A semiquantitative PCR technique for detecting human immunodeficiency virus type 1 (HIV-1) RNA in plasma was compared with quantitative viral culture and ...
JOURNAL OF CLINICAL MICROBIOLOGY, JUly 1994, p. 1669-1673 0095-1 137/94/$04.00+0
Vol. 32, No. 7
Copyright ©C 1994, American Society for Microbiology
Quantification of Human Immunodeficiency Virus in Plasma by RNA PCR, Viral Culture, and p24 Antigen Detection INGRID VAN KERCKHOVEN,* KATRIEN FRANSEN, MARTINE PEETERS, HANS DE BEENHOUWER, PETER PIOT, AND GUIDO VAN DER GROEN Division of Microbiology, Depatiment of Infection and Immulnity, Institlte of Tropical Medicine, Antwerp, Belgium Received 26 October 1993/Returned for modification 4 January 1994/Accepted 7 April 1994
A semiquantitative PCR technique for detecting human immunodeficiency virus type 1 (HIV-1) RNA in plasma was compared with quantitative viral culture and p24 antigen detection in plasma. Ninety-three samples from 20 symptomatic, 10 asymptomatic, and 10 seronegative individuals were tested. For most of the seropositive patients, consecutive samples were examined. Viral RNA was extracted from plasma by the method described by Boom et al. (R. Boom, C. J. A. Sol, M. M. M. Salimans, C. L. Jansen, P. M. E. Wertheim-van Dillen, and J. van der Noordaa, J. Clin. Microbiol. 28:495-503, 1990). The RNA PCR was the most sensitive method (100 and 74% sensitivity for symptomatic and asymptomatic patients, respectively) and produced less divergent results with the consecutive samples from individual patients compared with the other techniques. All samples positive by viral culture or p24 antigen assay were also positive in the RNA PCR. For each of the three assays, the number of positive results obtained correlated with the disease stage. The estimated mean number of HIV-1 RNA copies was significantly higher in symptomatic patients (22,750 copies per ml) than in asymptomatic patients (1,820 copies per ml). It was also higher in samples positive for viral culture than in culture-negative samples. No close co, elation was found between the amount of HIV-1 RNA and the amount of p24 antigen or the titer of infectious virus in plasma or between this titer and the level of p24 antigen. The plasma RNA PCR may be a useful additional marker of disease progression and may be valuable for monitoring the effects of antiviral therapy.
The determination of the host viral burden in human immunodeficiency virus (HIV) infection is fundamental in understanding the natural history of HIV infection, predicting disease progression, and assessing the efficacy of antiviral treatment. Methods for determining viral load include assays measuring viral components (p24 antigen) or infectivity (plasma culture). The prevalence of detectable p24 antigen in the plasma of HIV type 1 (HIV-1) antibody-positive individuals ranged from 8 to 93%, depending upon the patient population, disease stage, and methods employed (2, 3, 12, 21). Moreover, the clinical information obtained by testing for p24 antigenemia has been shown to be limited (10, 22). Plasma viremia determinations are very sensitive, and quantitative plasma cultures have been shown to correlate with disease progression (6, 8, 16, 19, 25). Culturing of virus is expensive, time-consuming, and labor-intensive, and it requires the handling of large volumes of infectious material. PCR has been used to measure the viral load by a quantitative detection of proviral HIV-1 DNA in peripheral blood mononuclear cells (PBMC) (18, 24, 29). However, detecting the proviral form of HIV provides no information on its replication status; HIV RNA has to be measured. HIV-1 RNA has been detected directly in PBMC and plasma by extraction of RNA, reverse transcription, and cDNA PCR (13, 23, 32). In the present study, we compared three techniques to measure the viral load of HIV-1 in plasma: a semiquantitative RNA PCR based on endpoint dilutions of cDNA, quantitative viral culture, and p24 antigen detection.
