Detection of parvovirus B19 DNA in blood: Viruses or DNA remnants?

BACKGROUND: Parvovirus B19 (B19V) DNA can be detected in blood over a long period after acute infection. Several reports associate the presence of B19V DNA with disease, irrespective of timing of the initial B19V infection. OBJECTIVES: This study aims to analyze the properties of B19V DNA in blood, differentiating between bare, non-infectious strands of DNA and B19V DNA in viable virions. STUDY DESIGN: Ten blood donors with asymptomatic acute B19V infection were followed and sampled up to 22 months after infection. The samples were treated with and without an endonuclease and tested for B19V DNA, to distinguish between DNA in virions and naked DNA. RESULTS: In the acute phase of infection, high levels of B19V DNA were detected, concurrent with B19V IgM antibodies. B19V DNA apparently was encapsidated, as indicated by resistance to endonuclease degradation. Subsequently, B19V DNA remained detectable for more than one year in all donors at low levels (<105 IU/mL). Approximately 150days after infection B19V DNA became degradable by an endonuclease, indicating that this concerned naked DNA. In some donors a second endonuclease-resistant peak occurred. DISCUSSION: Detection of B19V DNA in blood by PCR does not necessarily imply that B19V replication takes place and that infectious B19V virions are present. We propose that remnant B19V DNA strands can be released from tissues without active replication. This finding urges to reconsider an assumed role of B19V infection mainly based on B19V DNA detection in blood, a much debated subject in clinical syndromes such as myocarditis and arthritis.

Epidemiology of high-level parvovirus B19 viraemia among Dutch blood donors, 2003-2009.

BACKGROUND AND OBJECTIVES: Plasma derivatives and blood components with low levels of parvovirus B19 (B19) seem not infectious, but recently infected, highly viraemic donors may transmit B19. We studied the incidence of high-level B19 viraemia (B19 DNA>10(6) IU/ml) in 6.5 million Dutch blood donations. MATERIALS AND METHODS: Between 2003 and 2009, all Dutch blood and plasma donations were screened for the presence of B19 DNA, via pools of 480. Reactive pools were resolved and demographic parameters were obtained for all donors with B19 viraemia>10(6) IU/ml. In a subset, IgG and IgM antibodies to B19 were determined. RESULTS: Four hundred and eleven donations (1/15815) were identified with B19 DNA levels above 10(6) IU/ml, predominantly (83%) occurring in donors aged 18-47 years. Each year infection rates were elevated between December and July, with April accounting for 16% of infections. The years 2004 and 2009 were epidemic, with up to 1/4880 highly viraemic donations in May 2004. In a subset of 67 viraemic donations, 47/67 (70%) tested negative for IgG and IgM antibodies to B19; 16/67 (24%) showed isolated IgM and 4/67 (6%) contained IgG and IgM antibodies. The seasonal pattern of asymptomatic B19 infection in blood donors followed the notification rate of clinical cases. Geographically, B19 infection was randomly spread over the Netherlands. CONCLUSIONS: In epidemic seasons, blood donations with high levels of parvovirus, without concurrent antibodies, are common. They may infect immunocompromised and parvovirus-naïve recipients. The feasibility of preventive measures should be studied.

Real-time polymerase chain reaction detection of parvovirus B19 DNA in blood donations using a commercial and an in-house assay.

