Detection of human immunodeficiency virus type 1 provirus in mononuclear cells by in situ polymerase chain reaction.

BACKGROUND. Studies of human immunodeficiency virus type 1 (HIV-1) infection have attempted to quantitate the viral load correlate it with the degree of immune deficiency. In one study, only about 1 in 10,000 peripheral-blood mononuclear cells (PBMC) expressed HIV-1, but in other studies, at least 1 in 100 CD4-positive cells was infected and harbored the HIV-1 provirus. METHODS. We developed a new, highly sensitive in situ polymerase-chain-reaction (PCR) method that amplifies selected genetic regions within intact single cells. We used this technique to determine the proportion of PBMC carrying HIV-1 provirus in infected patients in different stages of disease. RESULTS. None of the PBMC from 11 HIV-1--seronegative patients were found to be positive for HIV-1 provirus by the in situ PCR method. In 56 patients infected with HIV-1, the percentage of PBMC with HIV-1 ranged from 0.1 percent to 13.5 percent. The mean percentage of infected mononuclear cells was greater in 13 patients with persistent generalized adenopathy (mean, 6.6 percent) and 19 with the acquired immunodeficiency syndrome (Stages IV-A to IV-C) (4.6 percent) than in 19 patients with asymptomatic HIV-1 infection (0.9 percent) (P less than 0.001). However, in five patients with Kaposi's sarcoma (Stage IV-D), an average of only 1.6 percent of mononuclear cells were infected. CONCLUSIONS. In HIV-1 infection, the proportion of PBMC that are infected appears to be at least 10 times higher than previously described. It is likely that most infected cells contain HIV-1 provirus in a latent or defective form that was not detected in some earlier studies.     

Nested polymerase chain reaction for detection of human immunodeficiency virus type 1 DNA in clinical specimens.

A highly sensitive two-step polymerase chain reaction (PCR) method was evaluated for detection of human immunodeficiency virus type 1 (HIV-1) DNA in clinical specimens. The product resulting from the first amplification reaction is used as the template for the second PCR with an internal (nested) primer pair. Even when starting from a single copy of HIV-1 DNA, the double PCR product was readily detected by direct visualization in ethidium bromide-stained agarose gels. Amplification of minute amounts of HIV-1 DNA was successful in a considerable excess of HIV-1 negative DNA than reported previously. All of 85 HIV-1-infected individuals were PCR-positive with at least two of the three sets of primers used, 252 of 255 amplifications allowing unambiguous visualization of a unique DNA fragment of the expected size. The two-step amplification protocol is simple and rapid and fulfills the requirements of sensitivity and specificity for use in a clinical laboratory.     

Quantitation of human immunodeficiency virus DNA by using the polymerase chain reaction.

The polymerase chain reaction was used to measure the DNA copy number of human immunodeficiency virus (HIV). Differences in polymerase chain reaction amplification efficiency were controlled by amplifying known amounts of HIV DNA in parallel with samples. This technique is a sensitive, accurate, and reproducible method for the quantitation of HIV DNA.     

Detection of human immunodeficiency virus type 1 DNA by polymerase chain reaction amplification and capture hybridization in microtiter wells.

We developed an improved microtiter-based assay for the detection of polymerase chain reaction (PCR)-amplified DNA sequences. The synthetic DNA sequences used to prime the PCR were labeled with biotin at their 5' ends so that the specific PCR product was labeled with biotin. Following amplification, an aliquot of the PCR product was denatured and hybridized to a capture DNA sequence immobilized in a microtiter well. The capture sequence was complementary to a portion of the sequence between the primers, so that only extended primers were captured. The captured PCR product was detected colorimetrically by using a streptavidin-peroxidase conjugate and tetramethylbenzidine substrate.     

Evaluation of infection with human immunodeficiency virus type 1 by using nonisotopic solution hybridization for detection of polymerase chain reaction-amplified proviral DNA.

