INTRODUCTION

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The first serological assay for detection of human immunodeficiency virus (HIV) type 1 (HIV-1) was implemented in 1985, 3 years after the recognition of HIV as the causative agent of AIDS (6, 17, 33). In 1986, a second HIV type, HIV-2, was isolated from patients with AIDS in West Africa (11), which led to the implementation of simultaneous screening for HIV-1 and HIV-2 (5, 41). In the following years, continuous improvements of the serological assays, along with donor education and deferral procedures, have greatly reduced the risks of transfusion-acquired HIV infection (16, 25, 39, 46). However, a residual risk that is extremely small still exists and remains a source of public concern over the safety of blood and blood products.

The residual risk of transfusion-acquired infection is attributed primarily to donations during the antibody-negative, preseroconversion window, the period between infection and detectable seroconversion. For HIV-1, that period is about 3 to 4 weeks for the contemporary serological tests (8, 9). Rare cases of HIV transmissions from seronegative blood and organ donors have been reported in the literature (42, 45; F. Coutlee, G. Delage, F. Lamothe, S. Cassol, and F. Decary, Lancet 340:59, 59, 1992, Letter). That residual risk led to investigations into newer technologies for direct detection of the virus to further close the HIV window. Several studies demonstrated the detection of HIV-1 p24 antigen prior to the detection of antibodies (4, 9, 43), which led to the implementation of p24 antigen screening, in addition to antibody screening, in the United States (19).

Direct detection of the virus can also be achieved by nucleic acid amplification techniques, which enable detection of the viral genome. Previous studies reported the detection of HIV RNA approximately 1 week earlier than detection of p24 antigen and HIV DNA and about 2 weeks earlier than antibody detection (9, 28, 31, 32). Here, we report on the performance of a PCR assay for the detection of both HIV-1 and HIV-2 RNAs. This multiplex assay for HIV-1 and HIV-2 (HIV-1/2) RNA detection is designed to detect both HIV-1 group M subtypes and group O. An internal control is incorporated to verify RNA isolation, amplification, and detection for each specimen.

	MATERIALS AND METHODS

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Primers, probes, and RNA transcripts. Two distinct sets of primers and probes were designed for HIV-1 and HIV-2 detection. Both HIV-1- and HIV-2-specific primer and probe sequences were targeted against conserved sequences located in the pol genes of the HIV-1 and HIV-2 genomes, respectively (23). The internal control primer and probe sequences were selected from regions in the pumpkin hydroxypyruvate reductase gene. HIV-1 transcripts (subtypes A to F) were generated from plasmids containing a fragment from the HIV-1 pol gene (provided by Steve Wolinski, Northwestern University, Chicago, Ill.). The plasmids were linearized with the restriction enzymes BamHI (for subtypes A, C, D, and F), HindIII (subtype E), and XbaI (subtype B) (Gibco BRL, Rockville, Md.). The RNAs were transcribed with the Ambion MegaScript T7 transcription kit (Ambion, Inc., Austin, Tex.) to yield a 1,693-base sequence. The HIV-2 transcript, of 1,643 bases, was produced by in vitro transcription of an XbaI-linearized plasmid that contained a fragment from the HIV-2 polymerase gene. The internal control transcript, of 1,242 bases, was obtained by in vitro transcription of a BssSI-linearized plasmid containing a 900-bp fragment of the pumpkin hydroxypyruvate reductase gene (provided by B. R. Andersen, University of Wisconsin-Parkside, Kenosha). All transcript RNA was purified from plasmid DNA by digesting the transcript mixture with RNase-free DNase followed by two phenol-chloroform-isoamyl alcohol extractions and one chloroform-isoamyl alcohol extraction. Unincorporated deoxynucleoside triphosphates were removed by passing the transcript over 5 Prime3 Prime Select Spin (5 Prime-3 Prime Inc., Boulder, Colo.) columns. RNA transcripts were quantitated by digesting 0.8 µg of RNA sample per µl with 100 µg of snake venom phosphodiesterase (Boehringer Mannheim, Indianapolis, Ind.) per ml plus 30 U of calf intestinal alkaline phosphatase (Gibco BRL) per ml in 1× hyperchromicity buffer (8 mM C₄H₆O₄Mg · 4H₂O, 3 mM MgCl₂, 40 mM K₂C₂H₃O₂, 8 mM Tris-acetate [pH 7.5]) at 32°C for 24 h. After digestion, the sample was heated for 10 min at 75°C. The nucleoside concentration was determined by measuring the optical density at 260 nm.

