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Journal of Clinical Microbiology, April 2003, p. 1594-1599, Vol. 41, No. 4
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.4.1594-1599.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Performance Characteristics of the TRUGENE HIV-1 Genotyping Kit and the Opengene DNA Sequencing System

Daniel R. Kuritzkes,1* Robert M. Grant,2 Paul Feorino,3 Marshal Griswold,4 Marie Hoover,5 Russell Young,1 Stephen Day,3 Robert M. Lloyd, Jr.,3 Caroline Reid,6 Gillian F. Morgan,6 and Dean L. Winslow6

Division of Infectious Diseases, University of Colorado Health Sciences Center, Denver, Colorado,1 Gladstone Institute of Virology and Immunology, San Francisco,2 Consolidated Laboratories, Van Nuys, California,4 Visible Genetics, Inc., Suwanee, Georgia,3 Advanced BioMedical Laboratories, Cinnaminson, New Jersey,5 Visible Genetics, Inc., Toronto, Ontario, Canada6

Received 11 February 2002/ Returned for modification 14 December 2002/ Accepted 9 January 2003


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ABSTRACT
 
The TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System are designed to sequence the protease (PR)- and reverse transcriptase (RT)-coding regions of human immunodeficiency virus type 1 (HIV-1) pol. Studies were undertaken to determine the accuracy of this assay system in detecting resistance-associated mutations and to determine the effects of RNA extraction methods, anticoagulants, specimen handling, and potentially interfering substances. Samples were plasma obtained from HIV-infected subjects or seronegative plasma to which viruses derived from wild-type and mutant infectious molecular clones (IMC) of HIV-1 were added. Extraction methods tested included standard and UltraSensitive AMPLICOR HIV-1 MONITOR, QIAGEN viral RNA extraction mini kit, and QIAGEN Ultra HIV extraction kit, and NASBA manual HIV-1 quantitative NucliSens. Sequence data from test sites were compared to a "gold standard" reference sequence to determine the percent agreement. Comparisons between test and reference sequences at the nucleotide level showed 97.5 to 100% agreement. Similar results were obtained regardless of extraction method, regardless of use of EDTA or acid citrate dextrose as anticoagulant, and despite the presence of triglycerides, bilirubin, hemoglobin, antiretroviral drugs, HIV-2, hepatitis C virus (HCV), HBV, cytomegalovirus, human T-cell leukemia virus type 1 (HTLV-1), or HTLV-2. Samples with HIV-1 RNA titers of >=1,000 copies/ml gave consistent results. The TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System consistently generate highly accurate sequence data when tested with IMC-derived HIV and patient samples.


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INTRODUCTION
 
The clinical significance of drug resistance in human immunodeficiency virus type 1 (HIV-1) infection is well-established (3, 13, 14). Patients treated with drugs to which their HIV-1 isolates are predicted to be resistant on the basis of genotypic or phenotypic assays are significantly less likely to achieve a virological response (4, 17). Conversely, use of drugs to which the virus is predicted to be susceptible enhances the likelihood of reestablishing virological control in patients in whom prior treatment regimens have failed (5). Randomized clinical trials demonstrate the clinical utility of drug resistance testing as a guide to the selection of subsequent antiretroviral regimens for patients with virological failure of their current regimen (1, 2, 7, 16). Guidelines recommending drug resistance testing in managing failure of antiretroviral therapy have been proposed by several expert panels (8, 10; see also guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents [http://www.atis.org]).

The routine use of genotypic resistance testing requires convenient assays with high reproducibility that can be performed by laboratories skilled in molecular diagnostic techniques. The TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System are designed to provide bidirectional sequencing of the protease (PR)-coding region (codons 10 to 99) and codons 41 to 237 of the reverse transcriptase (RT)-coding region of HIV-1 pol. A detailed description of this system is described in the accompanying work (9). In order to determine the effects of specimen characteristics on the performance characteristics of the TRUGENE HIV-1 Genotyping Kit and the OpenGene DNA Sequencing System, a series of studies was undertaken to assess the effect of RNA extraction methods, anticoagulants, sample freezing and thawing, and the presence of potentially interfering substances. Ability to detect drug resistance-associated mutations was also evaluated using a panel of plasma samples spiked with HIV-1 derived from infectious molecular clones carrying a variety of resistance-associated mutations in PR and RT.