MATERIALS AND METHODS Samples. A total of 93 plasma samples from 40 patients attending the clinic of the Institute of Tropical Medicine in Antwerp, Belgium, were examined. Twenty patients had AIDS or AIDS-related complex (500 CD4' cells per mm3), and 10 were HIV seronegative. Follow-up samples taken at monthly intervals from 24 of the seropositive patients were tested. Most of the patients came at regular intervals for a medical visit, and a sample was taken each time. A quantitative culture was processed for a limited number of patients (depending on the clinical examination) to decrease the cost. Nine patients yielded two follow-up samples each, I yielded three samples, and 14 patients yielded four samples each. Plasma samples were prepared from EDTA-treated blood, rapidly frozen, and stored at -70°C for several months. Most of the patients were receiving antiretroviral therapy such as zidovudine, alone or in combination with new antiretroviral drugs. RNA PCR. RNA was extracted from 50 p.l of plasma by the method of Boom et al. (5), which is based on the lysing and nuclease-inactivating properties of guanidinium isothiocyanate combined with the nucleic acid-binding properties of diatoms in the presence of this agent. The extracted RNA was suspended in 50 p.1 of Tris-EDTA buffer to which 0.1 p.1 of RNA Guard (32 U/,ul, Pharmacia) and 0.5 pL. of dithiothreitol (0.1 M) had been added. Positive and negative control plasma samples were extracted simultaneously as controls. Reverse transcription was performed by adding 9 p.1 of the 50 p.1 of RNA-buffer solution to 11 p.1 of reverse transcriptase reaction mixture (Moloney murine leukemia virus reverse transcriptase; Life Technologies) at 37°C for 1 h with random primers, as described by De Beenhouwer et al. (7). Five of the 20 pL. of cDNA product was amplified by nested PCR using
* Corresponding author. Mailing address: Institute of Tropical Medicine, Nationalestraat 155, 2000 Antwerp, Belgium. Phone: (32) 03 247 63 32. Fax: (32) 03 247 63 33. 1669
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TABLE 1. Comparison of the estimated mean number of RNA copies per milliliter of plasma with the mean titer of virus in plasma and the p24 antigen level in symptomatic and asymptomatic patients Patient population
RNA PCR
Sensitivitya (%)
Symptomatic Asymptomatic
100 (44/44) 74 (29/39)
Viral culture
p24 antigen
Geometric mean copy number (copies/ml)'
Sensitivitya (%)
Geometric mean titer (TCID/ml).c
Sensitivity' (%)
Mean p24 antigen level (pg/ml)b
22,750d
83 (34/41) 13 (5/39)
74
NDf
63 (22/35) 13 (5/38)
220e 121
1,820
The values in parentheses indicate the number of plasma samples positive of the total number tested. Calculated for all samples positive in the assay. c TCID, tissue culture infective doses. dp < 0.001. e p > 0.05. f ND, not done. a
b
primer pairs in the POL region as described previously (11). The PCR was done in a 50-pI reaction mixture containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 2.5 mM MgCl2, 0.01% gelatin, deoxynucleoside triphosphates (each at 0.2 mM), 1.25 U of Taq DNA polymerase (Perkin Elmer Belgium), and primers (each at 0.4 mM). A first round of 35 amplification cycles was done with the outer primers. With 1 RI of the amplified cDNA, a second round of 25 cycles was carried out with the inner primers. Cycling conditions for both the first and nesting PCR rounds were 1 min at 94°C, 1 min at 50°C, and 1 min at 72°C in a thermal DNA cycler machine (Biozyme). In the last cycle, the extension was prolonged to 7 min. The amplified product was subjected to electrophoresis on agarose gel and was stained with ethidium bromide, after which the DNA was visualized by transillumination at 254 nm. Negative and positive controls were included in each run. Three 10-fold dilutions were analyzed for each of the cDNA products with an initial positive result in the nested PCR system. The number of cDNA copies was estimated from the last dilution in the series that was reactive by PCR, assuming that at least one copy of RNA generated one cDNA sequence. Since the 5 pA (one-fourth of the 20 pA) of cDNA product, used for PCR amplification, corresponds to 2.2 p.1 of the original plasma sample (or one-fourth of the 9 p.1 of RNA-buffer solution contained in the 20 ,ul of cDNA product), and assuming that a positive PCR result was due to the presence of at least one copy of HIV-1 RNA in this 2.2 ,ul of plasma, the estimated minimum number of RNA copies detectable in 1 ml of plasma was 455. To assess the specificity of RNA amplification, PCR was performed with all extracted RNA samples without previous reverse transcription. To determine the reproducibility of the extraction method of Boom et al. and of the PCR, the method of Boom was performed repeatedly with 32 of the 83 samples tested, while PCR was repeated with 31 samples. Plasma culture. Blood was collected into EDTA-containing tubes, and plasma cultures were processed within 3 h. One milliliter of fresh plasma from the asymptomatic patients and decreasing volumes of plasma (1 ml, 200 p.1, 40 p.1, 10 ,ul, 2 p.l, and 0.5 p.1) from symptomatic patients were cocultured with 3 x 106 fresh phytohemagglutinin-stimulated donor PBMC from different donors by the method of Ho et al. (16). Consecutive plasma samples from the same patient were cultured on PBMC from different donors. Briefly, PBMC were cultured in 50-ml culture flasks in a 5% CO2 atmosphere at 37°C. The medium was replaced every 3 to 4 days. At the same time, cultures were monitored for the presence of p24 antigen in the supernatant by a p24 HIV antigen capture test (Innogenetics, Zwijnaarde, Belgium). Cultures were monitored for 5 weeks before a negative result was assigned. The smallest volume of plasma
