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Antimicrobial Agents and Chemotherapy, February 2004, p. 437-443, Vol. 48, No. 2
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.2.437-443.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Natural Variation of Drug Susceptibility in Wild-Type Human Immunodeficiency Virus Type 1

N. T. Parkin,^* N. S. Hellmann, J. M. Whitcomb, L. Kiss, C. Chappey, and C. J. Petropoulos

ViroLogic, Inc., South San Francisco, California 94080

Received 27 February 2003/ Returned for modification 11 June 2003/ Accepted 9 October 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The use of antiretroviral drug therapy has greatly improved the long-term clinical outcome for human immunodeficiency virus (HIV) type 1 (HIV-1)-infected individuals (18). However, loss of suppression of viral replication is often accompanied by the emergence of drug-resistant HIV-1 in treated patients (21). This complicates the selection of drugs for second- or third-line regimens, as cross-resistance within drug classes is a common phenomenon (11, 24). The use of phenotypic and genotypic assays for the detection and quantification of drug resistance is considered the standard of care when designing drug regimens following treatment failure (13a, 23). Retrospective and prospective studies have demonstrated the benefit of using such assays to guide the selection of treatment regimens that include a greater number of drugs to which the virus is susceptible [1, 5, 7-9, 15, 22; R. Haubrich, P. Keiser, C. Kemper, M. Witt, J. Leedom, D. Forthal, M. Leibowitz, J. Hwang, E. Seefried, J. A. McCutchan, N. Hellmann, D. Richman, and CCTG, Antivir. Ther. 6(Suppl. 1):63, 2001, abstr.].

Phenotypic drug susceptibility is measured as the concentration of drug required to inhibit virus replication by 50% (IC₅₀) and is commonly expressed as the fold change (FC) in the IC₅₀ between that for the patient virus and that for a well-characterized drug-sensitive reference virus (FC = IC₅₀ for patient virus/IC₅₀ for reference virus). The interpretation of phenotypic susceptibility assay results is enhanced by relevant thresholds, or cutoffs, that are intended to define the point above which the utility of a given drug begins to decline. Clinical cutoffs based on virologic response data from clinical trials provide the most clinically relevant threshold but are also the most difficult to define. To date, clinical cutoffs for the PhenoSense HIV assay have been defined for seven drugs [16; Haubrich et al., Antivir. Ther. 6(Suppl. 1):63, 2001, abstr.; E. R. Lanier, N. Hellmann, and J. Scott, Abstr. 8th Conf. Retrovir. Opportunist. Infect., abstr., 254, 2001; M. D. Miller, L. Zhong, S. Chen, N. A. Margot, and M. Wulfsohn, Antivir. Ther. 7(Suppl. 1):S12, 2000, abstr.; G. Skowron, J. Whitcomb, M. Wesley, C. Petropoulos, N. Hellmann, M. Holodniy, J. Kolberg, J. Detmer, M. T. Wrin, and K. Frost, Antivir. Ther. 4(Suppl. 1):55-56, 1999, abstr.; J. Szumiloski, H. Wilson, E. Jensen, R. Campo, N. Miller, H. Rice, A. Zolopa, D. Klein, M. Horberg, M. Coram, N. Hellmann, M. Bates, and J. H. Condra, Antivir. Ther. 7(Suppl. 1):S127, 2002, abstr.]: abacavir (4.5-fold), tenofovir (1.4-fold), stavudine (1.7-fold), didanosine (1.7-fold), lamivudine (3.5-fold), ritonavir-boosted lopinavir (10-fold), and ritonavir-boosted indinavir (10-fold). In the absence of clinical cutoffs, two alternative types of cutoffs have been used. The assay cutoff is defined by the intrinsic variability and technical limits of the assay during repeated testing of patient-derived viruses. The biological cutoff is defined by an upper limit of the distribution of susceptibility exhibited by wild-type viruses. Harrigan et al. (13), using the AntiVirogram assay, defined the biological cutoff as the mean FC plus 2 standard deviations (SDs) among a population of viruses from 1,000 drug-naïve patients. The use of the biological cutoff as a clinically relevant threshold is limited, since a higher or a lower FC value may be associated with declining virological responses. Importantly, the biological cutoff reflects both natural variations in viral susceptibility and inherent assay variability. Thus, such cutoffs may differ among assays that have different intrinsic variabilities.