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MATERIALS AND METHODS
 
Plasma samples. Plasma from HIV-1-infected subjects was collected by routine methods or by plasmapheresis. Specimen collection was conducted using protocols that complied with all relevant federal guidelines and that were approved by the appropriate institutional review boards. All subjects gave signed informed consent. HIV-1-negative human plasma was purchased from a commercial source (Boston Biomedica, Inc., Bridgewater, Mass.).

Viruses. Wild-type and mutant viruses were prepared from infectious molecular clones of HIV-1 or obtained from plasma samples collected from HIV-infected subjects by plasmapheresis as described elsewhere (9). Mutant variants of HIV-1 were prepared by site-directed mutagenesis of an infectious molecular clone of HIV-1LAI (pLAI.2) (kindly provided by L. Montagnier through the NIH AIDS Research and Reference Reagent Program). Mutations were introduced into the PR- or RT-coding region of pol using the Altered Sites II in vitro mutagenesis system (Promega, Madison, Wis.). The resulting plasmids were linearized by restriction enzyme digestion and electroporated into CEM-SS cells to generate infectious viruses, which were propagated to yield high-titer stocks. Table 1 lists the mutations associated with drug resistance that were present in the plasma specimens from HIV-1-infected subjects and infectious molecular clone (IMC)-derived viruses used in the test panels. HIV-2 (strain NIH-Z), human T-cell leukemia virus type 1 (HTLV-1), HTLV-2, and cytomegalovirus (CMV) were obtained from Advanced Biotechnologies, Inc., Columbia, Md. HIV-2, HTLV-1, and HTLV-2 were quantified by particle count. CMV was quantified by culture to determine the 50% tissue culture infectious dose. Hepatitis B virus (HBV) and HCV were obtained in the form of infectious plasma from the Cleveland Clinic, Cleveland, Ohio. The titer of HBV in plasma was determined using the HBV Hybrid Capture assay (Digene, Gaithersburg, Md.). The plasma HCV titer was determined using the Amplicor HCV MONITOR Test (Roche Molecular Systems, Branchburg, N.J.).


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  		TABLE 1. Resistance-associated mutations in HIV-1 PR and RT present in test samples

Test samples. Plasma was anticoagulated with EDTA except where indicated. The UltraSensitive AMPLICOR HIV-1 MONITOR Test (Roche Molecular Systems) was used to quantify the HIV RNA titer of patient plasma samples and culture supernatants, which were diluted as necessary into human plasma from seronegative donors to yield samples of the desired titer. For some tests, HIV-1-positive and HIV-1-negative plasma samples were spiked with triglycerides (1,024 mg/dl), hemoglobin (19.5 g/dl), or bilirubin (21 mg/dl) (values are final concentrations). Alternatively, samples were spiked with various antiretroviral agents, including abacavir (3.0 µg/ml), didanosine (1.31 µg/ml), lamivudine (1,500 µg/ml), stavudine (0.73 µg/ml), zalcitabine (0.007 µg/ml), zidovudine (0.55 µg/ml), amprenavir (5.36 µg/ml), indinavir sulfate (8.98 µg/ml), nelfinavir mesylate (3.5 µg/ml), ritonavir (11.2 µg/ml), saquinavir (2.05 µg/ml), delavirdine mesylate (19.34 µg/ml), efavirenz (4.07 µg/ml), nevirapine (4.4 µg/ml), and adefovir dipivoxil (0.211 µg/ml) (values are final concentrations). To test the effect of other viral sequences on performance of the TRUGENE HIV-1 Genotyping Kit, some samples were spiked with HIV-2 (105 virus particles [vp]/ml), HTLV-1 (105 vp/ml), HTLV-2 (105 vp/ml), HBV (2.5 x 106 vp/ml), HCV (5 x 105 vp/ml), or CMV (385 50% tissue culture infectious doses/ml).

Once prepared, test samples were stored frozen at -70°C as 1-ml aliquots. Samples were shipped on dry ice to the testing laboratories, where they were again stored at -70°C. Except for the study of the effects of sample freezing and thawing (see below), samples were thawed no more than three times prior to use.