BACKGROUND: European regulations require testing of manufacturing plasma for parvovirus B19 (B19) DNA to limit the load of this virus to a maximum acceptable level of 10 IU/µL. To meet this requirement, most manufacturers introduced a test algorithm to identify and eliminate high-load donations before making large manufacturing pools of plasma units. Sanquin screens all donations using a commercial assay from Roche and an in-house assay. STUDY DESIGN AND METHODS: Between 2006 and 2009, 6.2 million donations were screened using two different polymerase chain reaction (PCR) assays targeting B19 DNA. Donations with B19 DNA loads of greater than 1 × 10(6) IU/mL showing significant differences in viral load between the two assays were further analyzed by sequencing analysis. RESULTS: A total of 396 donations with B19 DNA loads of greater than 1 × 10(6) IU/mL were identified. Fifteen samples (3.8%) had discordant test results; 10 samples (2.5%) were underquantified by the Roche assay, two samples (0.5%) were underquantified by the in-house assay, and three samples (0.8%) were not detected by the Roche assay. Sequencing analysis revealed mismatches in primer and probe-binding regions. Phylogenetic analysis showed that 12 samples were B19 Genotype 1. The three samples not detected by the Roche assay were B19 Genotype 2. CONCLUSION: This study shows that 3.8% of the viremic B19 DNA-positive donations are not quantified correctly by the Roche or in-house B19 DNA assays. B19 Genotype 1 isolates showing incorrect test results are more common than B19 Genotype 2 or 3 isolates. Newly designed B19 PCR assays for blood screening should preferably have multiplexed formats targeting multiple regions of the B19 genome.

HBsAg-negative mono-infection with hepatitis B virus genotype G.

Infection with a genotype G strain of hepatitis B virus (HBV-G) often occurs as a co-infection with HBV genotype A. In mono-infection with HBV-G, the production of hepatitis B surface antigen (HBsAg), HBe antigen and anti-HBe seems diminished, hampering the serological diagnosis of HBV-G mono-infection. To corroborate this notion, we studied in detail a series of samples of a blood donor with transient HBV-G infection. In this donor, during the temporary presence of HBV DNA and the seroconversion to HBcore antibodies (anti-HBc), no HBsAg or hepatitis B e antigen was detected. During follow-up, no anti-HBe appeared. Multiple resistance mutations to lamivudine were present, demonstrating primary infection with a resistant HBV strain. Cloning and sequencing indicated that no other HBV genotype but genotype G was present. Like other HBV-G isolates, the DNA sequence of the HBsAg a-determinant showed no mutations that could explain the failure to detect HBsAg. Our findings demonstrate that HBV genotype G mono-infection occurs and that routine serology is unsuitable for its detection.

Development of an internally controlled PCR assay for broad range detection of bacteria in platelet concentrates.

A real-time PCR assay based on the 16S rRNA gene was optimized for the detection of a broad range of bacteria in plasma and platelet concentrates (PC). A lambda phage internal control was constructed and implemented in the assay, which made it suitable for diagnostic use. Spiking studies in plasma and PCs were performed to determine the analytical sensitivity of the assay. Thirty three colony forming units (CFU)/ml of E. coli and 72 CFU/ml of Staphylococcus epidermidis could be detected in plasma, and 97 CFU/ml of S. epidermidis in PCs. The assay detected all bacteria relevant for bacterial contamination of PCs. The short turn around time of the assay made it suitable for testing PCs for bacterial contamination prior to transfusion.

Multiple surface antigen mutations in five blood donors with occult hepatitis B virus infection.

Occult hepatitis B virus (HBV) infection is characterized by the presence of HBV DNA while the HBV surface antigen (HBsAg) remains undetectable. The HBV genomes in five asymptomatic blood donors with occult HBV infection and low viremia (<10 to 1,000 HBV DNA copies/mL, genotype D) were studied. An unusually large number of amino acid mutations was present in the immunodominant a-determinant of HBsAg (respectively 3, 6, 7, 10, and 10 mutations). Comparison of the HBV genomes in two donors to a consensus HBV genotype D sequence showed a most prominent hotspot of genetic variation in HBV nucleotides 480-570, encoding the HBsAg a-determinant. The phylogenetic comparison of separate donor HBV genes to the HBV genes of 11 reference strains (genotypes A-H) showed the donor HBV surface genes to form an outgroup, while the HBV polymerase, core and X genes closely cluster with the HBV genotype D reference strain. Maybe the HBV strains in this study represent a natural end-stage of seemingly cleared HBV infection, in which HBV maintains a low level of possibly non-infectious replication, after sacrificing its immunologically offending surface antigen, thus avoiding final clearance by the immune system.