A convenient assay combining solution hybridization and enzyme immunoassay for DNA-RNA hybrids (polymerase chain reaction-enzyme immunoassay [PCR-EIA]) was developed to detect human immunodeficiency virus type 1 (HIV-1) provirus amplified by the PCR and was compared with oligomer hybridization with 32P-labeled SK19. In PCR-EIA, a fragment of the HIV-1 gag gene from peripheral blood mononuclear cells was first amplified with primer pair SK38/SK39 or O1/O2. PCR-amplified material was reacted in solution with a biotinylated RNA probe. Biotinylated hybrids were measured in a microtiter-plate EIA with antibiotin antibody and a beta-D-galactosidase-conjugated monoclonal antibody to DNA-RNA hybrids. Ten copies of HIV-1 DNA could be detected by PCR-EIA by using two different sets of primers. HIV-1 DNA was detected in 104 of 108 peripheral blood mononuclear cell samples by using SK38/39 and oligomer hybridization, in 104 of 108 samples by using SK38/SK39 and PCR-EIA, and in 104 of 108 samples by using O1/O2 and PCR-EIA. HIV-1 provirus was detected in 107 of 108 samples by using a combination of two sets of primers. One sample from a seropositive patient was negative in all three PCR assays, and six samples gave discordant results between primer pairs. Six of the latter samples scored negative in a PCR for beta-globin but became positive when the sample was diluted before amplification. When applied to clinical samples, PCR-EIA generated results similar to those of an isotopic assay for detection of amplified DNA.     

Detection of polymerase chain reaction-amplified human immunodeficiency virus type 1 proviral DNA with a digoxigenin-labeled RNA probe and an enzyme-linked immunoassay.

An enzyme-linked immunoassay (EIA) combined with a solution hybridization (SH) reaction was devised to detect human immunodeficiency virus type 1 (HIV-1) provirus amplified by the polymerase chain reaction (PCR). In this nonisotopic PCR assay, designated PCR-EIASH, a fragment of the HIV-1 gag gene from peripheral blood mononuclear cells (PBMCs) was first amplified with biotinylated primers. The biotinylated amplified DNA segment was reacted in solution with an internal RNA probe labeled with digoxigenin-11-UTP. Hybrids were captured in a microtiter plate coated with streptavidin. Specific bound hybrids were quantitated by the addition of an enzyme-labeled antibody against digoxigenin and a fluorogenic substrate. The hybridization, immunological, and amplification parameters of PCR-EIASH were optimized as follows: 12.5 pmol of each primer was used in the PCR; the reannealing reaction of amplified products with the RNA probe, which was used at 0.30 microgram/ml, was completed in 30 min at 70 degrees C in 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Five copies of HIV-1 DNA diluted in a lysate of 100,000 PBMCs from a seronegative control could be detected by PCR-EIASH with a signal of 41 +/- 3 fluorescent units above a background noise of 13 +/- 2 fluorescent units. A total of 91 PBMC lysates from 91 seropositive patients sampled once and 20 PBMC lysates from 10 seropositive patients sampled twice were tested in duplicate in the PCR-EIASH; 107 samples were positive in duplicate tests, 1 sample was indeterminate, and 3 samples were negative. Of the latter three samples, one became positive by diluting the cell lysate, suggesting the presence of an inhibitor of Taq polymerase. The three samples negative for HIV-1 by PCR-EIASH were also negative when amplified with SK145-SK39 and detected with 32P-labeled SK102.     

Stability of dried blood spot specimens for detection of human immunodeficiency virus DNA by polymerase chain reaction.

Blood sampling on filter paper has many advantages for the detection of perinatal human immunodeficiency virus (HIV) infection by the polymerase chain reaction (PCR). However, if the method is to be widely used, an assessment of its performance under field conditions is required. To simulate conditions in the field, 50-microliters aliquots of whole blood containing low levels of HIV proviral DNA (4 to 1,024 copies per 100,000 nucleated cells) were spotted onto filter paper; dried; and subjected to heat, humidity, and prolonged storage at room temperature. After exposure, the DNA was recovered and amplified with primers to human leukocyte antigen DQ alpha- and HIV-specific sequences. Treatment at 37 degrees C and 60% humidity for 7 days, storage for 12 weeks at 22 degrees C, and freeze-thawing twice had no adverse effect on PCR reactivity when compared with the results obtained with reference spots stored at -20 degrees C. The lower limits of HIV detection in all tests ranged from 4 to 16 HIV copies per 100,000 cells. Fixation in 70% ethanol improved the amplification of low levels of HIV DNA and reduced biohazard risks. These findings suggest that dried blood spots will provide a powerful new resource for testing for HIV by PCR, especially in remote areas where refrigeration and immediate sample processing are unavailable.     