Extraction of HIV-1 RNA from plasma. For most studies, viral RNA was isolated from 140 µl of HIV-1-infected patient plasma using the QIAGEN Viral RNA Mini Kit (Qiagen, Santa Clara, Calif.). For the comparison of different extraction procedures, the following additional methods and sample volumes were used: QIAGEN Ultra HIV-1 RNA Extraction Method, 1.0 ml and 3 ml; AMPLICOR HIV-1 MONITOR Test (Roche Molecular Systems), 200 µl; UltraSensitive AMPLICOR HIV-1 MONITOR Test (Roche Molecular Systems), 0.5 ml; and NASBA Manual HIV-1 Quantitative NucliSens (Organon Teknika, Durham, N.C.), 1.0 ml. Extractions were performed according to the manufacturers' instructions, except that the positive control RNA from the AMPLICOR HIV-1 MONITOR Test kit was not added to any specimen. Only the QIAGEN Ultra HIV-1 RNA Extraction Method and UltraSensitive AMPLICOR HIV-1 MONITOR Test method were used for samples with low concentrations of HIV-1 RNA (<=1,000 copies/ml).

Sequence analysis of HIV-1 PR and RT. Sequence analysis of HIV-1 PR and RT reading frames was performed using the TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System according to the manufacturer's recommendations (Visible Genetics, Inc., Toronto, Canada), and the data were analyzed as described elsewhere (9).

Efficacy assessments. Efficacy assessments of the study device included identification of bases, codons, and resistance mutations in a sequence. For the study examining the effects of different anticoagulant media, test sample sequences collected in EDTA, acid citrate dextrose (ACD), or heparin were compared to sequences determined by the investigational system using samples collected in a VACUTAINER plasma preparation tube (PPT) from the same HIV-1-infected subject. Otherwise, the gold standard for the comparison of the test samples derived from HIV-1-infected subjects was the consensus sequence obtained from multiple independent molecular clones created from the each of the plasmapheresis samples and sequenced using universal primer sets. HIV-1 samples derived from IMC were compared to a gold standard sequence determined by sequencing of the progeny virus RNA generated from the IMC using standard sequencing chemistry and universal primers. For each test sequence, the proportion of agreement of the entire base sequence between the automatically or manually edited test sequence and the gold standard was determined by comparing the two sequences using the partial and exact match methods. For all analyses of base agreement, the "entire base sequence" refers to 920 bases of the sequence. The proportion of agreement was defined as the number of exactly matching bases between the test and gold standard sequences divided by the total number of bases in the gold standard sequence.

For comparison of different RNA extraction methods, analyses were based on intrasample comparisons, in which sequences obtained with each of the five extraction methods were compared to those of predetermined gold standards. Percent agreement of the bases and number and identity of mutations was calculated for the sequences generated from each test sample relative to the gold standard sequence using the following proportion: [(total number of resistance codons) - (number of resistance codons on which the two sequences disagree)]/(total number of resistance codons). The percent agreement of the bases was calculated based on exact matching of the bases.

Statistical analysis. Continuous variables, such as the accuracy rate for each sample, were summarized by presenting number, mean, and standard deviation. Categorical variables were summarized by presenting the frequency and percentage of samples or assays in each category. All statistical tests were two sided and were performed at the 5% level of significance using SAS (version 6.12) software on the Windows platform.

For comparison of different RNA extraction methods, percent agreement of the bases with the gold standard for each of the samples was summarized for each extraction method by testing site. Tests of significance of the difference in mean percent agreement between different extraction methods were performed using an analysis of variance (ANOVA) with a testing site factor, a method factor comprised of the five different extraction methods, method by testing site interaction, and a blocking factor for the samples. Interaction of extraction method by testing site was tested at the 0.1 level of significance.

Nucleotide sequence accession numbers. The PR and RT sequences of the plasma viruses have been deposited in GenBank (accession numbers AF472535 to AF472552).


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RESULTS
 
Effect of anticoagulants on sequence quality. To determine the effect of different anticoagulants on sequence data quality, blood was collected from 10 HIV-1-infected individuals (018-027-01 to 018-027-10) (Table 1) using plasma collection tubes containing heparin, EDTA, and ACD. Plasma was separated from cellular components and stored in aliquots at -70°C. A reference sample was also collected into a PPT (Becton-Dickinson, South Plainfield, N.J.), which contains EDTA. For each patient, sequence data from samples collected in heparin-, EDTA-, or ACD-containing tubes were compared to the reference sample. The median plasma HIV-1 RNA titer for these samples was 75,066 copies/ml (range, 6,288 to 2,535,030 copies/ml). As expected, no sequence data were obtained from the heparinized samples. In samples tested after storage at -70°C for 30 days the mean percent agreement between the EDTA and ACD samples and the reference samples was >=99.7% at both the nucleotide and codon level (Table 2). Mean agreement rates for mutation recognition and wild-type recognition were >=0.97. Similarly high agreement was observed in samples tested after storage for 3 months (Table 2). Rates of agreement with the reference sample were not significantly different for samples collected in EDTA or ACD.


