Comparative Evaluation of Three Human Immunodeficiency Virus Genotyping Systems: the HIV-GenotypR Method, the HIV PRT GeneChip Assay, and the HIV-1 RT Line Probe Assay

Abstract

Evaluation of drug resistance by human immunodeficiency virus (HIV) genotyping has proven to be useful for the selection of drug combinations with maximum antiretroviral activity. We compared three genotyping methods for identification of mutations known to confer drug resistance in the reverse transcriptase (RT) and protease genes of HIV type 1 (HIV-1). The HIV-GenotypR method (GenotypR; Specialty Laboratories, Inc., Santa Monica, Calif.) with the ABI 377 DNA sequencer (Applied Biosystems Inc.), the HIV PRT GeneChip assay (GeneChip; Affymetrix, Santa Clara, Calif.), and the HIV-1 RT Line Probe Assay (LiPA; Innogenetics, Alpharetta, Ga.) were used to genotype plasma samples from HIV-infected patients attending the University of Wisconsin Hospitals and Clinics and the Mayo Clinic. At the time of analysis, patients were failing combination therapy (n = 18) or were treatment naive (n = 6). Forty codons of the RT and protease genes were analyzed by GenotypR and GeneChip for resistance-associated mutations. LiPA analyzed seven RT codons for mutations. Each sample was genotyped by all three assays, and each assay was subjected to pairwise comparisons. At least 92% of the codons tested (by the three assays) in paired comparisons were concordant. GenotypR and GeneChip demonstrated 96.6% concordance over the 40 codons tested. GenotypR identified slightly more mutations than GeneChip and LiPA; GeneChip identified all primary mutations that corresponded to failing treatment regimens. Each assay identified at least 84% of the mutations identified by the other assays. Mutations that were discordant between the assays mainly comprised secondary mutations and natural polymorphisms. The assays had better concordance for mutations that corresponded to current failing regimens, present in the more predominant viral quasispecies. In the treatment-naive patients, GenotypR, GeneChip, and LiPA mainly identified wild-type virus. Only the LiPA identified K70R, a possible transmitted zidovudine resistance mutation, in the RT gene of a treatment-naive patient. We conclude that although discrepancies in results exist between assays, each assay showed a similar capacity to identify potentially clinically relevant mutations related to patient treatment regimens.

The human immunodeficiency virus (HIV) population in an infected patient includes genetically distinct viral variants called “quasispecies” that evolve from the initial inoculum (8). The propagation of certain novel mutant quasispecies is a function of existing environmental selective pressures created by drug therapy and immunologic responses of the host. Under this specific selection pressure, only viral strains that contain mutations that confer a survival advantage will propagate and emerge as a major quasispecies (U. Dietrich, H. Ruppach, S. Gehring, H. Knechten, M. Knickmann, H. Jager, E. Wolf, R. Husak, C. E. Orfanos, H. D. Brede, H. Rubsamen-Waigmann, and H. von Briesen, Letter, AIDS 11:1532–1533, 1997). HIV genomic mutations have been loosely categorized as primary and secondary mutations by their direct effects on drug susceptibility and the mechanism of resistance (10). Primary mutations generally decrease the level of drug binding to target enzymes and are selected for early as resistance mutations accumulate. They usually confer a severalfold decrease in drug activity and are relatively inhibitor specific. Secondary mutations usually have less discernible effects on viral resistance alone but can contribute to drug resistance by increasing the fitness of viral strains that already contain primary mutations. This compensatory effect of secondary mutations may spread and contribute to resistance to other drugs of the same class. In contrast to (acquired) mutations associated with drug resistance, naturally occurring polymorphisms exist as well (13; G. Myers, B. Korber, B. H. Hahn, K.-T. Jeang, J. W. Mellors, F. E. McCutchan, L. E. Henderson, and G. N. Pavlakis, Human retroviruses and AIDS: a compilation and analysis of nucleic acid and amino acid sequences, [http://hiv-web.lanl.gov/]). Such polymorphic variants apparently represent strains with enough viral fitness or replication capacity to exist in the absence of drug-induced selective pressure. Numerous polymorphisms have been described in the viral protease gene in HIV-infected patients who are treatment naive (11).

Evaluation of the genetic composition of HIV has evolved from a traditional epidemiological tool into an important clinical asset in the management of HIV infection. Genotypic analysis of HIV enables the identification of individual and combinations of nucleotide substitutions that are known to confer resistance to specific antiretroviral agents. Whether a patient has failed treatment with multiple drug combinations or is just beginning drug therapy, genotypic analysis can help identify drugs less likely to be therapeutically effective. This technology can help “individualize” drug combinations to attain maximum virus suppression and patient longevity.

