INTRODUCTION

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Diagnosis of human immunodeficiency virus (HIV) infection is commonly based on the detection of antibodies to HIV, but seroconversion, i.e., the appearance of specific anti-HIV antibodies, usually occurs 3 to 8 weeks after the infectious contact and 5 to 10 days after the onset of symptoms associated with early infection (4, 9, 12, 19). The window period between infection and seropositivity can be shortened by testing plasma or sera for the presence of HIV p24 antigen (4, 6, 9) and/or HIV type 1 (HIV-1) RNA (4, 8, 13). The presence of HIV-1 RNA in plasma is a more sensitive marker than the presence of p24 antigen in plasma (4, 8), and several commercial kits are now available for the detection of HIV-1 RNA, including quantitative PCR (Amplicor), nucleic acid sequence-based amplification, and branched DNA signal amplification (Quantiplex) (21). However, although these techniques are routinely used for the determination of viremia in HIV-1-infected individuals, they cannot be used for systematic screening for HIV-1 infection due to their high costs and labor-intensive nature. Assays for HIV-1 RNA with pooled sera instead of individual sera, as performed previously for HIV-1 antibody testing (15, 25), would be less expensive and time-consuming but would carry the risk of reduced analytical sensitivity. We recently developed a boosted version of the Amplicor assay with a lower detection limit of 20 HIV-1 RNA copies per ml (23). The increase in sensitivity was achieved by introducing a high-speed centrifugation step prior to purification of viral RNA. In this assay, standard high-speed centrifuges restrict the input volume of samples to 1.5 ml.

For the present investigation, which was aimed at detecting antibody-negative HIV-1 RNA-positive samples among sera sent to microbiological laboratories, we developed two modified formats of the Amplicor assay for the analysis of pooled sera. The first was designed to allow low-speed centrifugation of large serum pools by using a polyethylene glycol (PEG) precipitation step prior to viral purification. The second was based on high-speed centrifugation of smaller input volumes.

	MATERIALS AND METHODS

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Collection of specimens. Between November 1996 and March 1997 a total of 10,692 individual serum samples were collected at five centers including four university hospital laboratories (Basel, Bern, Lausanne, and Geneva, Switzerland) and one private laboratory (Bio-Analytique Institute [BAI], Geneva). The commercial kits used for the detection of anti-HIV antibodies were the following: HIV1/2 AxSYM, (Abbott, Delkenheim, Germany) (Basel, Bern, BAI, and Geneva), VIDAS (BioMérieux, Marcy-l'Etoile, France) (Basel and Bern), GENSCREEN HIV1/2 (Sanofi Pasteur, Marnes la Coquette, France) (Basel, Lausanne, and Geneva), Cobas core anti-HIV1/HIV2 EIA DAGS (Roche, Basel, Switzerland) (Lausanne), and MUREX HIV1/2 ICE 1.0.2 (MUREX, Dartford, England) (BAI). All of the centers except BAI used two different screening tests for each serum sample; BAI used only one, either the MUREX assay or the Abbott assay. Pools were prepared daily by mixing a maximum of 70 individual HIV-1 antibody-negative serum samples (200 µl of each sample), stored at 75°C, and sent on dry ice once a week to the Geneva Laboratory of Virology. Lists of the samples included in the pools were recorded on the daily protocol used to perform antibody screening. Individual serum samples were stored at 20°C according to the standard procedure in use in each of the participating laboratories and were available for further analysis on request. Assays for HIV-1 p24 antigen in individual serum samples were performed retrospectively with a commercial kit (HIVAG-1 monoclonal; Abbott).