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  		TABLE 2. Effect of anticoagulant on sequence agreementa

Comparison of HIV-1 RNA extraction methods. To determine the effect of HIV-1 RNA extraction method on sequence data quality, plasma samples from subjects VA-MH-001, VA-MH-003, VA-MH-004, VA-MH-007, and VA-MH-009 were each subjected to extraction by five different procedures in two different laboratories as described in Materials and Methods. Two samples had viral loads in the range of >1,000 to <2,000 copies/ml; two samples had viral loads in the range of >15,000 to <45,000 copies/ml, and one sample had a viral load of >350,000 copies/ml. The mean percent agreement of nucleotide bases between the test sequence and the gold standard sequence for the ten samples extracted with each extraction method was >=98.17% (Table 3). Similarly, when sequences were analyzed at the amino acid level, mean agreement rates of codons between the test sequence and the gold standard sequence were >=98.07% for each extraction method. No statistically significant differences in mean agreement rates were observed between extraction methods or between the two testing sites. Wild-type and mutant recognition rates ranged from 0.27 to 1.00. Mean mutation recognition agreement rates were >=0.773, and mean wild-type recognition agreement rates were >=0.993 for all extraction methods tested and across both testing sites (Table 3). No differences in sequence quality were observed as a function of sample HIV-1 RNA concentration (data not shown).


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  		TABLE 3. Effect of extraction method on sequence agreementa

Genotyping samples with low concentrations of HIV-1 RNA. A panel of plasma samples spiked with HIV-1 from an infectious molecular clone (PE-009-028) (Table 1) to yield final concentrations of 0, 30, 60, 125, 250, 500, and 1,000 HIV-1 RNA copies/ml was used to evaluate sequence reliability using extraction methods designed for low-concentration samples. This panel gave inconsistent results—9 of 28 (32%) samples processed by the two testing sites gave evaluable sequences (two samples for which genotypic data could not be obtained had no HIV-1 RNA). Neither laboratory obtained interpretable sequence data from the samples that contained 30 copies/ml, whereas three of four samples with titers of 1,000 copies/ml produced good sequence data. Similar results were obtained whether the QIAGEN Ultra HIV-1 RNA Extraction Method (3-ml sample volume) or the Roche UltraSensitive AMPLICOR HIV-1 MONITOR Test kit (0.5-ml sample volume) was used for RNA extraction from these samples.

Effect of freezing and thawing. To determine the effect of freezing and thawing on assay performance, plasma samples that were collected from two HIV-infected subjects (VA-MH-004 and VA-MH-009) (Table 1) through plasmapheresis were subjected to 10 full freeze-thaw cycles over 2 days at one laboratory. Nominal plasma HIV-1 RNA titers of the two samples were 1,200 and 43,000 copies/ml, respectively. The reference sequence for comparison was the exact sequence of bases and the presence of mutations determined by sequencing of multiple independent clones derived from HIV-1 in the plasmapheresis samples. The mean percentage base agreement for each of the 10 freeze-thaw cycles ranged from 98.1 to 98.5%, with no deterioration through 10 freeze-thaw cycles. Similarly, the mean agreement rate for all codons for each of the 10 freeze-thaw cycles ranged from 98.0 to 98.9%. ANOVA indicated no statistically significant differences in mean percent base agreement or mean percent codon agreement rates between the 10 freeze-thaw cycles (P = 1.000 and 0.858, respectively). Mutation recognition and wild-type recognition agreement rates likewise did not vary significantly between freeze-thaw cycles (data not shown).