Parvovirus B19 genotypes 1 and 2 detection with real-time polymerase chain reaction assays.

BACKGROUND AND OBJECTIVES: Parvovirus B19 (B19V) DNA screening has been introduced to comply with European regulations for certain plasma products. Current commercial and some in-house B19V DNA assays fail to detect or under-quantify the recently identified genotypes 2 and 3. In this report, we describe 2-year experience with B19V DNA screening using the commercial assay from Roche (detecting only genotype 1) combined with an in-house assay (detecting genotypes 1, 2 and 3). This dual testing approach enables the identification of molecular variants of B19V. MATERIALS AND METHODS: Between 2005 and 2007, approximately 2.6 million plasma donations were screened for B19V DNA loads exceeding 10(6) IU/ml using the Roche and the in-house real-time polymerase chain reaction assay. RESULTS: A total of 232 plasma units were identified with B19V DNA loads above 10(6) IU/ml. Concordant results were observed for the majority of B19V positive samples; however, three of these showed discrepant results between the two assay systems. One was a B19V genotype 2 strain not detected by the Roche assay; another was a B19V genotype 1 strain with a mismatch in the 3'-end of the reverse primer and therefore under-quantified by the Roche assay; and the third one was also a B19V genotype 1 strain that gave an unusual amplification plot in the in-house assay due to a mismatch in the probe-binding site. CONCLUSIONS: New, high viral load, B19V genotypes 2 and 3 infections are rare in blood donors tested by Sanquin. One case was found while testing 2.6 million donations. The prevalence of B19V genotype 1 variants not detected by commercial or in-house assays might be in the same range or even higher than the prevalence of B19V genotype 2 viruses, which remain undetected.

Improved detection of acute parvovirus B19 infection by immunoglobulin M EIA in combination with a novel antigen EIA.

BACKGROUND AND OBJECTIVES: Although parvovirus B19 is a significant blood product contaminant, few methods other than polymerase chain reaction (PCR) have been developed to detect the presence of the virus. MATERIAL AND METHODS: A B19 antigen enzyme immunoassay (EIA) has been developed and the sensitivity of detection is ascertained using dilutions of the B19 capsid protein VP2 and 10-fold dilutions of B19 viraemic serum. Once the assay cut-off was established, a panel of viraemic donations (n = 70) was screened by the antigen EIA. The B19 immunoglobulin M (IgM) and IgG status of these specimens was also determined. During screening of blood donor units by quantitative PCR, 70 individuals were identified with levels of B19 DNA greater than 10(6) IU/ml at the time of blood donation. RESULTS: The sensitivity of the B19 antigen EIA was estimated to be equivalent to between 10(8) and 10(9) IU/ml B19 DNA or 1-10 pg/ml of recombinant capsid protein. B19 detection was significantly enhanced when viraemic specimens were pretreated with a low pH proprietary reagent. Unlike other virus-detection assays, detection of the B19 antigen was not affected by the presence of B19 IgM or IgG antibodies. In addition, the assay was capable of detecting all three genotypes of human erythrovirus. Combined specimen analysis by the B19 antigen assay and a B19 IgM assay facilitated the detection of 91% of acute B19 infections in the test population. CONCLUSION: In combination with B19 IgM detection, application of the B19 antigen EIA is a flexible and efficient method of detecting recent B19 infection and can be used as an alternative to PCR.

No evidence of West Nile virus infection in Dutch blood donors.