Sample preparation. Samples were processed with a modified Qiagen QIAmp sample preparation kit (Qiagen, Chatsworth, Calif.). For each sample processed, 1 ml of plasma was added to 1 ml of Qiagen AL lysis buffer containing the internal control transcript. A total of 10⁴ copies of internal control transcript was added to each reaction mixture. One hundred twenty-five microliters of Qiagen protease solution was then added to each sample. The samples were heated for 10 min at 70°C. One milliliter of 100% ethanol was added to each sample. Each sample was then transferred to a Qiagen QIAamp spin column on a vacuum manifold to which a vacuum of 20 to 25 in. of Hg was applied. The columns were washed two times with 0.5 ml of Qiagen AW1 buffer, followed by two washes with 0.5 ml of Qiagen AW2 buffer. The columns were transferred to collection tubes, and the tubes were centrifuged at 13,000 × g for 4 min to remove the remaining wash buffer. After transferring the columns to new collection tubes, the nucleic acids were eluted by the addition of 170 µl of RNase-free water to each column and centrifugation at 13,000 × g for 4 min. The extracted RNA was stored frozen for up to 1 week before amplification.

Amplification. The master reagent mixture contained 100 µl of the following at pH 8.5: 100 mM bicine (Calbiochem, San Diego, Calif.), 164 mM K₂C₂H₃O₂, 66 mM KOH, 0.02 mg of acetylated bovine serum albumin (Gibco BRL) per ml, 0.2 mM EDTA, 0.4 mg of NaN₃ per ml, 16% molecular biology-grade glycerol (Gibco BRL), 1,700 µM deoxynucleoside triphosphates (Amersham Pharmacia, Picataway, N.J.), 300 nM HIV-1- and HIV-2-specific forward primers, 400 nM HIV-1- and HIV-2-specific reverse primers, 30 nM (each) HIV-1- and HIV-2-specific probes, 260 nM internal control primers, 18 nM internal control probe, and 11 U of rTth (Roche Molecular, Branchburg, N.J.). Preassembly of a master reagent mixture that includes all the components, including probes, eliminates the need to open the tubes after amplification and reduces potential contamination risk. Fifty microliters of processed specimen and 50 µl of a 14 mM MnCl₂ solution were added to each 100 µl of reaction mixture. Samples were vortexed and placed in a Perkin-Elmer 480 DNA Thermal Cycler (Perkin-Elmer, Foster City, Calif.) and were cycled by using the following parameters: 1 cycle consisting of 30 min at 60°C and 2 min at 94°C, followed by 43 cycles consisting of 30 s at 94°C, 40 s at 60°C, and 30 s at 72°C, followed by 1 cycle consisting of 15 min at 97°C, followed by a 4°C soak for a minimum of 5 min.

HIV capture and detection. After amplification, the tubes were transferred to the Abbott LCx analyzer (Abbott Laboratories, Abbott Park, Ill.) where a signal was generated by automated microparticle enzyme immunoassay detection (15). The HIV-1- and HIV-2-specific reverse primers were labeled with adamantane on the 5' end, yielding adamantane-labeled amplification products (amplicon) following the amplification cycles. The amplicons were hybridized to the carbazole-labeled HIV-1- or HIV-2-specific probes, and the hybrids were then captured by anticarbazole antibody-coated microparticles in the LCx instrument. The labeled amplicon was then fluorometrically detected with an alkaline phosphatase-labeled antiadamantane conjugate which reacts with the substrate 4-methylumbelliferyl phosphate (MUP).