The HIV-GenotypR method (GenotypR; Specialty Laboratories, Inc., Santa Monica, Calif.), the HIV PRT GeneChip assay (GeneChip; Affymetrix, Santa Clara, Calif.), and the HIV-1 RT Line Probe Assay (LiPA; Innogenetics, Alpharetta, Ga.) are three genotyping assays currently in use. Each system uses different methods to identify nucleotide mutations within the viral genome.

The GenotypR uses the ABI 377 DNA sequencing system (Applied Biosystems Inc.), which consists of an electrophoresis and detection unit, reagents, and a computer software program for sequencing analysis. The ABI 377 DNA sequencing system uses a modification of the Sanger dideoxynucleotide chain terminator chemistry, producing DNA amplicons labeled with four different fluorescent dyes, one specific for each nucleotide. DNA samples undergo electrophoretic separation on a polyacrylamide gel, and an argon laser excites the fluorescence-labeled nucleotides. The nucleotides are identified one by one as they migrate past the laser detector. The digital signal that represents the DNA sequencing data is analyzed by specialized software and is displayed as a graphic representation called an “electropherogram” or as a linear string of one-letter nucleotide codes. The single-letter code is then compared to a consensus sequence of wild-type HIV obtained from GenBank to identify the presence of drug resistance mutations at the regions of interest.

GeneChip consists of probe arrays, reagents, a fluidics station, a confocal laser scanner, and computer software. The probe array is a silicon-glass chip that contains over 16,000 unique oligonucleotide probes complementary to the viral reverse transcriptase (RT) and protease genes. The probes are laid out in a precise location or a grid pattern on the array and are available for hybridization with fluorescein-labeled target nucleic acids. Nucleotide and mutation identification is dependent upon the hybridization of labeled viral nucleic acid fragments to these oligonucleotide probes. The probe array is housed in a hollow plastic cartridge, through which RNA product and wash buffers are cycled. On the basis of the patterns of hybridization of the target sequences to the oligonucleotide probes, HIV genomic sequences and point-specific mutations are simultaneously identified.

LiPA consists of nitrocellulose strips that contain immobilized oligonucleotide probes, strip troughs, trough holding trays, and reagents. The LiPA test strip for the RT gene contains 33 oligonucleotide probes immobilized as 20 parallel bands. The probes contain both wild-type and single-base changes of the RT gene known to confer resistance to specific nucleoside RT inhibitors. Like GeneChip, mutation identification is dependent upon the hybridization of viral sequences to these probes; however, LiPA does not perform viral sequencing. It searches only for mutations in a preselected group of RT gene codons. Biotinylated target sequences hybridize to the probes attached to specific locations on the strip and form colored precipitate bands (14). The locations of these colored bands on the strip determine the presence of specific wild-type codons, mixtures of codons, and mutant codons.

We evaluated and compared each system with respect to (i) the concordance of the total nucleotide mutations identified and (ii) the identification of mutations with specific practical relevance to patients' antiretroviral drug programs. We describe further the practical feasibility of the assays, sources of assay data variation, and clinical applications of each assay.

MATERIALS AND METHODS

HIV genotyping assays basically use a two-step procedure: (i) PCR to amplify RNA or DNA fragments to sufficient quantities for mutation detection and (ii) mutation identification via nucleotide sequencing or hybridization of labeled nucleic acid fragments to oligonucleotide probes. Plasma samples with greater than 1,000 copies of viral RNA per ml are generally required for accurate results. Samples with “undetectable” viral loads (<400 copies/ml) are not suitable for genotyping. Accurate genotyping relies on laboratory quality assurance, skilled technique, and expert clinical interpretation of the mutations identified. The interpretation of resistance-associated mutations is dependent upon an up-to-date, expanding genomic database of sequence variations that correlate with phenotypic drug resistance.

Study population and specimen collection.