BACKGROUND AND OBJECTIVES: West Nile virus (WNV) can be transmitted by transfusion through infected blood components. This study aimed to determine the prevalence of WNV infection among Dutch blood donors to assess whether WNV is a possible threat for the Dutch blood supply. MATERIALS AND METHODS: Plasma samples from 61 992 blood donations were pooled in 7,749 test pools of eight donations using a Tecan robot. These samples were collected between April and October 2004. The pools were tested for the presence of WNV RNA by using the Procleix WNV assay. RESULTS: No WNV RNA-positive pools were detected. Based on Poisson distribution statistics, extrapolation of our data to all the Dutch donations in 2004 revealed that between 0 and 55 cases of WNV infection could be expected. CONCLUSIONS: No evidence of the presence of WNV RNA in Dutch blood donor samples from 2004 was found. However, surveillance of this emerging infection is of importance to safeguard the blood supply in the future because the transmission cycle of WNV is complex and hard to predict.

Evaluation of COBAS AmpliPrep nucleic acid extraction in conjunction with COBAS AmpliScreen HBV DNA, HCV RNA and HIV-1 RNA amplification and detection.

BACKGROUND AND OBJECTIVES: This report describes the evaluation of the COBAS AmpliPrep instrument for fully automated generic nucleic acid extraction in conjunction with hepatitis B virus (HBV) DNA, hepatitis C virus (HCV) RNA, and human immunodeficiency virus (HIV)-1 RNA COBAS AmpliScreen amplification and detection using serial dilutions of the WHO international standards (IS) and the PeliCheck reference panels. MATERIALS AND METHODS: Serial diluted samples of the WHO IS and the PeliCheck reference panels were tested 24 times to determine the HBV DNA, HCV RNA and HIV-1 RNA detection limits by Probit analysis. The existence and extent of cross-contamination were assessed by testing alternating high titre HBV DNA-positive and -negative samples. The specificity of the AmpliPrep-AmpliScreen test for HBV was determined by testing 232 minipools consisting of six donations, all negative for HCV/HIV-1 nucleic acid testing (NAT) and HBsAg. In addition, a HBV genotypes A-G panel was tested. RESULTS: The respective 95% detection limits (and 95% CI) on the WHO IS and on the PeliCheck reference panels were 6.7 (4.3-13) IU/ml and 123 (68-301) gEq/ml for HBV DNA, 23 (11-106) IU/ml and 126 (84-233) gEq/ml for HCV RNA, and 187 (108-422) IU/ml and 183 (108-434) gEq/ml for HIV-1 RNA. Based on the WHO IS and the PeliCheck reference panels, no significant differences in sensitivity for HBV and HCV were found between AmpliPrep and the licensed MultiPrep extraction method. The sensitivity of AmpliPrep-AmpliScreen for HIV-1 was probably twofold lower as compared to the MultiPrep-AmpliScreen method. No cross contamination was observed. All 232 minipools were HBV NAT-negative. The AmpliPrep-AmpliScreen test for HBV detected HBV genotypes A-G with equal sensitivity. CONCLUSIONS: The AmpliPrep instrument combined with the AmpliScreen assays for HBV, HCV and HIV-1 is robust and suitable for NAT donor screening. The sensitivity criteria for HIV-1 and HCV as defined by the Paul Ehrlich Institute and the Food and Drug Administration for minipool NAT screening are met by this system. SINGLE SENTENCE SUMMARY: Generic COBAS AmpliPrep nucleic acid extraction in conjunction with COBAS AmpliScreen detection for HBV, HCV and HIV-1.

Multicenter evaluation of the NucliSens EasyQ HIV-1 v1.1 assay for the quantitative detection of HIV-1 RNA in plasma.