Use of dried blood spot specimens in the detection of human immunodeficiency virus type 1 by the polymerase chain reaction.

Dried blood spots (DBSs) constitute a potentially valuable source of material for human immunodeficiency virus (HIV) serologic and molecular testing. To facilitate molecular testing, we have adapted the polymerase chain reaction (PCR) to the detection of HIV proviral DNA in DBS samples. The method is highly reproducible, with 75 microliters of whole dried blood providing sufficient DNA for duplicate testing with three primer sets. By using DBS PCR, 66 of 69 (95.6%) seropositive at-risk individuals tested positive by at least two primer sets and 85 of 85 (100%) low-risk seronegative blood donors tested negative by all three sets of primers. The frequency of HIV DNA detection in seronegative at-risk individuals was low, with only 1 of 58 (1.7%) individuals testing positive. These results show that in a clinical environment, HIV PCR analysis of DBS specimens is specific and sensitive. The method is cost effective and presents a useful alternative to the isolation of HIV from seropositive babies with an undefined infection status.     

Quantification of human immunodeficiency virus type 1 proviral DNA by using TaqMan technology

A protocol for quantification of human immunodeficiency virus type 1 (HIV-1) proviral DNA with the TaqMan technology was developed and validated. The assay was specific for HIV-1, with an analytic sensitivity of 10 copies and a linear dynamic range of >6 logs. Viral RNA levels, when at a stable state, were highly correlated with proviral DNA levels in 80 specimens of 18 HIV-infected children.     

PCR-Based assay to quantify human immunodeficiency virus type 1 DNA in peripheral blood mononuclear cells

An assay that quantifies the amount of human immunodeficiency virus type 1 (HIV-1) DNA in peripheral blood mononuclear cells has been developed. PCR amplification of the HIV-1 DNA is performed in the presence of an internal quantitation standard, and colorimetric detection of the amplified product is performed with microwell plates. The copies of HIV-1 DNA are normalized to total genomic DNA input. The assay has an analytical sensitivity of 10 input copies per amplification reaction and a three-log detection range. In an analysis of sequential samples from patients on combination therapy, HIV-1 DNA was quantifiable for all individuals tested, including those with undetectable plasma HIV-1 RNA. In a separate study, a comparison of HIV-1 DNA levels was made with a group of long-term survivors and progressors. The mean HIV-1 DNA levels were lower in the long-term survivors than in the progressors (P, 0.04). The mean HIV-1 RNA levels were also lower, but the difference was not statistically significant (P, 0.164). A quantitative DNA assay will provide an additional tool to gain insight into the natural history of infection and the continued efficacy of potent antiretroviral therapies.     

Quantitative detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA in peripheral blood mononuclear cells by SYBR green real-time PCR technique.

BACKGROUND: The persistence of proviral human immunodeficiency virus type 1 (HIV-1) DNA reservoir represents one of the major drawbacks to the total eradication of HIV-1. The quantitative determination of proviral HIV-1 DNA load offers significant therapeutic information, especially when the HIV-1 RNA levels drop below the detectable limits during the highly active retroviral therapy (HAART) treatment. Moreover, the detection of HIV-1 proviral DNA is an important diagnostic marker in the evaluation of HIV-1 infection of newborns of HIV-1 seropositive women. OBJECTIVE: We evaluated a real-time PCR based on LightCycler technology revealed through SYBR green fluorochrome (SYBR green real-time PCR) to quantify the HIV-1 proviral DNA load in peripheral blood mononuclear cells (PBMC) of HIV-1 seropositive patients. STUDY DESIGN: Firstly, we assessed the SYBR green real-time quantitative PCR for HIV-1 proviral DNA load detection determining the specificity and sensitivity of the assay using the LightCycler system. Secondly, we tested the performance of our SYBR green real-time PCR on 50 HIV-1 seropositive patients under HAART and 20 blood donors. RESULTS/CONCLUSIONS: The results of this study showed that our SYBR green real-time PCR is able to detect five copies of the HIV-1 genome. Moreover, our method revealed HIV-1 proviral DNA in all the 50 HIV-1 seropositive patients ( 627 +/- 1068 HIV-1 proviral DNA copies per 10(6) PBMC, with a range of 30-6300 copies), whereas no positive signal was observed in any PBMC blood donors. Our SYBR green real-time PCR represents a sensitive and useful approach that could be applied both in HIV-1 proviral DNA reservoir determination and in HAART monitoring, particularly when the HIV-1 plasmatic RNA is undetectable.     