Twenty-four plasma samples from 22 HIV type 1 (HIV-1)-positive patients (confirmed by enzyme-linked immunosorbent assays and Western blot assays) were collected from August 1998 through March 1999. Patients were recruited with informed consent at the University of Wisconsin Hospitals and Clinics, Madison, Wis. (n = 19), and at the Mayo Clinic, Rochester, Minn. (n = 3). Thirteen of the patients enrolled in the study were failing their first triple-therapy regimen, as defined by a rising “detectable” viral load (>400 copies/ml), despite combination antiretroviral therapy (4). Sixteen patients had received prior drug therapy, including one patient who was off therapy at the time of sample collection. Six patients were treatment naive at the time of sample collection. Plasma from two treatment-naive patients was collected and genotyped a second time after both were failing an initial three-drug regimen. The plasma viral load of each patient was determined by the Roche-Amplicor HIV Monitor Test (12) and ranged from 1,027 to more than 10⁶ copies/ml. Upon collection, each sample was spun and separated into three plasma aliquots (one for each assay) and frozen to −20°C. GenotypR was performed at Specialty Laboratories, Inc., while GeneChip and LiPA were both performed at the Mayo Clinic.

Target preparation.

Viral RNA isolation was conducted by the Qiagen (Qiagen Inc., Valencia, Calif.) extraction protocol at Specialty Laboratories, Inc., and the RNAzol B (Biotech Laboratories, Inc., Houston, Tex.) nucleic acid extraction protocol was conducted at the Mayo Clinic. Target cDNA was generated by reverse transcription-PCR with specific primers from the HIV-1 pol gene, as described by the manufacturers. The cDNA amplicons were then subjected to DNA sequencing or probe hybridization, depending upon the assay used.

GenotypR by DNA sequencing.

For DNA sequencing, amplification of viral RNA was performed with RT and by various nested PCR methods. Briefly, reverse transcription was done in one cycle of 10 min at 25°C, 30 min at 42°C, and 5 min at 99°C with the primer NE1 (5′-CCT ACT AAC TTC TGT ATG TCA TTG ACA GTC CAG CT-3′). The first PCR was done in 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 60 s with primers NE1 and pol A (5′ TTG GTT GCA CTT TAA ATT TTC CCA TTA GTC CTA TT 3′). These primers flank the first 266 amino acids (800 bp) of the HIV polymerase gene. Conditions similar to those used for the first PCR were used for the second PCR with primers pol A and Brev (5′-GGT GAT CCT TTC CAT CC-3′) or primers NE1 and B (5′-GGA TGG AAA GGA TCA CCG-3′). These primers generated fragments of 157 (471 bp) and 113 amino acids (339 bp), respectively. The amplified products were visualized by agarose gel electrophoresis, purified on a Qiagen QIAquick 8 PCR Purification Kit, and stored at −20°C for DNA sequencing.

Sequencing reactions of double-stranded PCR products were performed with the Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Branchburg, N.J.) (ABI Prism dRhodamine Terminator Cycle Sequencing Ready Reaction Kit, kit protocol; PE Applied Biosystems). Briefly, 8 μl of Qiagen kit eluant were mixed with 8 μl of the Terminator Ready reaction mixture and 4 μl of 0.8 pmol of one of the 4 primers, NE1, B, Brev, or pol A, per liter. For the sequencing reactions 25 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min were used. The final product was cleaned in a Qiagen QIAquick 8 Purification Kit, lyophilized, and resuspended in a solution of formamide-blue dextran (6 μl) before loading of the sequencing gel. Automated DNA sequencing was performed in 4% acrylamide gels with the ABI 377 DNA Sequencer (Applied Biosystems Inc.). The DNA sequences obtained from the HIV-infected patient samples were compared to the HIV consensus sequences derived from GenBank sequences of accession numbers MZ7323, M26727, and X70613 and were analyzed by using the clustal alignment format of the Sequence Navigator Software Program (Applied Biosystems Inc.).

GeneChip assay.

In GeneChip, target HIV nucleic acids were converted into fluorescein-labeled, fragmented RNA copies by an in vitro transcription reaction in a mixture that consisted of 5× transcription buffer, 5× HIV PRT labeling mix containing fluorescein-UTP, MgCl₂, dithiothreitol, T3 or T7 RNA polymerase, RNA guard, and 1 to 6.5 μl of the PCR cDNA template. The mixture was incubated at 37°C for 60 min to denature the cDNA and allow bidirectional transcription to occur. The cRNA obtained from this reaction was then fragmented by adding concentrated MgCl₂, and the mixture was incubated at 94°C for 30 min. A hybridization mix was created by combining the cRNA transcripts with 5× SSPE (1× SSPE is 0.18 M NaCl, 10 mM NaH₂PO₄, and 1 mM EDTA [pH 7.7])–Triton X-100 solution and a control oligonucleotide.