Interfering substances. To determine the effect of clinically relevant biochemicals, antiretroviral compounds, or viruses on assay performance, plasma samples containing virus derived from an infectious molecular clone (HIV-1NL4-3) of HIV-1 at a titer of 10,000 copies/ml and HIV-negative plasma were spiked with hemoglobin, bilirubin, triglycerides, antiretroviral drug compounds, HIV-2, HTLV-1, HTLV-2, CMV, HBV, and HCV as described in Materials and Methods. None of the HIV-1-negative samples spiked with a potentially interfering substance gave a positive test result. The HIV-1-positive controls showed agreement at the nucleotide sequence level of >=99.9%. The mean mutant and wild-type recognition agreement rates for the HIV-1 positive control samples were 1.00 for all panels.

Each biochemical was tested in triplicate. For the manually edited output based on exact matching of the nucleotide sequence, the base agreement rate of the HIV-1-positive test sequences with the gold standard sequence was >99.9% (data not shown). The mean mutation agreement rate for all codons among the test sequences was 1.00, and the mean wild-type recognition agreement rate ranged from 0.999 to 1.000 (data not shown).

The effect of each antiretroviral compound on test accuracy was tested once in HIV-1-positive and -negative samples. For the manually edited output based on exact matching of the nucleotide sequence, the base agreement rate of the HIV-1-positive test sequences with the gold standard sequence was 100% for the spiked drug samples and >=99.9% for the positive controls. The mutation agreement rate and mean wild-type agreement rate for all codons among the test sequences was 1.00.

Similarly, the effect of each pathogen was tested once in HIV-1 positive and negative samples. For the manually edited output based on exact matching of the nucleotide sequence, the base agreement rate of the HIV-1 positive test sequences with the gold standard sequence was >=99.3%. The mutation agreement rate for all codons among the test sequences was 1.00, with the exception of the sample spiked with HIV-2NIH-Z. For HIV-2-spiked specimens, no agreement was found between the test sequence and the gold standard sequence at any of the five known mutation sites. The codons detected in the assay were not present in the HIV-2NIH-Z sequence present in these specimens, and the sequence detected clustered phylogenetically with HIV-1 rather than HIV-2. Moreover, phylogenetic analysis demonstrated a closer relationship to wild-type HIV-1LAI, than to HIV-1NL4-3, suggesting that contamination had occurred in the testing laboratory. The wild-type recognition agreement rate for all codons among the test sequences ranged from 0.99 to 1.00.


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DISCUSSION
 
The studies reported here assessed the performance of the TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System under a variety of conditions. Mean agreement at the nucleotide level between test samples and the reference sequences was >=97.5% overall for plasma samples in which the HIV-1 RNA titer was >1,000 copies/ml. Mean agreement at the amino acid level was >=98%. Comparable results were obtained using either EDTA or ACD as an anticoagulant and using a variety of HIV-1 RNA extraction methods. No significant loss of sample integrity was observed over 10 freeze-thaw cycles. Presence of bilirubin, hemoglobin, triglycerides, and antiretroviral compounds had no significant effect on sequence quality. Similarly, presence of other viruses including HBV, HTLV-1, and HIV-2 did not interfere with the assay. Results of this evaluation demonstrated that the TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System provide highly accurate and reproducible sequence data for HIV-1 PR and RT.

Plasma samples with HIV-1 RNA titers <=1,000 copies/ml gave inconsistent results. No sequence data were obtained from plasma samples with an HIV-1 titer of 30 copies/ml, and only 50% of samples with HIV-1 titers of 125 to 500 copies/ml generated a sequence result. However, all of the positive results for samples with 60 to 500 HIV-1 RNA copies/ml came from one laboratory, suggesting that operator-specific factors may have played an important role. More precise definition of the utility of different methods for extracting HIV-1 RNA from low-copy number samples for use in the TRUGENE assay requires evaluation at a larger number of test sites. Nevertheless, these data are consistent with the general recommendation that performance of resistance testing be limited to patients with plasma HIV-1 RNA levels >=1,000 copies/ml (10).