HIV-1 RNA assays. (i) PEG Amplicor assay. The PEG Amplicor assay is based on modifications of the Amplicor HIV-1 Monitor test (Roche) (17, 23) including (i) a PEG precipitation step prior to the extraction of viral RNA in order to accommodate larger input volumes and allow initial centrifugation at low speed (1,500 × g); (ii) an increase in the PCR amplification step from 30 to 40 cycles in order to achieve a higher sensitivity (qualitative assay); and (iii) a modification of reagent volumes in order to reach a final concentration of the internal quantitation standard (IQS) identical to that used in the standard Amplicor HIV-1 Monitor and to increase the concentration of RNA introduced in the PCR. Briefly, the pools of sera from the same participating center prepared daily were thawed, the tubes were centrifuged at 1,500 × g for 10 min to remove fibrin deposits, and half of the pooled volume (corresponding to 100 µl of each serum sample) was used for the detection of HIV-1 RNA. When the pools contained less than 20 serum samples they were mixed with other pools from the same center to yield an input volume of 3 to 7 ml. A 50% commercial PEG solution (Polyethylene glycol 6'000 Solution; Fluka Chemie AG, Buchs, Switzerland) and a 3 M NaCl solution (final concentration of PEG, 3%; final concentration of NaCl, 0.15 M) were added, and the tubes (15-ml Falcon tube; Becton Dickinson, Franklin Lakes, N.J.) were placed on ice for 30 min and spun at 1,500 × g for 30 min at 4°C. The resulting pellet, corresponding to about 100 µl, was mixed with 1,650 µl of Working Lysis Reagent (containing the IQS) and kept at room temperature for 10 min, and then 1,750 µl of isopropanol was added. The tubes were centrifuged at 1,500 × g for 15 min at room temperature, and the pellet was washed with 1 ml of 70% ethanol and then transferred to a 1.5-ml tube and spun at 12,500 × g for 5 min with a microcentrifuge. The ethanol was then removed completely, and the pellet was resuspended in 100 µl of Specimen Diluent. For the PCR, 50 µl of the processed specimen was mixed with 50 µl Master Mix and was amplified according to the manufacturer's instructions with a Perkin-Elmer 9600 thermal cycler (Roche, Branchburg, N.J.) with 36 cycles of 10 s at 95°C, 10 s at 60°C, and 10 s at 72°C instead of 26 cycles (40 total cycles instead of 30 total cycles). The detection of amplified sequences was performed according to the manufacturer's instructions, except that amplified samples were diluted only 1/1, 1/5, and 1/25 for the detection of HIV-1 RNA and 1/5 for the detection of the IQS. This allowed the inclusion of two samples per slot and thus a reduction in the cost. Pools were considered positive when the optical density for HIV-1 was >0.2 and when the OD for the IQS was >0.3. When HIV-1 RNA was detected, the pools of sera prepared daily were analyzed separately, and positive pools were split for further analysis (they were first split into pools of 10 serum samples, and then the individual serum samples were tested).

(ii) Centrifugation Amplicor assay. The centrifugation Amplicor assay is based on modifications of the Amplicor HIV-1 Monitor test and was performed with pools of 1.5 ml containing 59 HIV-negative serum samples and either 25 µl of an individual serum sample with a known HIV-1 RNA copy number or 25 µl of one serum sample from a seroconverting patient. Pools were centrifuged at 27,000 rpm (50,000 × g) for 80 min at 4°C with a Biofuge 28 RS centrifuge with a 3740 rotor with 12 positions (Heraeus AG, Osterode, Germany). The pellet was resuspended in 518 µl of Lysis Reagent and 82 µl of Working Lysis Reagent, and the mixture was then incubated for 10 min at room temperature. HIV-1 RNA was precipitated with 620 µl of isopropanol, spun at 12,500 × g for 15 min, washed with 1 ml of 70% ethanol, and spun again at 12,500 × g for 5 min. The ethanol was removed completely with a disposable transfer pipette, and the tubes were left open for 15 min under a closed box to remove traces of ethanol. The box was then treated with UV light overnight. The pellet was suspended in 55 µl of Specimen Diluent, and the processed specimen was treated as recommended by the manufacturer. Pools were considered positive when the OD for HIV-1 was >0.2 and when the OD for the IQS was >0.3.