The hybridization and chip washings were conducted in the automated, temperature-regulated GeneChip Fluidics Station 400 (Affymetrix). Standard 1.5-ml tubes that contained the cRNA-hybridization mixtures were inserted into the fluidics station along with corresponding PRT 440S (sense) or 440A (antisense) probe arrays. The 440S probe array hybridization occurred for 30 min at 35°C, while the 440A probe array hybridization occurred for 30 min at 30°C. The arrays were then washed with SSPE–0.005% Triton X-100–RNase-free water. The wash program consisted of four cycles of 10 mixes per cycle, which removed nonhybridized RNA fragments from the array.

After completion of the hybridization-wash cycles, the arrays were scanned one at a time in a Hewlett-Packard confocal laser scanner. The probes that best matched the target viral sequences yielded the highest fluorescence intensity when they were exposed to the laser scanner. Specialized software calculated the average signal intensity for each probe cell and identified matching nucleotide base sequences. Since the position and sequence of the specific cells on the array are known, the sequences of the target nucleic acids can be determined with the software. A report sheet that listed all nucleotide substitutions within the RT and protease genes identified with respect to the reference wild-type strain sequence, the codon locations, and subsequent amino acid substitutions was generated.

LiPA system.

LiPA is based on the principle of reverse hybridization. After reverse transcription of HIV RNA, which was done according to the manufacturer's specifications, target viral sequences were amplified with biotinylated nested PCR primers (14). The amplification mixture contained 5 μl of cDNA, 4.5 μl of 10× Taq buffer, 0.3 μl of each deoxynucleoside triphosphate at a concentration of 25 mM, 1 μl (10 pmol) of each biotinylated primers, 38 μl of H₂O, and 0.2 μl (1 U) of Taq (Stratagene, La Jolla, Calif.). The annealing temperature was set at 57°C, extension was at 72°C, and denaturation was at 94°C. Each step was 1 min, the outer PCR was performed for 40 cycles, and the nested reactions were performed for 35 cycles.

After amplification, equal amounts (10 μl) of biotinylated amplification products and a denaturation mixture that contained 0.4 N NaOH and 0.1% sodium dodecyl sulfate were mixed and incubated at room temperature for 5 min. Following this denaturation step, 2 ml of prewarmed hybridization solution containing Tris-HCl, sodium dodecyl sulfate, and standard saline citrate was added to a membrane strip and hybridization was carried out at 39°C for 30 min. The hybridization mixture was then replaced with a stringent wash solution to wash the membrane strips at room temperature and then at 39°C for 10 min. The wash solution was replaced by the streptavidin-alkaline phosphatase conjugate, and after a 30-min incubation at room temperature, the conjugate was rinsed away and was replaced by the substrate nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. After a 30-min incubation at room temperature, the probes became visible as a purple-brown precipitate. The locations of the colored bands on the membrane strips enabled the identification of selected target codons.

RESULTS

Details of the treatment history of each patient with HIV infection and measurements of each patient's viral load at the time of genotyping are presented in Table 1, along with a list of mutations discordantly identified by the three genotyping systems. The analysis of the results is described below.

TABLE 1.