The range in FC observed among viruses from treatment-experienced patients varies for different drugs and is considerably narrower for some drugs than for others. For example, FC values over 10-fold are much less common for stavudine, didanosine, and tenofovir than for zidovudine, lamivudine, or the nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs), for which FC values over 100 are often seen. The clinical cutoffs for these drugs (stavudine, didanosine, and tenofovir) are also low (FC < 2). Thus, definition of the natural variation in drug susceptibility in wild-type viruses is necessary to determine the prevalence of innate drug resistance and to assess the capability of the assay to reliably measure subtle reductions in drug susceptibility. In this study, we sought to describe the variability in drug susceptibility in wild-type viruses and to assess the contribution of assay variability to the defined biological cutoffs.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Phenotypes and genotypes were determined in the Clinical Laboratory Improvement Amendments-approved ViroLogic clinical reference laboratory (ViroLogic, Inc., South San Francisco, Calif.). The drug susceptibility phenotypes of HIV-1 isolates from patient plasma samples was determined by the PhenoSense HIV assay (20). This assay is performed by amplifying the protease (PR)-RT segment of the pol gene from patient plasma and inserting it into a genomic HIV-1 vector. The vector contains a luciferase reporter gene to monitor recombinant virus infection in cell culture. Results are expressed as the FC in the IC₅₀ for the patient-derived virus compared to that for a reference control virus, NL4-3. Assay techniques have been optimized to minimize variability, and assay performance has been extensively validated (ViroLogic, data on file). Several recent modifications have further enhanced assay performance. Drug dilutions are arranged to maximize curve-fitting accuracy for the range of wild-type virus susceptibilities and over clinically relevant ranges of increased and decreased susceptibilities. Microtiter plates are incubated in customized incubators in which the temperature, CO₂ level, and humidity are controlled to minimize variation in cell growth and medium composition changes throughout the plate (i.e., edge effects).

Genotypes were determined by the GeneSeq HIV assay. This assay uses the resistance test vectors constructed for the phenotype assay as the template, dye-terminator reaction chemistry, and automated capillary electrophoresis to determine the sequences of the patient-derived HIV-1 PRs and RTs (amino acids 1 to 99 in PR and amino acids 1 to 305 in RT). The electropherograms were analyzed and reviewed by trained clinical laboratory personnel to detect all mutations, mixed bases, insertions, and deletions. The deduced amino acid sequences of patient viruses were compared to the sequence of a reference virus strain (strain NL4-3; GenBank accession no. AF324493). In assay validation studies, the GeneSeq HIV assay detected minor virus populations in plasma samples present at concentrations as low as 10% of the total virus population (ViroLogic, GeneSeq HIV validation data on file).

ViroLogic maintains a database of phenotype and genotype results for all patient viruses submitted to the clinical reference laboratory for phenotypic and genotypic testing. Prior to analysis, repeat samples from the same patient were removed on the basis of coded unique identifiers. To avoid inclusion of resistant viruses transmitted to drug-naïve individuals (17), wild-type viruses were defined as any virus lacking a drug-selected mutation (DSM) in either PR or RT. Any amino acid substitution at the following positions in PR was considered a DSM: 23, 24, 30, 32, 46, 47, 48, 50, 54, 84, 88, and 90. In addition, L33F and V82A, V82F, V82S, or V82T were considered DSMs. Any substitution at the following positions in RT was considered a DSM: 41, 65, 67, 69, 70, 74, 75, 100, 151, 181, 184, 188, 190, 210, 215, 219, 225, 227, 230, and 236. In addition, A98G, K101E or K101P, K103N or K103S, and V106A or V106M were considered DSMs (2, 6, 14, 19). The data set primarily comprises subtype B HIV-1 isolates from the United States but also contains 7% non-clade B virus isolates. The number of non-B clade isolates and isolates from outside the United States was too small to permit meaningful subanalyses by clade or country of origin. FC data were log transformed and analyzed by using Statview (version 5.0) software (SAS, Inc., Cary, N.C.) to characterize the distribution of FC values for each drug. Two alternative definitions for the biological cutoff were used: the mean FC plus 2 SDs (calculated by using the log-transformed data) or the 99th percentile of the FC distribution.

Genotypic correlates of reduced susceptibility were determined for certain drugs (delavirdine and nelfinavir) with broader FC distributions among wild-type viruses by using the Fisher exact test after categorization of samples on the basis of phenotype (categorization as "high" if the FC was over the threshold or "low" if the FC was below the threshold) and genotype (categorization as "mutant" if a given mutation was present or "wild type" if the mutation was absent). An arbitrary phenotypic threshold corresponding to the 90th percentile of the FC distribution for a drug was selected to define a high FC in order to provide sufficient numbers of samples in that phenotypic category. Mixtures of wild-type and mutant variants were scored as mutants. Odds ratios to define the strength of association between a mutation and isolates for which FC were high were calculated as the fraction of isolates for which FC were high and which bore a certain mutation divided by the fraction of isolates for which FC were low and which bore the mutation. Mutations for which the odds ratios (ORs) were >1.5 and the P value was <0.05 by the Fisher exact test were considered significant.