Detection of human immunodeficiency virus-1 by polymerase chain reaction and virus cultivation

Peripheral blood of 57 patients with antibodies to human immunodeficiency virus 1 (HIV-1) and of five HIV-1 seronegative subjects at risk for HIV-1 infection were analysed by polymerase chain reaction (PCR) and virus isolation. The virus was recovered from peripheral blood cells in 89% and from plasma in 75% of the HIV-1 seropositive cases. In contrast, proviral HIV-1 DNA was detected in all HIV-1 seropositive patients by dot blot hybridization of the amplified fragments. The intensities of the dot blot reactions were less pronounced in asymptomatic HIV-1 seropositive individuals than in patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC), suggesting an increase in proviral DNA with advancing disease. Three of five seronegative patients with signs or symptoms suggesting HIV-1 infection, but none of the controls, were positive for HIV-1 DNA by one or two primer pairs. These results show a high sensitivity of the PCR for detecting HIV-1 DNA in patients of all stages of HIV-1 infection. Proviral DNA can also be detected in some individuals without detectable antibodies to the virus. The virus load in peripheral blood, as determined by virus cultivation and PCR, seems to increase with progression of the infection.     

Stability of human immunodeficiency virus type 1 proviral DNA in whole-blood samples

The Roche Amplicor HIV-1 Test requires that whole blood be processed within four days of collection. However, this requirement may be too limiting for use in international settings. Thus, we demonstrate that blood may be processed up to 10 days after collection if maintained under ambient conditions (2 to 25 degrees C).     

Diagnosis of human cytomegalovirus-induced retinitis in human immunodeficiency virus type 1-infected subjects by using the polymerase chain reaction.

In 13 of 16 AIDS patients with retinitis, a herpesviruslike infection was diagnosed by clinical investigation. In 12 of the 13 patients, human cytomegalovirus (HCMV) DNA was detected in 5 microliters of aqueous humor by using the polymerase chain reaction (PCR). In the aqueous humor of 12 control patients HCMV DNA could not be detected by PCR. PCR may be used to monitor specific antiviral long-term therapy in HCMV retinitis.     

Detection of human immunodeficiency virus type 1 proviral DNA by PCR using an electrochemiluminescence-tagged probe.

We have developed a rapid, pseudohomogeneous assay for the detection of PCR amplicons, based on the use of electrochemiluminescence generated from a Tris-bipyridine ruthenium(II) label. PCR amplification of highly conserved human immunodeficiency virus type 1 (HIV-1) gag gene sequences was performed with SK38 and SK39 primers, the latter of which was 5' biotinylated. Post-PCR reaction mixtures were combined with 10(12) copies of the SK19 probe-Tris-bipyridine ruthenium(II) conjugate, denatured by heating at 100 degrees C for 5 min, and hybridized at 55 degrees C for an additional 15 min. Hybridization to the biotinylated strand of the amplified DNA was determined by the addition of streptavidin-conjugated magnetic particles and analyzed by using an Origen-1 electrochemiluminescence analyzer. Our results demonstrated a sensitivity of fewer than five copies of HIV-1 (pre-PCR), by using either purified plasmid DNA containing one complete copy of the HIV-1 cDNA genome or lysed, proteinase K-treated 8E5 cells as the starting material. In an evaluation of actual clinical specimens (peripheral blood monocytes from both healthy and HIV-1-infected children), the electrochemiluminescent detection assay correlated 100% with both our standard method (solution hybridization with a radiolabeled probe followed by polyacrylamide gel electrophoresis [PAGE] and autoradiography) and a commercial method (Roche Amplicor). The electrochemiluminescent method was substantially easier to perform than either the PAGE or microtiter plate assays and was considerable faster to perform than either of these alternative formats.     