Mutations discordantly identified by GenotypR, GeneChip, and LiPA

Patient	Treatment historya	Viral load (no. of copies/ml)	Mutation identifiedb
			GenotypR	GeneChip	LiPA
201	Naive	470,000		P-L10I
204	Naive	82,710			RT-K70R
204 (II)	D4T, DLV, NLF	127,640			RT-K70R
205	Naive	>1,000,000
205 (II)	3TC, DLV, SQV	3,457
206	Naive	39,420	P-A71A/I	P-V77V/I
207	3TC, ADF, NVP, NLF, AZT, D4T, ddC	6,882	RT-D67N		RT-L74V
208	3TC, D4T, NLF, d4T, ddC, AZT, NVP, ADV	3,940	P-V77I RT-T215Y	RT-T215Y	—
209	ddC, IDV, NVP, 3TC, D4T, AZT, ddI, ADV	1,106–2,125	RT-D67N	P-L10R	RT-K70R
210	3TC, D4T, NFV, AZT, ddC, SQV	∼13,500	P-V77I
			RT-D67N
			RT-T69D	RT-T69D	—
211	3TC, D4T, NFV, NVP	3,387–3,643
217	3TC, ddI, IDV, D4T, AZT, NLF, NVP	∼1,400	—	RT-L74V	RT-L74V
			P-N88S
219	AZT, 3TC, RTV, D4T, SQV	2,149	P-L33F	P-L101
			RT-D67N
			RT-T215F	RT-T215F	—
222	ABA, 3TC, NLF, D4T, ddI, 3TC	15,437
226	3TC, ABA, SQV, AZT, RTV, IDV, D4T, NLF, EFV, ddI, HU	432,100		P-L10I
			RT-M41L	RT-M41L	—
			RT-D67N
			RT-V75I
227	3TC, EFV, IDV, D4T, ddC, ddI, AZT, ADV, RTV, SQV, NLF, NVP, DLV	318,520	P-M36I P-V77I	P-K20R RT-K70R
229	3TC, D4T, ddI, NLF, IDV	30,172	P-A71V RT-M41L
				RT-M41L RT-Y188L RT-L210W	—
232	Off treatment, AZT, 3TC, ddI, D4T, IDV, RTV	21,180
234	Naive	213,280
235	ABA, EFV, SQV, AZT, 3TC, D4T, ddI, ddC, IDV, RTV, NFV	9,800
236	Naive	27,636
237	AZT, 3TC, ABA, NLF, AZT, D4T, ddI, EFV, DLV, RTV, SQV, NLF		P-L90M K219Q	RT-K65R
238	3TC, NVP, NLF, AZT, ddI, ddC, D4T, IDV, RTV, SQV	55,661	P-M36I P-M46M/L RT-D67N RT-M184V	P-L10I P-K20R
				RT-M184V	—
239c	ddI, D4T, NLF	1,027	P-L90L/M

^aBoldface type represents current treatment regimen; all other designations represent previous drug therapies. AZT, zidovudine; ddI, didanosine; ddC, zalcitabine; D4T, stavudine; 3TC, lamivudine; ADV, adefovir; RTV, ritonavir; IDV, indinavir; NLF, nelfinavir; SQV, saquinavir; ABA, abacavir; EFV, efavirenz; NVP, nevirapine; DLV, delavirdine; HU, hydroxyurea. 

^bOnly discordant mutations detected between the three assays are listed. —, mutation detected by two of the three assays. The codons studied were selected by Specialty Laboratories, Inc., to identify drug resistance-associated mutations. HIV genes analyzed: protease gene (P) and RT gene. LiPA searched for 41L, 69D/N/R, 70R, 74V, 184V, and 215Y/F mutations encoded in the RT gene. 

^cInsufficient sample was available for testing by LiPA. 

The performances of HIV genotyping by GenotypR and GeneChip were compared for the viral open reading frames represented in both systems; 40 codons in the RT and protease genes were evaluated. LiPA identified mutations in seven codons within the RT gene alone, six of which represent known drug resistance-associated mutations. All samples were genotyped by all three assays, and the results were subjected to pairwise comparisons. In comparisons that involved LiPA, only the six resistance-associated codons evaluated by LiPA were considered.

A total of 960 codons (40 codons per patient sample × 24 samples) were evaluated by using GenotypR and GeneChip (Table 2). Of these codons, 927 were identified identically (concordant codons) by both methods (96.6% concordance). Of the 33 mutant codons that were not identified identically by both methods (discordant mutant codons) (3.4% discordance), 20 were identified only by GenotypR and 13 were identified only by GeneChip. Of the mutations identified by GenotypR alone, eight corresponded to secondary mutations and natural polymorphisms (RT gene, D67N and K219Q; protease gene, L33F, M36I, V77I, and L90M) associated with patients' current failing drug regimens. The other 12 mutations identified by GenotypR alone correlated with past drug regimens. Among the mutations identified by GeneChip alone, five mutations corresponded to patients' current failing drug regimens (RT gene, K65R and L74V; protease gene, L10I, L10R, and K20R). The L74V mutation in the RT gene, a primary mutation conferring resistance to didanosine, was found in a patient failing a didanosine-based regimen. The other eight mutations identified by GeneChip alone included five mutations related to previous drug regimens and three mutations unrelated to current or past therapies.

TABLE 2.

Examination of 960 codons by GenotypR and GeneChip^a

Test and correspondence of mutation	No. of discordant mutations	Discordant mutations
		RT gene	Pr gene
GenotypR
Current failing therapy	8	D67N, K219Q	L33F, M36I, V77I (n = 2), L90M (n = 2)
Previous therapy	12	D67N (n = 5), V75I, K219Q	M36I, M46M/L, A71A/V, V77I, N88S
GeneChip
Current failing therapy	5	K65R, L74V	L10I, L10R, K20R
Previous therapy	5	K70R, L210W	L10I (n = 2), K20R
Unrelated to therapy	3

^aA total of 927 concordant codons were identified, for 96.6% concordance. A total of 33 discordant mutations were identified, for 3.4% discordance. 