Internal control capture and detection. The internal control probe was labeled with two distinct haptens: carbazole on the 5' end and adamantane on the 3' end. When amplification failed to occur, the internal control probe was captured with the anticarbazole-coated microparticles in the LCx instrument and was detected with a beta-galactosidase-antiadamantane conjugate which reacts with the substrate 4-methylumbelliferyl galactopyranoside (MUG) to produce a fluorescent molecule. When amplification of the internal control target did occur, the probe was degraded by the 5' exonuclease activity of the recombinant Tth (rTth), which separates the capture and detection haptens, leading to a decrease in signal indicating that amplification had occurred. After the detection step, amplified products are chemically destroyed by automated addition of copper(II) phenanthroline and hydrogen peroxide to prevent contamination (1, 22).

Data analysis. Samples were evaluated for amplification efficiency and HIV positivity. The internal control signal was used to evaluate amplification efficiency, with a decrease in the MUG signal of 70% or greater from the background MUG signal being indicative of efficient amplification (2). The MUP signal generated indicated amplification of HIV sequences present in the sample, with a positive sample defined as having a signal/cutoff ratio (S/CO) greater than 1.

	RESULTS

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Sensitivity. The analytical sensitivity of the assay for HIV-1 detection was assessed by using quantitated HIV-1 virion panels. In vitro-cultured HIV-1 virions were quantified by using the Viral Quality Assurance (Viral Quality Assurance Laboratory, Rush Presbyterian-St Luke, Chicago, Ill.) standards and were further diluted in anti-HIV-1 and anti-HIV-2 antibody-negative and HIV-1 PCR-negative plasma to the concentrations shown in Fig. 1A. One milliliter of each panel member along with an internal control was processed by using the modified Qiagen system described in the Materials and Methods section. Each panel member was tested in replicates of six. One-third of the processed samples (equivalent to about 0.3 ml of plasma) was amplified and was then detected in the LCx analyzer as described above. As few as 20 copies per ml, which is equivalent to 6 copies per reaction mixture, were detectable. The mean S/CO for the six replicates at this level was 1.5, and five of the six replicates were positive, i.e., S/CO of >1.0. For the panel member with 50 copies per ml, which is equivalent to 15 copies per reaction mixture, all the replicates were positive, with a mean S/CO of 2.8 (Fig. 1A).

View larger version (33K): [in this window] [in a new window]   FIG. 1.   (A) Analytical sensitivity of the LCx HIV-1/2 qualitative RNA assay with virion panels. HIV-1 virion panels quantitated against viral quality assurance standards were diluted in negative human plasma to the indicated concentrations. One milliliter of each sample was processed as described in Materials and Methods, and one-third of each of the processed samples (0.3 ml plasma equivalent) was tested. Six replicates of each concentration were tested. Percent positive samples are indicated above the bars. An S/CO of >1 is considered a positive result. (B) Analytical sensitivity of the LCx HIV-1/2 qualitative RNA assay with transcript panels. HIV-1 and HIV-2 transcripts quantitated by hyperchromicity were tested with the indicated number of copies per reaction mixture as described in the Materials and Methods section. Six replicates of each concentration were tested. Percent positive samples are indicated above the bars. An S/CO of >1 is considered a positive result.

