CSF HIV-1 infection (indeed more effectively than that of plasma) and restrain lymphocyte traffic into the CSF. Simultaneous change of resistance mutations in CSF and plasma was likely due to re-emergence and overgrowth of pre-existing strains with ready exchange of virus between these two compartments, either facilitated by or provoking a local CSF lymphocytosis.

Sequencing of protease inhibitor therapy: insights from an analysis of HIV phenotypic resistance in patients failing protease inhibitors.

OBJECTIVE: To characterize the pattern of HIV-1 susceptibility to protease inhibitors in patients failing an initial protease inhibitor-containing regimen. DESIGN: A cross-sectional analysis of antiretroviral susceptibility. SETTING: HIV clinics in six metropolitan areas. PATIENTS: Eighty-eight HIV-infected adults with HIV RNA > 400 copies/ml after > or = 6 months of antiretroviral therapy, including the use of one protease inhibitor for > or = 3 months. MEASUREMENTS: The frequency and magnitude of decreased susceptibility, measured with a phenotypic assay using recombinant constructs, to five protease inhibitors. Decreased susceptibility was defined as > 2.5-fold increase in the 50% inhibitory concentration (IC50) compared with drug sensitive control virus. RESULTS: At study entry, patients were being treated with nelfinavir (63%), indinavir (25%), or another protease inhibitor (11%). HIV isolates from these patients were susceptible (fold change < 2.5) to all five protease inhibitors in 18% of patients and to none in 8%. Isolates from patients receiving nelfinavir were less likely to have reduced susceptibility to other protease inhibitors than isolates from patients treated with indinavir (P < 0.001) or one of the other three agents (P < 0.001), even after adjustment for the duration of prior protease inhibitor use. Reduced susceptibility to saquinavir and amprenavir was observed significantly less frequently than for the other protease inhibitors. CONCLUSION: The frequency of protease inhibitor cross-resistance and the magnitude of changes in susceptibility varied according to the initial protease inhibitor used in the failing treatment regimen. Significantly less protease inhibitor cross-resistance was demonstrated for isolates from patients failing a nelfinavir-containing regimen compared with those from patients receiving other protease inhibitors.

The allosteric effector l-lactate induces a conformational change of 2x6-meric lobster hemocyanin in the oxy state as revealed by small angle x-ray scattering.

Hemocyanins are multisubunit respiratory proteins found in many invertebrates. They bind oxygen highly cooperatively. However, not much is known about the structural basis of this behavior. We studied the influence of the physiological allosteric effector l-lactate on the oxygenated quaternary structure of the 2x6-meric hemocyanin from the lobster Homarus americanus employing small angle x-ray scattering (SAXS). The presence of 20 mm l-lactate resulted in different scattering curves compared with those obtained in the absence of l-lactate. The distance distribution functions p(r) indicated a more compact molecule in presence of l-lactate, which is also reflected in a reduction of the radius of gyration by about 0.2 nm (3%). Thus, we show for the first time on a structural basis that a hemocyanin in the oxy state can adopt two different conformations. This is as predicted from the analysis of oxygen binding curves according to the "nesting" model. A comparison of the distance distribution functions p(r) obtained from SAXS with those deduced from electron microscopy revealed large differences. The distance between the two hexamers as deduced from electron microscopy has to be shortened by up to 1.1 nm to agree well with the small angle x-ray curves.

Binding of urate and caffeine to haemocyanin analysed by isothermal titration calorimetry.

Haemocyanin serves as an oxygen carrier in the haemolymph of decapod crustaceans. The oxygen-binding behaviour of the pigment is modulated by the two major anaerobic metabolites, l-lactate and urate. The binding of these two metabolites to haemocyanin has been investigated mainly indirectly by following the effector-induced changes in the oxygen-binding properties of the respiratory pigment. Only a few direct investigations of effector binding, employing ultracentrifugation techniques and equilibrium dialysis, have been carried out. No evidence for cooperative binding for either effector was detected using these methods. However, isothermal titration calorimetry (ITC) offers a useful tool to gain additional insight into the binding of effectors to these highly allosterically regulated macromolecules. By applying the ITC method to the fully oxygenated dodecameric haemocyanin of the lobster Homarus vulgaris, cooperativity in binding has been found for the urate analogue caffeine but not for urate itself: using urate and the urate analogue caffeine as ligands, two conformations of the oxygenated pigment were detected.

Virologic and immunologic consequences of discontinuing combination antiretroviral-drug therapy in HIV-infected patients with detectable viremia.

BACKGROUND: In many patients with human immunodeficiency virus (HIV) infection, therapy with potent antiretroviral drugs does not result in complete suppression of HIV replication. The effect of cessation of therapy in these patients is unknown. METHODS: Sixteen patients who had a plasma HIV RNA level of more than 2500 copies per milliliter during combination antiretroviral-drug therapy were randomly assigned, in a 2:1 ratio, to discontinue or continue therapy. Plasma HIV RNA levels, CD4 cell counts, and drug susceptibility were measured weekly. Viral replicative capacity was measured at base line and at week 12. RESULTS: Discontinuation of therapy for 12 weeks was associated with a median decrease in the CD4 cell count of 128 cells per cubic millimeter and an increase in the plasma HIV RNA level of 0.84 log copies per milliliter. Virus from all patients with detectable resistance at entry became susceptible to HIV-protease inhibitors within 16 weeks after the discontinuation of therapy. Drug susceptibility began to increase a median of six weeks after the discontinuation of therapy and was temporally associated with increases in plasma HIV RNA levels and decreases in CD4 cell counts. Viral replicative capacity, measured by means of a recombinant-virus assay, was low at entry into the study and increased after therapy was discontinued. Despite the loss of detectable resistance in plasma, resistant virus was cultured from peripheral-blood mononuclear cells in five of nine patients who could be evaluated. Plasma HIV RNA levels, CD4 cell counts, and drug susceptibility remained stable in the patients who continued therapy. CONCLUSIONS: Despite the presence of reduced drug susceptibility, antiretroviral-drug therapy can provide immunologic and virologic benefit. This benefit reflects continued antiviral-drug activity and the maintenance of a viral population with a reduced replicative capacity.

Phenotypic drug susceptibility testing predicts long-term virologic suppression better than treatment history in patients with human immunodeficiency virus infection.

To assess the value of phenotypic drug susceptibility testing as a predictor of antiretroviral treatment response in human immunodeficiency virus (HIV)-infected people, drug susceptibility testing was performed retrospectively on plasma samples collected at baseline in a cohort of 86 antiretroviral-experienced, HIV-infected people experiencing treatment failure and initiating a new antiretroviral treatment regimen. Two separate criteria for reduced drug susceptibility were evaluated. In multivariate analyses, phenotypic susceptibility was an independent predictor of time to treatment failure (adjusted hazards ratio [HR], 0.70; 95% confidence interval [CI], 0.55-0.90; and adjusted HR, 0.76; 95% CI, 0.61-0.95, with reduced drug susceptibility cutoffs defined as 4.0-fold and 2.5-fold higher than reference virus IC(50) values, respectively). Previous protease inhibitor experience was also a significant independent predictor. Notably, drug susceptibility predicted on the basis of treatment history alone was not predictive of time to treatment failure. In this cohort, phenotypic testing results enhanced the ability to predict sustained long-term suppression of virus load.