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.

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.

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.

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.

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.

Proliferation-dependent HIV-1 infection of monocytes occurs during differentiation into macrophages.

Requirements for the establishment of productive infection with the human immunodeficiency virus type 1 (HIV-1) in primary monocytes were investigated. In vitro, monocytes rendered susceptible for infection after at least a 2-d culture, but when cultured in the presence of differentiation-inducing agent IL-4, accelerated susceptibility was seen. Complete resistance to HIV-1 infection was observed in monocytes that had been treated for 5 d with rIL-4, and comparable results were obtained with other differentiation inducers such as dexamethasone or 1,25(OH)2 vitamin D3 (1,25(OH)2vitD3). The inhibition of productive infection was not caused by downregulation of CD4 expression or HIV-1 transcription, nor by intracellular accumulation of virions. Since treatment with rIL-4, dexamethasone, or 1,25(OH)2vitD3 also resulted in complete inhibition of monocyte proliferation, we studied whether establishment of productive infection in monocytes is proliferation dependent. Irradiation or mitomycin-C treatment within 24 h after inoculation prevented productive HIV-1 infection of monocytes, suggesting a proliferation-dependent step early in the virus replication cycle. Polymerase chain reaction (PCR) analysis revealed the presence of only incomplete proviral DNA species in non-proliferating monocytes, indicating restriction of viral replication at the level of reverse transcription. Thus, in analogy with HIV-1 infection of CD4+ T cells, proliferation of monocytes during differentiation into macrophages is a prerequisite for productive infection with HIV.

Enhanced detection of HIV-1 sequences using polymerase chain reaction and a liquid hybridization technique. Application for individuals with questionable HIV-1 infection.

In this report we describe a sensitive HIV-1 detection method which is applicable for confirmation testing of donors whose blood sample gives indeterminate viral-serology results. The method involves performing a polymerase chain reaction (PCR) and detecting the generated fragments using liquid hybridization and gel retardation. We found that it is as specific as blotting on a filter and hybridization with an internal probe but at least tenfold more sensitive. After applying it on DNA samples of a panel of 11 persistent indeterminate anti-p24gag-reactive donors, none was found to be PCR positive. Considering other negative virological and biochemical test results and case-historical data, these donors are not likely to be HIV-1 infected.

Efficiency of white cell filtration and a freeze-thaw procedure for removal of HIV-infected cells from blood.

Strategies for diminishing the risk of blood transfusion-associated transmission of HIV-1 were evaluated. HIV-1-infected peripheral blood mononuclear cells were added to blood that was subsequently filtered by using different white cell (WBC) filters (cellulose acetate and polyester). The average log reduction of infected cells with polyester filters was at least 2.5 as measured by ID50 titration and polymerase chain reaction. In two WBC filtration experiments with blood from seropositive donors diluted 1:4 with seronegative blood, log reductions of 2.4 and greater than 2.5 were observed. No cell-free virus was retained by the filter used. A freeze-thaw procedure applied to HIV-1-contaminated blood resulted in a minimal log reduction. These results indicate that the reduction of HIV-1 infectivity as a result of filtration is mainly due to the removal of HIV-1-infected WBCs, and that complete removal of infected WBCs cannot be achieved by the current filtration or freeze-thaw procedures. However, the development of filters with enhanced ability to remove (possibly infected) WBCs may have the added benefit of improving the safety of donor blood, especially in multiply transfused patients.

Expression in Escherichia coli and purification of a translocation-competent precursor of the chloroplast protein ferredoxin.

The precursor of the chloroplast protein ferredoxin from Silene pratensis was expressed in Escherichia coli. When a low copy number plasmid was used, the preferredoxin level was low, and the protein was soluble. The expression level was increased by using a high copy number plasmid. In protease-deficient cells transformed with the latter plasmid, the preferredoxin accumulated up to 1% of total protein, and it was found in insoluble aggregates. These aggregates were dissolved in 4 M urea, and the protein was purified to homogeneity. Amino-terminal sequencing confirmed the amino acid sequence as deduced from the copy DNA. However, the first methionine residue of the expected sequence was absent in E. coli. The purified precursor was readily imported by isolated chloroplasts and processed to the mature size.