The Nuclisens EasyQ HIV-1 v1.1 assay (Biomerieux) is a real-time detection method combined with NASBA technology designed to measure plasma HIV-RNA. Its performance was assessed in 1008 clinical specimens collected from individuals infected with clade B (774) and non-B (234) HIV-1 variants at four European laboratories. The results were compared with those obtained using three other commercial viral load assays: Cobas Amplicor Monitor HIV-1 v1.5 (Roche), Versant HIV-1 RNA assay (Bayer) and Nuclisens HIV-1 QT (Biomerieux). Overall, the linearity, specificity and reproducibility of the EasyQ assay was comparable with that from the other tests. The correlation coefficient (R) between methodologies was 0.85 for Amplicor; 0.87 for Versant; and 0.91 for Nuclisens. The specificity of the assay was 99.4%. Of note, Versant missed 17% of specimens with non-B subtypes which could be detected by EasyQ, while Amplicor provided similar results than EasyQ. HIV-1 group O specimens were only detected by the EasyQ assay. In conclusion, the performance of the EasyQ assay seems to be similar to that of other HIV-1 viral load tests currently on the market, but it is more sensitive than Versant for HIV-1 non-B subtypes and shows a wider dynamic range than Amplicor. Moreover, as it incorporates the advantage of real-time detection procedures, it facilitates high throughput and short turnaround time.

Diversity and origin of hepatitis B virus in Dutch blood donors.

Two considerations led us to study the genetic diversity and origin of hepatitis B virus (HBV) in Dutch blood donors. Firstly, an HBV-infected Dutch blood donor was found negative by four assays used commonly for detection of HBV surface antigen (HBsAg). How variable is HBsAg among HBV infected blood donors? Secondly, the WHO recommends universal vaccination against HBV, but north-west European countries limit vaccination to groups at risk of HBV. This policy may reduce hepatitis B among low-risk, unvaccinated persons if HBV strains that infect low-risk persons stem from local at-risk groups. Studying the nucleotide sequence of the S-gene of HBV from 63 Dutch blood donors, considerable variation was found. The majority of the donor strains (52/63, 83%) appears closely related to local HBV isolates as present in intravenous drug users, immigrants, and homosexual men. The remaining 11 (17%) HBV strains belong to various non-Western genotypes. This implies that an indigenous Dutch HBV strain (heterosexually transmitted, not associated with intravenous drug abuse, or immigrants) does not exist, and it supports the policy in low endemic countries to limit vaccination to at-risk groups. On the other hand, it must be realised that, after 20 years of vaccination of at-risk groups, HBV still circulates in the at-risk groups and Dutch blood donors acquire the HBV strains involved.

Parvovirus B19 viraemia in Dutch blood donors.

Blood, donated by asymptomatic donors, may contain and transmit parvovirus B19. To investigate the dynamics of parvovirus viraemia in asymptomatic blood donors, we studied the amounts of parvovirus DNA in pools of donor plasma, the prevalence of parvovirus antibodies among blood donors in relation to age, and the seasonal and year-to-year variation of the incidence of parvovirus infection in The Netherlands. The incidence of parvovirus infection follows a seasonal cycle and a cycle of several years. Among Dutch blood donors the incidence was estimated to be 0.56% per year. Forty seven out of 100 pools of 5000 plasma donations tested positive for parvovirus DNA. We inferred that the course of viraemia in asymptomatic donors shows a short peak (> 10(9) copies parvovirus DNA/ml), followed by viraemia below 10(6) copies/ml for about 2 weeks.

Recognition properties of V3-specific antibodies to V3 loop peptides derived from HIV-1 gp120 presented in multiple conformations.

To identify structural constraints and amino acid sequences important for antibody recognition of the third variable domain (V3) of HIV-1 gp120, we have studied the solution conformation of three 35-mer circular V3 loop peptides derived from HIV-1 strains which differ in syncytium- (SI) and non-syncytium-inducing (NSI) capacity. In addition to 2D NMR and CD analyses, fluid- and solid-phase immunoassays were performed using V3-specific antibodies to V3 peptides and gp120 derived from different strains of HIV-1. NMR and CD spectroscopy indicated that circular and linear V3 loops exist in water as a dynamic ensemble of multiple conformations. Amino acid substitutions and biochemical modifications of the V3 loop were found to affect antibody binding depending on the presentation of the antigens. From NMR observations and immunological experiments, we provide evidence for a V3 loop specific monoclonal antibody interaction which is directed predominantly against linear epitopes rather than against discontinuous epitopes. The absence of a single defined solution conformation of 35-mer circular V3 peptides should be taken into account when using V3-related peptides to investigate structural elements in the V3 domain of the gp120 envelope protein of HIV-1 involved in biological processes of the virus.