To determine assay reproducibility, 10 clonal viruses (whole-assay controls [WACs]) with diverse patterns of drug resistance mutations (Table 1) were tested repeatedly through the entire assay. Each WAC represents a single variant derived from a clinical plasma sample. WAC viral stocks were prepared by transfecting HEK293 cells with WAC vector DNA, harvesting the cell culture supernatant after 48 h, and treating the virus stocks with DNase I. An aliquot of each virus stock was added to HIV-negative human plasma and then processed through the entire PhenoSense HIV assay multiple times on different days. The mean FC and coefficient of variation (CV) for each WAC were calculated for each drug. CVs were not determined when the IC₅₀ for the WAC virus exceeded the highest drug concentration tested in the assay.

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TABLE 1. WAC virus genotypes (resistance-associated mutations)

Top Abstract Introduction Materials and Methods Results Discussion References

 
Phenotypic drug susceptibility and PR and RT genotype data for 2,924 wild-type HIV-1 test vector pools were included in the analyses (data for all drugs were not available for all isolates) (Table 2). The viruses were derived from patient plasma samples submitted for either routine resistance testing or research studies and were collected between September 1985 and March 2003. The great majority (96%) of the samples were collected between 1999 and 2003.

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TABLE 2. Median drug susceptibilities by FC and IC50 for wild-type viruses and upper biological cutoffs as defined by two separate criteriaa

Histograms of log-transformed FC values for each drug (except zalcitabine, for which the distribution was similar to that for didanosine) are shown in Fig. 1 (RT inhibitors) and Fig. 2 (PR inhibitors [PIs]). FC values were essentially log-normally distributed (the normal curve is shown for reference). The median FC for each drug as well as the corresponding IC₅₀ is shown in Table 2. The median FC was below 1 for several drugs, particularly the PIs, indicating that many wild-type viruses are slightly more susceptible than the NL4-3 reference virus. Two alternative definitions for biological cutoff are presented: the mean FC plus 2 SDs or the 99th percentile of the FC distribution (Table 2). The former permits comparison to data from other phenotypic assays (12, 13), while the latter provides a more conservative estimate of the upper limits of the distributions (i.e., the 99th percentile FC values are always higher than the mean FC plus 2 SD values). In the nucleoside or nucleotide RTI (NRTI) class, the 99th percentile values ranged from 1.2-fold for tenofovir to 1.8-fold for zidovudine. Wider distributions were observed for the NNRTIs, with 99th percentile values ranging from 3.0-fold for efavirenz to 6.2-fold for delavirdine. The FC distributions for PIs were intermediate, with 99th percentile values ranging from 1.6-fold for lopinavir to 3.6-fold for nelfinavir.

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FIG. 1. Histograms of FC values for RT inhibitors for wild-type viruses obtained by the PhenoSense HIV assay. The FC in IC50 were log transformed and divided into 50 bins. The theoretical normal distribution curves are shown for comparison. The vertical lines represent FC of 1.0. See Table 2 for the numbers of samples included and the statistical parameters for each drug, and see footnote b of Table 2 for definitions of the drug abbreviations.

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FIG. 2. Histograms of FC values for PIs for wild-type viruses obtained by the PhenoSense HIV assay. The FC in IC50 were log transformed and divided into 50 bins. The theoretical normal distribution curves are shown for comparison. The vertical lines represent FC of 1.0. See Table 2 for the numbers of samples included and the statistical parameters for each drug, and see footnote b of Table 2 for definitions of the drug abbreviations.

Univariate analyses were performed to determine the genotypic correlates of phenotypic FC values above the 90th percentile of the distribution for two drugs with the broadest FC distributions: nelfinavir (2.0-fold) and delavirdine (2.6-fold). Eleven mutations were significantly associated with elevated FC values for nelfinavir (Table 3). Five of these mutations are recognized secondary PI resistance mutations that also exist as natural polymorphisms (L10I and L10V, A71V and A71T, and V77I). Another mutation associated with reduced nelfinavir susceptibility, Q61N, is often found in subtype F HIV-1 (4, 10), indicating that clade F HIV-1 may possess natural reductions in NFV susceptibility. Twelve mutations (K49R, A98S, K101Q, V108I, I135L, I135T, I142T, I142V, I178L, V179D, V179E, and V179I) were associated with elevated FC values for delavirdine (data not shown), including polymorphisms at positions 98, 101, 108, 135, and 179, which have previously been suggested to play a role in determining NNRTI susceptibility (3, 19).