Standardized nested polymerase chain reaction-based assay for detection of human immunodeficiency virus type 1 DNA in whole blood lysates.

The routine detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA in clinical samples requires a standardized, simple, and sensitive test. To identify the HIV-1 proviral DNA in blood, we used a solid-phase assay based on the affinity capture and the gamma counting of the amplified product after a nested polymerase chain reaction (AMPLICIS test). In order to simplify the general process, whole-blood lysates rather than peripheral blood mononuclear cell lysates were used for the amplifications. The solid-phase capture and counting of the final amplified products allowed us to define precise interpretive criteria to determine the positivity level of the test. Three new primer sets located in the gag and pol structural genes and in the tat regulatory gene of HIV-1 were studied. The results obtained in 54 seropositive and 120 seronegative individuals demonstrated the ability of the AMPLICIS test to be used for HIV-1 provirus detection: 53 of 54 of the seropositive specimens were found to be positive with at least two primer sets. We also assessed the usefulness of this test for the estimation of the HIV-1 DNA load by the end point dilution method with serial dilutions of blood lysates from 26 HIV-1-seropositive patients.     

Detection of vertical transmission of human immunodeficiency virus type 1 by a commercial polymerase chain reaction assay

In a prospective study, a commercial polymerase chain reaction (PCR) system was compared with a conventional procedure, based on PCR and hybridization with a radio-labeled probe, for the detection of human immunodeficiency virus (HIV) infection in 131 blood samples from 80 children born to HIV-seropositive mothers. Twenty-three of these children were HIV infected. The sensitivity and specificity of the commercial assay as compared with the conventional PCR procedure were 100% and 95.1%, respectively. This commercial method simplifies the performance of the conventional PCR technique and can be used to detect HIV type 1 vertical transmission.     

Application of a commercial kit for detection of PCR products to quantification of human immunodeficiency virus type 1 RNA and proviral DNA.

Quantitative tests for human immunodeficiency virus type 1 (HIV-1) RNA in plasma and proviral DNA in peripheral blood mononuclear cells (PBMC) provide valuable information on the status of HIV-1 infection. This paper describes tests that were carried out with commercially available materials and an enzyme-linked immunosorbent assay reader for detecting spectrophotometric changes. Samples consisted of 100 microliters of plasma or 200,000 PBMC. The procedure involved sample preparation, PCR-based amplification with the primer pair SK39 (biotinylated at the 5' end) and SK38, hybridization of the cDNA PCR product to an RNA probe, capture of the RNA-DNA hybrid on a solid phase by means of strepavidin, binding to an alkaline phosphatase-conjugated antibody directed against RNA-DNA hybrids, and incubation with p-nitrophenylphosphate. Spectrophotometric changes were recorded at four intervals over a period of 20 h. The inclusion of HIV-1 RNA or proviral DNA standards in each run was an integral part of the procedure. The dynamic ranges afforded by these assays--500 to 1 million RNA copies per ml and 10 to 5,000 proviral DNA copies per 10(6) PBMC--were applicable to most plasma specimens and to all PBMC specimens from HIV-1-infected patients. Correlations of log-transformed HIV-1 RNA and proviral DNA concentrations with those found by reference methods were, respectively, 0.88 and 0.80. The between-run coefficients of variation for the detection method were < or = 25% (range, 9.1 to 24.7) and < or = 15% (range, 10.9 to 15.1), respectively, for HIV-1 RNA and proviral DNA. The reproducibility of the overall procedure for HIV-1 RNA in plasma (including sample preparation, amplification, and detection) was given by a duplicate standard deviation of log10 copies per ml of 0.11. Thus, the method was sufficiently precise to allow the detection of fourfold changes in plasma HIV-1 RNA concentrations, with a power of 0.95.