required to produce a positive culture was taken as the end point, and HIV-1 titers were expressed as 1, 5, 25, 100, 500, or 2,000 tissue culture infective doses per ml of plasma. p24 antigen detection in plasma. Plasma p24 HIV antigen was detected by a commercial antigen capture enzyme-linked immunosorbent assay (Abbott). p24 antigen levels were expressed in picograms per milliliter of plasma. The limit of detection of the assay was 20 pg/ml of plasma. To eliminate the possibility that viral antigens are undetectable because of binding to antibodies, antigen-antibody complexes were dissociated by acid treatment (20) and samples were retested. Statistics. Statistical significance of differences found was tested with the chi-square test or the Student t test. RESULTS The PCR results for the 83 plasma samples from seropositive patients were compared with those obtained by virus isolation from plasma and p24 antigen detection (Table 1). Viral RNA was detected by PCR in all 44 samples (100%) from symptomatic patients, while virus culture and p24 antigen detection yielded positive results in 83% (34/41) and 63% (22/35), respectively. For the asymptomatic patients, RNA PCR had a sensitivity of 74% (29/39), while the other two techniques had a sensitivity of only 13% (5/39 and 5/38). After dissociation of the antigen-antibody complexes, two more samples from asymptomatic patients were positive in the p24 antigen assay (data not shown). This increased the sensitivity of this assay from 13% to 18% in asymptomatic patients. All specimens that were positive for virus culture or p24 antigen (assay) were also positive in the reverse transcriptase PCR. With 8 of the 29 samples initially negative by RNA PCR from asymptomatic patients, three independent Boom extractions as well as two to eight PCRs were done. They yielded about 50% positive and negative results in the different PCR runs and were therefore considered as being at the limit of detection and regarded as positives. All 10 seronegative samples tested in the reverse transcriptase PCR yielded a negative result. The negative controls for the extraction procedure also remained negative in the RNA PCR. All three assays yielded a significantly higher rate (P < 0.001) of positive results for symptomatic patients than for asymptomatic patients (Table 1). The mean HIV RNA copy number was about 13 times higher in the plasma of patients with more advanced disease than in the plasma of asymptomatic patients (P < 0.001) (Table 1, Fig. 1). Considering only virus culture-positive samples, the mean HIV RNA copy number found in symptomatic patients was comparable (P > 0.05) to that found in the
VOL. 32, 1994
HIV QUANTIFICATION IN PLASMA
455000
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0 0 0* 000 *
z
455
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FIG. 1. Relationship between plasma HIV-1 RNA tiiter and clinical stage in 93 samples from patients infected with HIV-1 0.*, minimum RNA copies per milliliter; , geometric mean titer of the positive samples.
asymptomatic counterparts (Table 2). The meani p24 antigen level was only about two times higher in symptonnatic patients and was not significantly different from the meari p24 antigen level in asymptomatic patients (Table 1). The HIV-1 RNA copy number per milliliter oof plasma was significantly higher in samples positive for viral Clalture than in samples negative for viral culture but positive for PCR (Table 2). The differences in viral copy number between p24 antigenpositive specimens and p24 antigen-negative spc-cimens were not significant (Table 2). A minimum of 45,500 RNA copies per ml were found in 62% (24/39) of samples pos,itive for viral culture compared with 8% (3/41) of samples negzative for viral culture (P < 0.001) and in 48% (13/27) of samjples with p24 antigen compared with 22% (10/46) of samples without p24 antigen (P < 0.02). No close correlation could be found between tlhe amount of viral RNA in plasma by PCR and the amount of pi24 antigen or the titer of infectious virus in plasma, or betwee n the titer of infectious virus in plasma and the level of p24 aintigen, when the results of the three assays, obtained for indivi(dual samples, were compared. TABLE 2. Comparison of the geometric mean HIN RNA copy number detected per milliliter of plasma, in sympt omatic and asymptomatic patients, for virus culture- or p24 antigen -positive samples and virus culture- or p24 an tigen -negative samples positive in PCR RNA PCR result (mean viral copy nt imber/ml)
population
Viral culture Positive (n) Negative (n)
p24 antigen Negative (n) Positive (n)
Symptomatic 22,750" (34) 9,100 (7) 22,750"' (22) 18,200 (13) Asymptomatic 18,200" (5) 1,365' (23) 4,550" (5) 1,365' (22) "P < 0.001. P > 0.05. 11 samples, negative in the PCR, were not included.
The number of RNA copies per milliliter in PCR obtained for the consecutive samples from one patient showed less fluctuation than the corresponding titers obtained by the other two techniques. Moreover, for 7 of 22 patients (32%) whose plasma was tested repeatedly at monthly intervals by virus culture, discrepant results were obtained by virus isolation. The RNA PCR yielded discrepant results among the consecutive samples of only 3 of 24 patients (12.5%), all of whom were asymptomatic. HIV-1 Proviral DNA was detected in only 3 (4%) of the 83 plasma samples tested. A 10-' dilution of these samples was negative for HIV DNA. For the samples repeatedly subjected to Boom extraction or PCR, 50% (16/32) and 61% (19/31), respectively, showed an identical level of positivity in the subsequent repetitions, while for the remaining 50% (16/32) and 39% (12/31) of the samples, respectively, a 10-fold difference in the level of positivity was found.
symptomatics
CLINICAL STAGE
Patient
1671
DISCUSSION The RNA PCR, based on endpoint dilutions of cDNA, was a more sensitive method and yielded less divergent results among the consecutive samples from individual patients than virus culture from plasma or p24 antigen assay. All samples positive in one of the latter two techniques were also positive in the RNA PCR. Symptomatic patients, who all had CD4-' counts of