Mechanism of anti-HIV activity of succinylated human serum albumin.

In the present study, we described the interaction of succinylated human serum albumin (Suc-HSA), a negatively charged anti-HIV-1 active protein, with HIV-1 gp120 and in detail with the third variable domain of gp120 (V3 loop). To this end, different assay formats were tested in which gp120- and V3-related peptides were presented in various configurations in order to investigate the effect of the conformational structure of the V3 loop on the interaction with negatively charged albumins. When gp120 presented via a lectin was used, it was observed that Suc-HSA bound to native gp120. The binding site appeared to be located at or near the thrombin digestion site (GPGRAF sequence) in the V3 loop of gp120, since the cleavage of the loop resulted in decreased binding of Suc-HSA. In addition, Suc-HSA was able to protect the V3 region of gp120 from cleaving with thrombin. In contrast, significant binding of Suc-HSA to V3 loop or gp120 peptides was not observed when both were presented in a fluid phase system, suggesting the involvement of a monovalent-low affinity binding of Suc-HSA. Using overlapping peptides delineating the whole V3 loop immobilized to CNBr-Sepharose, we noticed that the interaction of the V3 loop with Suc-HSA was predominantly induced by electrostatic interactions between positively charged linearized peptide fragments and Suc-HSA and was positively influenced by the presence of hydrophobic amino in the V3 loop fragments as well. Moreover, the highest affinity site was located at sites near the GPGRAF sequence. These observations add to the evidence, collected earlier, that Suc-HSA interferes at the level of virus entry, independent of interaction with the CD4 receptor. Since the recently discovered chemokine receptors are negatively charged, we can hypothesize that Suc-HSA is able to prevent the positively charged V3 loop from interacting with these types of receptors, thereby inhibiting virus entry.

The epidemiology of chronic fatigue in San Francisco.

Despite considerable research on chronic fatigue syndrome (CFS) and conditions associated with unexplained chronic fatigue (CF), little is known about their prevalence and demographic distribution in the population. The present study describes the epidemiology and characteristics of self-reported CF and related conditions in a diverse urban community. The study used a cross-sectional telephone screening survey of households in San Francisco, followed by interviews with fatigued and nonfatigued residents. Respondents who appeared to meet case definition criteria for CFS, based on self-reported fatigue characteristics, symptoms, and medical history, were classified as CFS-like cases. Subjects who reported idiopathic chronic fatigue (ICF) that did not meet CFS criteria were classified as ICF-like cases. Screening interviews were completed for 8,004 households, providing fatigue and demographic information for 16,970 residents. Unexplained CF was extremely rare among household residents <18 years of age, but was reported by 2% of adult respondents. A total of 33 adults (0.2% of the study population) were classified as CFS-like cases and 259 (1.8%) as ICF-like cases. Neither condition clustered within households. CFS- and ICF-like illnesses were most prevalent among women and persons with annual household incomes below $40,000, and least prevalent among Asians. The prevalence of CFS-like illness was elevated among African Americans, Native Americans, and persons engaged in clerical occupations. Although CFS-like cases were more severely ill than those with ICF-like illness, a similar symptom pattern was observed in both groups. In conclusion, conditions associated with unexplained CF occur in all sociodemographic groups but appear to be most prevalent among women, persons with lower income, and some racial minorities.

Use of competitive polymerase chain reaction to determine HIV-1 levels in response to antiviral treatments.