(iii) Amplicor assay. The standard Amplicor HIV-1 Monitor test (Roche) was performed with individual serum samples according to the manufacturer's instructions.

	RESULTS

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PEG Amplicor assay. We first determined the optimal concentrations of PEG and NaCl required to achieve maximal recovery and ease of resuspension of the pellet and found that the addition of final concentrations of 3% PEG and 0.15 M NaCl was a good compromise (data not shown). A major goal was to achieve a degree of analytical sensitivity for individual samples within pools similar to that achieved by the Amplicor assay for individual samples tested separately. Table 1 reports the results for three series of pools of 6 ml each tested in quintuplicate and containing serum with known numbers of copies of HIV-1 RNA from the same infected individual. HIV-1 RNA was detected down to an input of 50 copies per pool, corresponding to a detection limit of 5 to 10 copies/ml. Because the pools contained only 0.1 ml of each serum sample, the detection limit for an individual serum sample within a pool was 500 copies/ml. A similar sensitivity was achieved when the same experiment was repeated twice with pools mixed with HIV-1 RNA containing sera from two other infected individuals (data not shown).

Screening of HIV-1/2 antibody-negative sera. A total of 234 pools containing 10,692 individual serum samples negative for antibodies to HIV-1 and HIV-2 (HIV-1/2 antibody-negative sera) were analyzed for the presence of HIV-1 RNA by the PEG Amplicor assay. Details concerning the number and size of the serum pools are reported in Table 2.

Centrifugation Amplicor assay. Since the PEG Amplicor assay is relatively complex, we developed a second, simpler assay for the detection of HIV-1 RNA in pooled sera. This assay is based on a previously described boosted Amplicor assay characterized by high-speed centrifugation prior to purification of viral RNA (23). The centrifugation Amplicor assay allows centrifugation of pools containing as many as 60 individual serum samples (25 µl of each sample) in 1.5-ml tubes, accommodated by standard high-speed centrifuges (e.g., Heraeus Biofuge 28 RS). The analytical sensitivity of the centrifugation assay is reported in Table 4. Four pools of 1.5 ml each containing 60 individual serum samples, 1 of which was from an HIV-1-infected individual and which had a known HIV-1 RNA copy number, were tested in quintuplicate. The results indicate that HIV-1 RNA was detected in all five pools containing approximately 12.5 copies and in four of five pools containing approximately 6.25 copies. No inhibition of PCR amplification was observed. Thus, the theoretical lower detection limit for individual sera within pools (input volume, 25 µl per serum sample) can be estimated to be 12.5 copies × 40 = 500 copies/ml, where 40 is a correction factor taking in account the 25 µl of volume of each serum sample introduced into the pool. Similar results were obtained when the same experiment was repeated twice with pools mixed with HIV-1 RNA containing either 10 or 5 copies provided by sera from two other HIV-1-infected individuals. HIV-1 RNA was detected in all pools containing 10 copies and in 59% of pools containing 5 copies (data not shown).

	DISCUSSION

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The window period between HIV infection and its detection by laboratory methods poses a challenge for the diagnosis of HIV-1 infection (4, 6, 26, 27). The infectious window period defines the time during which an individual is both infected and infectious prior to seroconversion. While the sensitivities of HIV antibody detection assays have significantly improved since the tests were first licensed in 1985 (2, 4, 5, 10, 28), the infectious window period is still longer for the assays based on the detection of anti-HIV antibodies than for the assays based on the detection of viral nucleic acids. Indeed, the assays based on the detection of HIV-1 RNA by PCR gave positive results 7 to 11 days before the assays based on the detection of anti-HIV antibodies (4). However, PCR for HIV-1 RNA is too expensive and labor-intensive for routine diagnostic screening of single serum specimens. A reduction in cost might be achieved by using an assay with pooled samples, which reduces the amounts of both laboratory materials and the supplies required. The Amplicor HIV-1 Monitor kit for the detection of HIV-1 RNA costs about US$600. The kit allows 24 assays to be performed; i.e., each assay costs the laboratory about US$25. The patient pays about US$50 for an assay for HIV-1 RNA performed with a single serum sample (US$25 for the Amplicor HIV-1 Monitor kit and US$25 for additional costs). Considering a mean pool size of 45 serum samples, the assays with pooled sera described here reduce the cost of screening of a single serum specimen for HIV-1 RNA by a factor of 45, since only 1 assay (instead of 45 assays) needs to be performed. Moreover, the assays with pooled sera can be performed in a time-efficient manner. In 1 working day, using the PEG Amplicor assay, a trained technician can test 15 to 20 pools containing approximately 1,000 individual serum samples, i.e., 20 times more serum samples than can be tested individually.