Detection of HIV seroconversion samples. Twenty-five seroconversion panels (Boston Biomedica Inc., Boston, Mass., and Bioclinical Partners Inc., Franklin, Mass.) consisting of 151 total members were tested by the qualitative assay for detection of HIV-1 and HIV-2 RNA (HIV-1/2 qualitative RNA assay) to determine the earliest date of detection. Due to the limited quantities of the samples, 0.2 ml of each panel member was processed through Qiagen columns and was eluted with 120 µl of water, and 50 µl of the eluate was tested per reaction mixture. This is equivalent to 0.083 ml of plasma sample, in contrast to the 0.3 ml of plasma equivalent that was used in the experiment described above. Table 1 lists the commercial seroconversion panels tested and the bleed day at which the individual became positive by the HIV-1/2 qualitative RNA assay, a p24 antigen detection assay (HIV AG-1 monoclonal EIA; Abbott Laboratories), and an HIV-1/2 antibody detection assay (HIV1/HIV2 third-generation antibody test; Abbott Laboratories). In general, RNA detection preceded antigen detection, which in turn preceded antibody detection. For 22 of 25 panels, detection of HIV infection by the RNA test preceded detection by the antigen test, and for 24 of 25 panels, RNA detection preceded antibody detection.

Subtype detection. Detection of HIV-1 subtypes by the HIV-1/2 qualitative RNA assay was assessed with samples from HIV-positive patients from Uganda, HIV isolates from cultures, and transcript RNAs of HIV-1 isolates of different subtypes. Twenty-one samples were obtained from HIV-positive patients from Uganda, with the samples containing subtype A (n = 8), subtype C (n = 3), and subtype D (n = 10) HIV-1 isolates. Each sample was processed, and 0.25 ml of plasma equivalent of each sample was tested by the HIV-1/2 qualitative RNA assay. They were all positive, with S/COs ranging from 5.6 to 7.7, all well above the cutoff value of 1.0 (Table 4).

View larger version (33K): [in this window] [in a new window]   FIG. 2.   Sensitivity of the LCx HIV-1/2 qualitative RNA assay with HIV-1 subtype A to F clones. Cloned fragments containing a region of the pol genes from HIV-1 subtypes A to F were transcribed in vitro. The transcripts were quantitated by hyperchromicity, diluted to the indicated concentrations, and tested by the HIV-1/2 qualitative RNA assay. An S/CO of >1 is considered a positive result. rxn, reaction.

Specificity and inhibition rate. Seven hundred fifty-one HIV-negative samples were tested by the HIV-1- and HIV-2-specific assay to assess the specificity of the assay. Three samples tested positive initially, resulting in an initial false-positive result frequency of 0.4% and an assay specificity of 99.6%. These samples were retested in duplicate and each was negative upon repeat testing, resulting in a confirmed false-positive result frequency of 0%.

	DISCUSSION

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The value of amplification-based assays in improving the safety of the blood supply will depend on the extent to which these assays can identify HIV-positive units of blood that escape detection by current serological screening methods due to the window period between infection and the time at which current assays can detect the infection. We demonstrate in this report that the multiplex HIV-1/2 qualitative RNA assay detects HIV RNA before the p24 antigen test and the antibody detection tests do for a majority of samples in the seroconversion panels tested. Analysis of well-characterized panels with frequently obtained samples from the same individual suggests that the PCR assay detects HIV RNA approximately 6 days before the p24 antigen detection test does and 11 days before antibody detection assays do. These results are consistent with previous reports in which the reduction in the preseroconversion window period was estimated with a mathematical model. In that analysis, viral RNA detection preceded p24 antigen detection by 5 days and seroconversion by 11 days (9).

The ability of nucleic acid-based amplification assays to further reduce the window period is due to the high degrees of sensitivity of such assays. Analysis of seroconversion panels in this study indicated that a viral load greater than 80,000 copies per ml was required for p24 antigen detection. In contrast, less than 100 copies of RNA per ml was sufficient for detection of the viral genome by the RNA detection assay. In the multiplex HIV-1/2 qualitative RNA assay, HIV-1 can be detected 100% of the time when it is present at 50 copies per ml and 83% of the time when it is present at 20 copies per ml. While similar sensitivities for HIV-1 RNA detection have been reported for other amplification-based assays, none have incorporated HIV-2 RNA detection in their assays (30, 37).