OBJECTIVE: To develop a competitive polymerase chain reaction technique with which to evaluate the usefulness of HIV-1 level as a marker of response to antiviral treatment. DESIGN: HIV-1 sequences were assessed by competitive polymerase chain reaction in four subjects participating in a double-blind study of monotherapy versus combination therapy with nucleoside analogues. METHODS: We inserted a mutant construct of the HIV-1 pol sequence into a commercial vector, enabling us to generate known amounts of mutant DNA and RNA for competitive polymerase chain reaction. To measure HIV-1 DNA copies in cells, the mutant DNA fragments were allowed to compete in a 10-fold dilution series with a constant amount of nucleic acid from the subject. To measure HIV-1 RNA copies in plasma, in vitro synthesized mutant RNA was added in a 10-fold dilution series to a constant amount of subject RNA and copy DNA was synthesized. DNA and copy DNA were used as the input for nested pol polymerase chain reaction. Mutant and wild-type amplimers were discriminated by size. RESULTS: The competitive polymerase chain reaction technique has been validated in model experiments and can be used over a broad range (at least 6 logs) of levels. Three of the four subjects showed a decline of 1 log in proviral DNA levels in cells after beginning antiviral treatment. All four showed a decline of at least 1 log in viral RNA levels in plasma, but this decline was transient in one subject. CONCLUSION: The HIV-1 sequence level is a useful marker in antiviral treatment studies.

Detection of HIV-1 distribution in different blood fractions by two nucleic acid amplification assays.

A new amplification procedure, NASBA (nucleic acid sequence-based amplification), was used together with the polymerase chain reaction (PCR) to detect HIV-1 sequences in different blood fractions of both HIV-infected and uninfected samples. We tested whole blood, plasma, peripheral blood mononuclear cells (PBMCs), and platelets. No HIV-1 sequences were found in blood fractions of 37 uninfected persons either by PCR, reverse transcriptase-PCR (RT-PCR), or NASBA. We found that none of the infected plasma samples contained HIV-1 DNA sequences. However, a high percentage of these plasma samples was positive for HIV-1 RNA: 86% by NASBA and 80% by RT-PCR. The concordance on a sample-to-sample basis of NASBA and RT-PCR was 91%. Only 33% of the plasma samples was HIV-1 p24-antigen positive, demonstrating the superior sensitivity of amplification procedures. We found that almost all PBMC fractions were positive for HIV-1 (pro)viral sequences (99% HIV-1 DNA positive, 91% HIV-1 RNA positive). A large proportion of the platelet fractions contained HIV-1 RNA, as demonstrated by positive RT-PCR and NASBA results. We found an inverse relation between CD4+ T cell count and T cell reactivity on the one hand and detection of HIV-1 sequences by PCR, RT-PCR, and NASBA on the other hand in all blood fractions. Quantification of the HIV-1 PCR signal in PBMCs revealed an inverse relation of proviral titers with CD4+ levels. This finding supports earlier observations that clinical disease and low CD4+ cell counts are related to an increased viral burden.

Concordance of human immunodeficiency virus detection by polymerase chain reaction and by serologic assays in a Dutch cohort of seronegative homosexual men.

In three subgroups of a clinically and socially well defined group of Dutch homosexual men, the prevalence of human immunodeficiency virus type 1 (HIV-1) sequences in seronegative blood samples was studied using the polymerase chain reaction (PCR). In 19 seronegative partners of seropositive persons, no HIV-1 sequences were found by PCR in either early (1984/1985) or more recent (1987) samples. In 42 seronegative persons selected by their high risk for HIV-1 infection, none harbored HIV-1 sequences in either early (1985/1986) or late (1989) samples. In 15 people who seroconverted for HIV-1, only 2 samples collected 3 months before seroconversion were PCR-positive. These persons were also HIV antigen-positive at this time. These data suggest that a latent infection greater than 6 months does not occur and that the combination of HIV antibody and HIV antigen tests is appropriate and conclusive in most cases of HIV-1 infection.