The difficulty in obtaining sequence data from samples with low copy numbers was surprising, as previous studies had shown that sequence data could be generated from patient samples with virus loads as low as 60 copies/ml provided specimens underwent a centrifugation step to concentrate virions prior to RNA extraction (J. Lawrence, R. M. Lloyd, Jr., L. M. Hough, P. M. Feorino, and M. A. Thompson, Abstr. 7th Conf. Retrovir. Opportun. Infect., abstr. 795, 2000; D. R. McClernon, A. L. Matthews, L. Salter, M. Cronin, and M. St. Clair, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother, abstr. I-1965, 2001). It is possible that the homogeneous template present in test samples containing virus derived from infectious molecular clones of HIV-1 disturbs the stoichiometric relationship of the primers to the virus template binding sites. Some of the primers provided in the TRUGENE HIV-1 Genotyping Kit are degenerate; thus, if the ratio of primer with appropriate binding sites to template is too low, the sample will fail to amplify (at the RT-PCR stage) or CLIP (at the sequencing stage). Alternatively, blood plasma-associated HIV-1 may differ from infectious molecular clones in ways that affect sedimentation during centrifugation, release of viral RNA during lysis, binding of viral RNA to activated silica during extraction, and extent of RNA fragmentation, all of which would affect the yield of RNA templates available for analysis.

Comparison of assay results from samples collected into EDTA- or ACD-containing VACUTAINER tubes suggests that either anticoagulant is suitable for plasma collection. As expected, samples anticoagulated with heparin gave negative results, most likely due to interference of heparin with the reverse transcription and amplification of HIV-1 RNA (11) Previous studies have shown that quantitative yields of plasma HIV-1 RNA are greater when EDTA is used as the anticoagulant compared to ACD, particularly if plasma is not separated from blood for more than 6 h after collection (6, 12). No significant difference in results was observed in the present study between samples anticoagulated with EDTA or ACD. Nevertheless, careful attention should be given to prompt specimen processing after blood collection in order to optimize assay results.

Despite the excellent level of agreement between the test samples and reference sequences overall, mutation recognition agreement rates varied from 0.27 to 1.00. By contrast, wild-type recognition agreement rates were usually >=0.9. Because the number of mutations present in any sample was considerably smaller than the number of wild-type codons, small changes in the number of mutations identified in the test sample could result in large changes in the mutation agreement rate. A better measure of the sensitivity of the assay to detect presence of mutations comes from experiments that tested the ability to detect specific mutations introduced into molecular clones of HIV-1 by site directed mutagenesis. In those experiments in which the ability to detect an individual mutant allele was tested 9 or 10 times, overall sensitivity was 97.5%.

The results of several assays in our study were invalidated due to the presence of a contaminating HIV-1 sequence. Sequence contamination was detected using the Genetic Fingerprint function of the OpenGene software. This software function compares the pattern of polymorphisms and mutations of a sequence to all other sequences in the software library's database and looks for identity between samples. Sequences showing identity with another sequence in the database (other than those from the same patient) suggest the possibility of contamination and should be investigated prior to reporting the result. The occurrence of sequence contamination in this study is a reminder of the need to take stringent precautions against contamination when performing PCR-based tests. Phylogenetic analysis of HIV-1 sequences provides a helpful way to screen for contamination (C. Kuiken and B. Korber [http://www.hiv.lanl.gov/seq-db.html]).

In summary, the TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System have robust performance characteristics and are capable of reliably and reproducibly detecting the mutations in the PR and RT regions of HIV-1 that confer resistance to currently available antiretroviral agents. Reports of the high prevalence of drug-resistant HIV-1 among patients under care for HIV/AIDS in the United States and the increasing transmission of drug-resistant virus among newly infected individuals heighten the need for resistance testing as part of the clinical care of infected individuals (15; D. D. Richman, S. Bozzette, S. Morton, S. Chien, T. Wrin, K. Dawson, and N. Hellmann, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. LB-17, 2001). Results of the studies reported here demonstrate that the TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System are well-suited for this purpose.


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ACKNOWLEDGMENTS
 
This work was funded by Visible Genetics, Inc., and received partial support from the Virology Advanced Technology Laboratory contract of the Adult AIDS Clinical Trials Group (to D.R.K.) (NIH grant U01 AI-38858).

The assistance of John Baxter and Mark Holodniy in obtaining plasma samples is gratefully acknowledged. We also thank Kristin Doherty for expert editorial assistance.


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FOOTNOTES
 
* Corresponding author. Present address: Partners AIDS Research Center, Brigham and Women’s Hospital, 65 Landsdowne St., Rm. 449, Cambridge, MA 02139. Phone: (617) 768-8371. Fax: (617) 768-8378. E-mail: dkuritzkes{at}partners.org. Back


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Journal of Clinical Microbiology, April 2003, p. 1594-1599, Vol. 41, No. 4
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.4.1594-1599.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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