A total of 138 RT codons (6 codons per patient sample × 23 patients) were evaluated by LiPA and compared to the same codons evaluated by GenotypR (Table 3) and GeneChip (Table 4). A total of 127 codons were identified identically by LiPA and GenotypR and also by LiPA and the GeneChip (92% concordance for both pairs). Among the mutations confirmed by both LiPA and GeneChip, GenotypR failed to identify the L74V mutation in the RT gene for a patient failing a didanosine-based regimen. Among the mutations confirmed by both GenotypR and GeneChip, LiPA failed to identify the primary mutations T215F and M184V in the RT gene for two patients failing current drug regimens that containing zidovudine and lamivudine, respectively. All mutations confirmed by both GenotypR and LiPA were also identified by GeneChip. Two mutations identified by LiPA alone, L74V and R70K in the RT gene, were associated with patients' previous drug regimens. The 11 discordant mutant codons (8.0% discordance) between LiPA and GenotypR represented six mutations identified only by GenotypR and five mutations identified only by LiPA (Table 3). Similarly, the 11 discordant mutant codons between the LiPA and the GeneChip represented seven mutations identified only by GeneChip and four mutations identified only by LiPA (Table 4).

TABLE 3.

Examination of 138 codons by GenotypR and LiPA^a

Test and correspondence of mutation	No. of discordant mutations	Discordant mutation(s) in RT gene:
GenotypR
Current failing therapy	2	M184V, T215F
Previous therapy	3	M41L, T69D, T215Y
Unrelated to therapy	1
LiPA
Current failing therapy	1	L74V
Patients' previous therapy	2	K70R, L74V
Unrelated to therapy	2

^aA total of 127 concordant codons were identified, for 92.0% concordance. A total of 11 discordant mutations were identified, for 8.0% discordance. 

TABLE 4.

Examination of 138 codons by GeneChip and LiPA^a

Test and correspondence of mutation	No. of discordant mutations	Discordant mutations in RT gene
GeneChip
Current failing therapy	2	M184V, T215F
Previous therapy	4	M41L, T69D, K70R, T215Y
Unrelated to therapy	1
LiPA
Current failing therapy	0
Previous therapy	2	K70R, L74V
Unrelated to therapy	2

^aA total of 127 concordant codons were identified, for 92.0% concordance. A total of 11 discordant mutations were identified, for 8.0% discordance. 

Because no single assay may be capable of identifying all drug resistance-associated mutations present in a heterogeneous viral population, we reanalyzed the data using the total number of mutations identified by each assay as an independent reference or “gold standard” for comparison (Table 5). By using GenotypR as the reference method, GeneChip identified 85.4% and LiPA identified 85.7% of all mutations identified by GenotypR. Similarly, GeneChip identified 88.1% and LiPA identified 88.9% of the mutations associated with resistance to patients' current treatment programs as identified by GenotypR. GeneChip identified 100% of the primary mutations identified by GenotypR.

TABLE 5.

Pairwise comparisons by use of each assay as a gold standard

Reference method of mutation identification	% Concordance with mutations identified by the corresponding reference method
	Total mutation concordance			Corresponding to patients' current therapy
	GeneChip	LiPA	GenotypR	GeneChip	LiPA	GenotypR
GenotypR	85.4	85.7		88.1a	88.9
GeneChip		84.1	90.0		88.9	92.2a
LiPA (within 6 RT codons)	90.2		87.8	100		94.1

^aThe GeneChip and GenotypR identified 100 and 96.2%, respectively, of the primary mutations seen by the reference method. 

By using GeneChip as the reference method, GenotypR identified 90% and LiPA identified 84.1% of all mutations identified by GeneChip. Similarly, GenotypR identified 92.2% and LiPA identified 88.9% of the mutations associated with resistance to patients' current treatment programs as identified by GeneChip. GenotypR identified 96.2% of the primary mutations identified by GeneChip.