HIV-2 has been found predominantly in West Africa and to a lesser extent in Portugal and France. Despite its rare occurrence in the United States, serological screening for HIV-2, in addition to serological screening for HIV-1, has been implemented in an effort to increase the safety of blood and blood products (5). Similarly, once the RNA assays are implemented, the requirement to test for both HIV-1 and HIV-2 RNAs could be imminent. Despite its overall similarities to HIV-1, HIV-2 shares only 60% overall nucleotide homology with HIV-1, even in the most conserved genes, gag and pol (20). Therefore, a distinct set of PCR primers was needed to detect the HIV-2 RNA in the HIV-1/2 qualitative RNA assay. Multiplexing in PCR, that is, detection of several markers by the use of multiple, distinct primer sets, has been shown to be a challenging task (7, 36). Multiple primers tend to cause a loss of sensitivity, mainly due to the formation of primers-dimers and extension products of nonspecifically paired primers. These nonspecific reactions compete for and deplete the limiting reagents. In the multiplex HIV-1/2 qualitative RNA assay described here, these difficulties were overcome by the meticulous choice of primers and cycling conditions, without compromising sensitivity for HIV-1 detection. The multiplex assay for HIV-1 and HIV-2 detection has a sensitivity of 20 to 50 copies per ml for HIV-1, which is similar to the sensitivities of ultrasensitive versions of assays that detect only HIV-1 (22, 30, 37). Sensitivity is also critical for detection of HIV-2, especially since lower HIV-2 RNA levels in plasma have been demonstrated (29, 41). The multiplex HIV-1/2 qualitative RNA assay can detect 10 copies of HIV-2 per reaction mixture, which is equivalent to 34 copies per ml.

The sample processing procedure described above uses about 0.3 ml of plasma equivalent. One milliliter of sample is processed and one-third of the processed sample is used for the multiplex HIV-1/2 qualitative RNA assay. The remaining two-thirds can be used for any other hepatitis or retrovirus detection assays, giving the flexibility of running three distinct assays from a single processed sample. Alternatively, all of the processed sample can be used for the HIV-1/2 qualitative RNA assay to further increase the sensitivity for detection of HIV-1 and HIV-2 by eluting the entire sample from the column in a smaller volume.

The ability of an HIV-1-specific PCR assay to detect diverse subtypes is a critical requirement. Until recently, data on subtype distribution indicated that in Europe and the United States, infections primarily occurred with subtype B. The subtype pattern is changing quite drastically in Europe. Reports from Germany, England, and France on the prevalence of non-subtype B infections indicate that it ranges from 10 to 30% (18, 40). In some selected populations, such as French soldiers deployed overseas, the prevalence of non-subtype B infections is as high as 63% (26). Worldwide, subtype B accounts for only 16% of infections, while subtype C accounts for 36%, subtype A accounts for 23%, subtype D accounts for 13%, and subtype E accounts for 7%. Group O, which was originally found in Cameroon and Gabon (13, 27), has now been identified in the United States and Europe as well (34).

All the nucleic acid detection assays developed so far have been based primarily on subtype B strains from North America and Europe. The sensitivities of these tests for detection of divergent strains were evaluated in several studies (3, 14, 22, 35, 44). Evaluation of the HIV-Monitor, version 1.0, and the nucleic acid sequence-based amplification (NASBA) assays revealed that 56 and 44% of subtype A-containing samples tested were negative by these assays, respectively (3). This poor sensitivity was likely due to the number of mismatches in the selected primers. It was reported that a decrease in amplification efficiency occurs with five to six mutations in the primer-binding region (10, 24), and this amount of divergence occurs most frequently with subtypes A and E. Similar results were reported when samples from Uganda, where subtypes A and D predominate, were tested with the Amplicor HIV-1 DNA test; the sensitivity for the samples from Uganda was 74% (22). Replacement of the primer-probe set selected from the gag gene with a primer-probe set from the better conserved pol gene increased the sensitivity of the DNA detection assay to 98% (22). Similarly, Respess et al. (35) reported 100% sensitivity for detection of subtypes A and E with pol-based primers but only 87 and 67% sensitivities, respectively, when gag-based primers were used. Moreover, with gag-based primers, none of the five group O isolates were detected, while with the pol-based primers all five were detected (35). It has recently been reported that detection of subtypes A and E was significantly improved with the Monitor, version 1.5, assay, which uses primers different from the primers used for version 1.0 of the assay (44). Group O-containing specimens were still not detectable with the Monitor, version 1.5, assay. The branched DNA assay, which uses 49 specific probes, has been shown to be more robust to the diversity of different subtypes of the M group (14).