This study demonstrates that PCR for HIV-1 RNA can be performed with large pools of sera for diagnostic purposes and that the sensitivities of the PEG and centrifugation Amplicor assays for the detection of HIV-1 RNA in individual serum samples within pools are equivalent to the sensitivity of the Amplicor assay for the detection of HIV-1 RNA in single serum samples, i.e., 500 copies/ml. Both assays with pooled sera have a detection limit of between 5 and 10 copies/ml for total HIV-1 RNA in pools (23) (Tables 1 and 4) and thus are 5 to 10 times more sensitive than the recently modified, ultrasensitive Amplicor assays, which have detection limits of 50 copies/ml (18, 23). Of note, a very high analytical sensitivity is important for the detection of suppressed viremia in patients treated with potent antiretroviral drugs but not for the identification of HIV-1 RNA in individuals with primary HIV infection prior to seroconversion. These patients have elevated HIV-1 RNA levels, ranging between 10³ and 10⁸ copies/ml (3, 20).

The assays with pooled sera represent an alternative to additional diagnostic procedures selected to complement diagnoses based on a single antibody test. Thus, in countries such as France and Switzerland, it is recommended that microbiology laboratories screen individual serum samples by two commercial antibody-based assays. However, the assays with pooled sera for the detection of HIV-1 RNA are likely to reduce the window period during which infection can be detected to a greater extent than additional assays for p24 antigen and/or HIV antibodies. One of the temporary limitations of the present pooled serum assays based on the Amplicor assay is their inability to detect some of the non-B HIV-1 subtypes (1), but new PCR primers with a wider range of detection for non-B HIV-1 subtypes are being released by the Amplicor assay manufacturer (16).

According to the locally available equipment and local needs, laboratories may give preference to one of the pooled serum assays described here. The centrifugation Amplicor assay requires a high-speed centrifugation and the pipetting of small volumes (25 µl), and the pool size is limited to 60 serum samples. The PEG Amplicor assay may accommodate larger pools containing up to 80 serum samples, allows for easier pipetting due to the use of larger volumes (100 µl), and does not require additional equipment for centrifugation. Its main drawback is the necessity to transfer the pellet from a large 15-ml tube to a 1.5-ml tube.

Early recognition of primary HIV-1 infection has important implications for treatment, prognosis, prevention of disease dissemination in the community, and the safety of blood and tissue donations (4, 6, 7, 22, 24, 26, 27). The pooled serum assays described here with 10,692 consecutive HIV-1/2 antibody-negative serum samples sent to microbiology laboratories for HIV-1 antibody testing led to the identification of five individuals suffering from primary HIV-1 infection. This corresponds to an average of 1 infected serum sample per 2,138 serum samples. There may be two explanations for this relatively high incidence: first, the higher incidence of HIV-1-infected individuals in a population of people screened for HIV-1 infection compared to the incidence in the general population and, second, the relatively high prevalence of HIV-1 infection in Switzerland compared to that in other Western countries (14). All five individuals and their sexual partners likely involved in the transmission of the infection are receiving potent antiviral combination therapy. They have been informed of their infection status and how to protect their sexual partners. The early identification of subjects with primary HIV infection might contribute to a reduction in the spread of HIV.