By using LiPA as the reference method, GenotypR identified 87.8% and GeneChip identified 90.2% of all mutations identified by LiPA. Similarly, GenotypR identified 94.1% and GeneChip identified 100% of the mutations associated with resistance to patients' current treatment programs as identified by LiPA. For two patients, mutations K70R and L74V in the RT gene were identified by LiPA alone. Both of these mutations correlated with the patients' previous drug programs and most likely represent minority quasispecies.

For the treatment-naive patients, GenotypR, GeneChip, and LiPA identified mainly wild-type virus. Certain polymorphisms and secondary mutations were identified by GenotypR (A71A/V and V77I in the protease gene) and GeneChip (L10I and V77I in the protease gene). LiPA identified the K70R mutation in the RT gene of one treatment-naive patient, the first mutation to appear with zidovudine treatment and a possible transmitted mutation.

DISCUSSION

The 96.6% concordance of codon identification between GenotypR and GeneChip and the 92% concordance between LiPA and the other two assays lend support to the premise that the three assays are comparable in terms of mutation identification. The few discordant mutations present generally corresponded to secondary mutations and natural polymorphisms with unrecognized clinical significance. The discordant mutations most commonly identified were D67N in the RT gene, which was missed six times by GeneChip, and L10I/R in the protease gene, which was missed five times by GenotypR. Intrinsic assay sensitivity could be a factor, but LiPA could neither confirm nor refute the presence of these mutations (which were not included on the test strip), raising the additional possibility of false-positive calls. These mutations did not appear to significantly alter the drug resistance profiles of the isolates from the patients. GeneChip identified all primary mutations that corresponded to patients' current drug regimens as identified by either GenotypR or LiPA. GenotypR and GeneChip identified slightly more mutations than LiPA (among the six codons evaluated with all three systems). With few exceptions, the rate of discordance between the three assays for the detection of mutations that corresponded to patients' current failing drugs was minimal.

By using each assay as a reference for comparison, at least 84% of the total mutations identified by one assay were confirmed by another assay (Table 5). The <16% rate of discordance among the assays was mainly seen for secondary mutations and polymorphisms of unrecognized clinical significance. Mutations associated with patients' current failing therapy were more consistently identified by each assay, with a resulting >88% concordance. By using GenotypR as the reference method, the percentages of overall mutations identified by GeneChip and LiPA were similar. By using GeneChip as the reference method, GenotypR identified a higher percentage of both total and current treatment-associated mutations than LiPA. In a comparison of GenotypR and GeneChip, each assay identified at least 96% of current treatment-associated primary mutations identified by the other assay. By using LiPA as the reference method, GeneChip identified more mutations than GenotypR.

No genotyping assay is 100% sensitive for the detection of mutations present in all quasispecies, and these results indicate that each assay has the capacity to identify mutations not detected by one or both of the other assays. The number of total codons analyzed by LiPA must be considered within the context of the other assays. The reduced number of total codons analyzed by LiPA enabled each missed mutation to have a more profound impact on the degree of concordance. Nevertheless, when taken together, each system demonstrated a similar capacity to identify clinically relevant mutations associated with contemporaneous drug therapies. Mutations associated with resistance to failing treatment regimens are present in the predominant circulating viral quasispecies (R. W. Shafer and D. Stevenson, Primer on HIV resistance [http://hivdb.stanford.edu]). In this study these predominant mutations were more readily identified among the three assays. Because discrepancies in mutation identification can be found between genotyping assays, ultimate decisions regarding antiretroviral therapy should also incorporate information regarding patient clinical and virologic response to therapy and a thorough ascertainment of treatment history.

A general understanding of fundamental assay technology, protocol feasibility, data interpretation, and sources of data variation all play a role in the selection of the optimal genotyping assay for the clinical microbiology laboratory. Identification of the limitations of each assay is important for the clinician to appropriately interpret and apply test results to patient care (Table 6).

TABLE 6.

Features and limitations of each assay

Feature	GenotypR	GeneChip	LiPA
Time requirement (h)a	7	2.5–3	2–3
Sequencing function	Yes	Yes	No
Ability to detect base pair insertions, deletions	Yes	No	No
System automationb	+	++	+c
Assay sensitivityb	++	++	++d
Data interpretation	Subjective; expert opinion required	Subjective; expert opinion required	Objective; band identification
Required trainingb	++	+	++

^aTime required to obtain nucleotide information for one patient sample after target amplification by PCR. 

^b+, less; ++, more. 

^cAn automated reagent dispenser with aspiration manifold is available with LiPA to simplify and expedite the workload but is not included with the standard assay and poses an additional cost. 