For the multiplex HIV-1/2 qualitative RNA assay described in this report, the HIV-1-specific primers and probe were chosen from the highly conserved pol region of the genome after an analysis of 29 available pol sequences from the database which included the pol sequences of group M subtypes A, B, and D and group O isolates (23). The probe and the reverse primer had no mismatches or a single base mismatch with the sequences of all subtypes including group O. The forward primer had no mismatches or a single mismatch with subtype A, B, and D sequences and four mismatches with group O sequences. Our study with samples from Uganda demonstrates that the HIV-1/2 qualitative RNA assay detects all (100%) isolates of subtypes A, C, and D. Moreover, to ensure that even very low levels of diverse subtypes could be detected, 21 HIV isolates consisting of subtypes A, Thai B, D, E, F, and G from cultures were diluted to 100 copies per reaction mixture; they were detected 100% of the time. Also two group O isolates diluted 1,000- and 10,000-fold were detected by the multiplex HIV-1/2 qualitative RNA assay. To better assess the analytical sensitivity of the HIV-1/2 qualitative RNA assay for the detection of multiple subtypes, one isolate each of subtypes A, B, C, D, E, and F was cloned to generate transcripts. These transcripts were quantitated by an independent and accurate method (12), and all subtypes were positive when they were present at 50 copies per reaction mixture, which is equivalent to 170 copies per ml.

Several levels of specificity are built into the HIV-1/2 qualitative RNA assay: specific primers for amplification of the target, a specific probe to which the amplified target can hybridize, and specific immunoassay reagents for detection of the amplified target-probe complex. In nucleic acid-based tests, false-positive results that affect "assay specificity" are usually caused by contamination with low levels of target sequences combined with the high degrees of sensitivity of the tests and do not necessarily reflect true assay specificity. The system described in this report includes several measures that control for contamination, such as the use of a single reaction vessel, which does not require opening after amplification, and the automatic chemical destruction of the amplified product after detection. These measures were previously shown to be extremely effective (1, 21). Of the 751 HIV-negative samples tested by the HIV-1/2 qualitative RNA assay, three samples tested positive initially, resulting in an initial frequency of false positivity of 0.4%. Upon retesting in duplicate, all three were negative, resulting in a confirmed frequency of false positivity of 0%.

The internal control included in the HIV-1/2 qualitative RNA assay is critical to achieving a high degree of confidence in negative values. The internal control, which is added prior to sample processing, controls for sample recovery, impurities in extracted samples that may inhibit the amplification reaction, and technical errors during the testing process. A negative result for the internal control indicates that the test result may be falsely negative, and such a sample should be retested. A positive result for the internal control validates the test result and ensures that a negative result is truly negative (38). Of the 751 samples tested by the HIV-1/2 qualitative RNA assay, 13 samples were initially inhibited (1.7%); that is, the result for the internal control was negative. Without inclusion of an internal control, these samples would have been called negative and would not have been identified as inhibited.

In conclusion, we have described a qualitative multiplex assay for the detection of HIV-1 and HIV-2 RNAs in plasma. The LCx HIV-1/2 qualitative RNA assay detects HIV RNA in samples prior to p24 antigen detection and before seroconversion and antibody detection. Furthermore, HIV-1 subtypes A, B, C, D, E, and F and group O were detected with high degrees of sensitivity. This assay demonstrated excellent specificity due to effective contamination control measures and the use of highly selective primers and probes. Moreover, the inclusion in each reaction mixture of an internal control that controls for sample inhibition ensures that the negative specimens are truly negative.