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TABLE 3. Mutations associated with reduced susceptibility to nelfinavira

To determine whether drug-specific differences in assay variability account for the different distributions that were observed, 10 clonal patient-derived viruses with various patterns of drug resistance were assayed repeatedly (8 to 30 replicates each) (Tables 1 and 4). Assay variations (median CVs) were similar for all drugs except zidovudine, ranging from 12% (for tenofovir and efavirenz) to 18% (for lamivudine and zalcitabine); the median CV for zidovudine was 32%. Thus, the wider distributions of FC values for certain drugs (NNRTIs and PIs) among wild-type viruses were not associated with increased assay variability for those drugs. Notably, the drug with the broadest distribution of FC for wild-type viruses, delavirdine (99th percentile, 6.2-fold), also had one of the lowest CVs (13%). The susceptibility curves for zidovudine typically had reduced slopes compared to the slopes of the curves for the other drugs, resulting in reductions in the precisions of IC₅₀ and FC determinations (data not shown) and a higher CV than those for the other drugs.

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TABLE 4. Mean FC and median CVs for WAC viruses

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have defined the natural variation in drug susceptibility among a large population of wild-type HIV-1 isolates using the PhenoSense HIV assay. The natural variation in drug susceptibility in wild-type HIV varies across drugs and drug classes. The greatest variation was observed among the NNRTIs (biological cutoff range, 3.0- to 6.2-fold), and the least variation was observed among the NRTIs (biological cutoff range, 1.2- to 1.8-fold). The measured biological variability in the present study is considerably lower than that previously reported by use of a different recombinant virus assay and a collection of >1,000 wild-type viruses (12, 13). For example, the mean FC plus 2 SDs for tenofovir is 1.2-fold by the PhenoSense HIV assay, whereas it is 3.0-fold by the Antivirogram method (12). Assuming that the extent of natural variation in susceptibility in the two virus populations studied is equivalent, the better precision of the PhenoSense HIV assay likely accounts for the lower biological cutoffs; however, as this was not a comparative study, the differences may be due to the viruses analyzed. Enhanced assay precision is important when susceptibility to drugs with clinical cutoffs that are close to the limits of assay reproducibility, such as tenofovir (1.4-fold), is measured. Notably, for each of the seven drugs with defined clinical cutoffs, the biological cutoff was lower than the clinical cutoff, indicating that there is no significant overlap among wild-type viruses and viruses with clinically relevant levels of reduced susceptibility to those drugs. Clinical cutoffs below twofold (i.e., for didanosine, stavudine, and tenofovir) are well above the 99th percentile of the distribution for wild-type virus, demonstrating the ability of the PhenoSense HIV assay to reliably measure subtle but clinically relevant changes in drug susceptibility. Differences between the biological and clinical cutoffs highlight the critical need to define clinical cutoffs for all drugs in order to provide the most clinically relevant thresholds for therapy guidance.

As with any biological assay, the accuracy of dichotomous categorization, such as "drug sensitive" and "drug resistant," can never be 100% when the assay result is close to the defined resistance threshold. Thus, the closer that an FC value is to the cutoff, the lower the confidence level in the categorization of the result will be. Greater assay precision reduces the uncertainty associated with results close to the assay cutoff. Repeated testing of a virus for which the mean FC for tenofovir was 1.54 produced FC results over the clinical cutoff (1.4-fold) for 29 of 39 (74%) replicate results; similarly, the FC result for a virus for which the mean FC was 1.73 was above the cutoff for 34 of 36 (94%) replicate results (data not shown). The use of an assay with high precision provides an accurate indication of the biological cutoffs for wild-type viruses and an accurate classification of viruses that are drug sensitive (drug susceptibility below the cutoff) or drug resistant (susceptibility above the cutoff). Proper interpretation of phenotypic assay results should take into account both the quantitative nature of the result and the assay reproducibility.

This study demonstrates that the natural variation in drug susceptibility observed among wild-type HIV-1 isolates is not caused by assay variation. Assay reproducibility was similar for all drugs except zidovudine. Natural variation in drug susceptibility was greater for NNRTIs and some PIs than for NRTIs. Whether the variation in drug susceptibility for NNRTI and some PIs has clinical significance remains to be determined. The results of the few small studies that have attempted to assess the likelihood of clinical failure of NNRTI-based regimens in patients infected with viruses possessing low-level reduced susceptibility are inconclusive [L. Bacheler, L. Ploughman, K. Hertogs, and B. Larder, Antivir. Ther. 5(Suppl. 3):70, 2000, abstr.; P. R. Harrigan, W. Verbiest, B. Larder, K. Hertogs, J. Tilley, J. Raboud, and J. Montaner, Antivir. Ther. 5(Suppl. 3):68, 2000, abstr.]. The clinical cutoffs for these drugs are not yet defined. Notably, the FC for some viruses isolated after NNRTI treatment failure are as low as 5 to 10, despite the presence of well-recognized drug resistance mutations, and overlap the range of FC values observed for wild-type viruses.