^dLiPA has the potential to detect mutations present in less than 25% of the total circulating viral population (14). 

General observations.

The ability to sequence the entire RT and protease genes enables GenotypR and GeneChip to identify essentially any referenced nucleotide mutation present in the region of interest. GenotypR is the only method at present able to identify insertions, deletions, and frame shifts implicated in resistance to specific drugs. In this study, we used the first-generation LiPA method, which identifies mutations in primary codons that correspond to resistance to four antiretroviral drugs. LiPA strips that identify mutations that correspond to resistance to the nonnucleoside RT inhibitors and protease inhibitors have recently become available. The relevant quality control procedures for these three assays have recently been described (1–3).

Assay feasibility.

The automated hybridization and wash cycles performed by the GeneChip fluidics station along with the automated laser scanner and computer software data analysis allow efficient sample handling and shortened processing time. Laboratory output can be maximized while minimizing sample mishandling. An automated reagent dispenser with an aspiration manifold is available for use with LiPA; however, it is not included with the standard assay and poses an additional cost. Manual performance of LiPA wash and rinse cycles requires additional effort and may require additional training of technicians.

Data interpretation.

The identification of colored precipitate bands on the LiPA strip enables more simplified, objective, and consistent data interpretation. Bands specific for wild-type and mutant codons enable identification of true viral mixtures. Both sequencing with the sequencer from Applied Biosystems Inc. and GeneChip produce a complete list of mutations identified in the RT and protease genes but require additional expert interpretation to define clinically relevant mutations.

Sources of data variation.

There are numerous potential sources of data variation between the three assays. Patient samples contain numerous heterogeneous viral strains variably present as quasispecies. The prevalence of each strain is a product of the specific environmental selective pressures, both immunologic and drug induced (7). Variable levels of some of these viral quasispecies may not be consistently identified by each assay. LiPA may provide enhanced identification of some of these low-prevalence strains and assist in the detection of emerging drug-associated mutations (14). For our patients, discrepancies in mutation identification occurred predominantly among secondary mutations and polymorphisms. The extraction procedures for any assay can be technically demanding for the inexperienced technician and pose an enduring risk of sample contamination. The automated systems used in GeneChip and the optional LiPA reagent dispenser can minimize errors due to sample handling and maximize product output.

Management of HIV infection has advanced considerably during the past few years and has largely benefited from the emergence of new antiretroviral drugs. Although current combinations of nucleoside RT inhibitors, nonnucleoside RT inhibitors, and protease inhibitors can substantially suppress viral replication, the duration of suppression is not indefinite (6). The high replicative and mutagenic rates of HIV, despite the use of combination therapy, remain the primary obstacle to continued successful treatment. Even when successful combination drug therapy suppresses viral loads to undetectable levels, resistance will eventually emerge. Multidrug- and drug class-resistant HIV isolates have been identified in heavily treated patients (13). The risk of drug resistance increases with patient noncompliance and with suboptimal drug programs that contain just one or two drugs. Additionally, HIV-1 variants with multiple drug resistance mutations can be transmitted from the treatment-experienced patient to another individual (9). With the growing widespread use of antiretroviral therapy, the acquisition of drug-resistant HIV is now becoming an additional hazard. The identification of specific viral mutations that confer drug resistance provides clinicians with an increasing opportunity to select combination drug programs with maximum virus suppression potential. For these reasons, the value and utility of HIV genotyping as a clinical tool will continue to increase and an understanding of the technologies and applications of the different systems available will become a necessity for investigators, clinicians, and laboratorians.

Conclusion.

GenotypR, GeneChip, and LiPA are three systems currently in use for HIV genotyping. Each presents a different approach to mutation identification. Overall, the total mutations identified among the codons evaluated were concordant by the three assays. There was a higher concordance rate among the assays when mutations associated with patients' current failing regimens were identified. Most mutations that were not concordantly identified by the assays were secondary mutations and polymorphisms with minimal clinical significance. Among the three assays, GenotypR identified the largest number of mutations and polymorphisms. GeneChip identified all primary mutations associated with patient's current drug regimens, and LiPA was the only one that identified a possible transmitted zidovudine resistance mutation. Mutations associated with failing treatment regimens were occasionally missed by each assay. Nevertheless, for the vast majority of isolates in patient samples genotyped, each assay correctly identified the most relevant mutations associated with patients' current treatment programs. Aside from equipment costs, each system can readily be adapted to most clinical microbiology laboratories and used efficiently by the experienced technician (5).