The median FC value for wild-type viruses for most PIs (all PIs except NFV) was slightly below 1. The most likely explanation for this observation is that the reference virus containing PR and RT sequences from NL4-3 is slightly less susceptible to PIs than the average clinical isolate. The median FC value for wild-type viruses does not affect the interpretation of assay results, as long as the drug susceptibility cutoffs for the PhenoSense HIV assay are defined on the basis of data obtained with the same reference virus. For example, the clinical cutoff for ritonavir-boosted lopinavir (10-fold) was determined by the PhenoSense HIV assay (16), and so the PhenoSense HIV assay results do not require adjustment because the FC values for wild-type viruses tend to be less than 1. However, the cutoffs defined by using data from one assay or for one reference virus may require adjustment before they can be applied to another phenotypic assay that has different performance characteristics or that uses a different reference virus.

It is important to appreciate the potential drawbacks of using biological cutoffs to help interpret phenotypic test results for patient management. Natural variation in the drug susceptibilities of wild-type viruses may not relate to treatment response if an antiretroviral drug is not uniformly active against all wild-type viruses. A priori, an FC value lower than the biological cutoff does not necessarily indicate that the virus will respond to a drug; conversely, an FC value greater than the biological cutoff does not indicate that the drug will be ineffective. For example, the clinical cutoffs for abacavir and lopinavir were higher than their respective biological cutoffs. Thus, biological cutoffs are somewhat arbitrary and without clear clinical relevance for many drugs. Nevertheless, in the absence of a clinical cutoff, the natural variability in drug susceptibility among wild-type viruses provides a threshold for defining abnormal reductions in drug susceptibility and an indicator of an increased probability of drug resistance.

This analysis has several limitations. Our definition of wild-type virus may lead to the inclusion of drug-resistant test vector pools containing unrecognized DSMs or the exclusion of wild-type test vector pools that contain natural polymorphisms that have been defined as DSMs. However, the large size of the patient population and the use of standard genotypic guidelines to define DSMs minimize the impact of the occasional misclassification of viruses. Also, the use of the 99th percentile or the mean plus 2 SDs rather than the maximum of the distribution of FC values for wild-type viruses to define the biological cutoff reduces the potential impact of inclusion of a small number of resistant viruses in the analyses. Comparison of our results to those of Harrigan et al. (12, 13) is complicated by the different amplification strategies (different lengths of the RT sequences of the test viruses were sequenced) and cell-based assays (single-cycle versus multiple-cycle replication) used. With regard to the extent of RT sequence captured by RT-PCR, since lower biological cutoffs were also seen for PIs, this is unlikely to account for all of the observed differences. In addition, previous studies may have included samples from therapy-naïve patients infected with drug-resistant virus.

Examination of the natural variation in drug susceptibility provides a useful, albeit imperfect, means of defining the upper limit of expected drug susceptibility among wild-type viruses. However, biological cutoffs are not a substitute for clinical cutoffs rigorously defined by treatment outcome data obtained from carefully conducted clinical trials. The availability of a phenotypic assay with enhanced accuracy and precision permits the confident assessment of subtle changes in drug susceptibility. The use of carefully defined clinical and/or biological cutoffs further enhances the ability of phenotypic assays to provide relevant assessments of drug activity.

 
We are grateful for the contributions of the members of the ViroLogic Clinical Reference Laboratory for performing phenotypic and genotypic testing and the ViroLogic Information Technology group for database infrastructure support. We also thank Soumya Nidtha for assistance with the genotypic analysis.

The customized software for genotype analysis was provided by GeneCodes, Inc. (Ann Arbor, Mich.).

 
* Corresponding author. Mailing address: ViroLogic, Inc., 345 Oyster Point Blvd., South San Francisco, CA 94080. Phone: (650) 866-7438. Fax: (650) 616-3687. E-mail: nparkin{at}virologic.com.

Present address: Roche Molecular Systems, Pleasanton, CA 94588.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Antimicrobial Agents and Chemotherapy, February 2004, p. 437-443, Vol. 48, No. 2
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.2.437-443.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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