Levels Of B19 DNA Research Articles

Seroprevalence, molecular epidemiology and quantitation of parvovirus B19 DNA levels in Iranian blood donors.

Human parvovirus B19 (B19) infection is common among blood donors, and healthy blood donors can transmit virus via transfusion. Due to resistance of B19 to viral inactivation methods, there is a potential concern regarding transfusion safety in blood products. We aimed to determine the seroprevalence, molecular epidemiology, and quantitation of B19 DNA levels in blood donors in Tehran, Iran. A total of 500 blood donors from Blood Transfusion Research Center were studied. ELISA was used for detection of B19 IgG and IgM and nested PCR was carried out for detection of B19 DNA. PCR products were subjected to direct sequencing. B19 viral load was determined by real time PCR. B19 IgG, IgM, and DNA were detected in 27.6, 2.6, and 1.2% of donors respectively. Ten samples (2%) were positive for both antibodies while in four cases (0.8%), B19 IgG and DNA detected simultaneously. One case had B19 IgM, IgG, and viremia concurrently. The titers of B19 DNA in four of six donors were more than 106 IU/mL (high level viremia) and all four cases had IgG simultaneously. All B19 isolates categorized in genotype 1A. Our findings indicated that prevalence of B19 DNA in Iranian blood donors was comparable with previous studies throughout the world. High level B19 viremia found in 0.8% of our donors and all viremic donors revealed neutralizing B19 antibody. Therefore implementation of a B19 screening test for each volunteer blood donor does not appear to be necessary but B19 testing for plasma-derived products seems important in Iranian donors.

Epidemiology of high‐level parvovirus B19 viraemia among Dutch blood donors, 2003–2009

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. 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. 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. 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.

Parvoviruses and blood transfusion

Parvoviruses and blood transfusion

Prevalence and quantitation of parvovirus B19 DNA levels in blood donors with a sensitive polymerase chain reaction screening assay

Blood donor parvovirus B19 DNA prevalence with sensitive nucleic acid test assays has recently been demonstrated to be higher than that found with assays designed to detect high viral titers in the plasma manufacturing sector. Stored plasma aliquots from 5020 donations collected between 2000 and 2003 at seven US blood centers were tested. Testing was performed with a real-time B19 DNA polymerase chain reaction (PCR; TaqMan, Applied Biosystems) assay with a 50 percent limit of detection (LOD) of 1.6 IU per mL (95% confidence interval [CI], 1.2-2.1 IU/mL) and a 95 percent LOD of 16.5 IU per mL (95% CI, 10.6-33.9 IU/mL). Confirmation and quantitation of B19 DNA was accomplished by retesting of two additional subaliquots. Confirmed-positive specimens were tested for the presence of anti-B19 immunoglobulin M (IgM) and IgG with FDA-licensed assays. B19 DNA prevalence was 0.88 percent (95% CI, 0.64%-1.2%). Among the 23 donations with B19 DNA titers of at least 20 IU per mL, the median DNA concentration was 105 IU per mL with an interquartile range of 42 to 481 IU per mL; the highest value was 1869 IU per mL. All B19 DNA-positive donations were positive for the presence of IgG and 10 (23%) were also positive for the presence of IgM; IgM seropositivity was associated with increasing DNA levels (p = 0.0013). Low-level B19 DNA was detected in nearly 1 percent of donations. The 23 percent of DNA-positive donations with both IgM and IgG B19 antibody most likely represent acute resolving infection, whereas those with IgG but no IgM are most consistent with a more chronic and possibly persistent phase of B19 infection.

Parvovirus B19 DNA in Factor VIII concentrates: effects of manufacturing procedures and B19 screening by nucleic acid testing

Parvovirus B19 (B19) is a common contaminant, especially in coagulation factors. Because of B19 transmission by pooled plasma, solvent/detergent treated in 1999, some fractionators initiated minipool nucleic acid testing (NAT) to limit the B19 load in manufacturing pools. In this study, the extent of B19 DNA contamination in commercial Factor VIII concentrates, that is, antihemophilic factor (human) (AHF), manufactured before and after B19 NAT screening was implemented, was determined. A total of 284 lots representing six AHF products made during 1993 to 1998 and 2001 to 2004 were assayed for B19 DNA by an in-house NAT procedure. Anti-B19 immunoglobulin G (IgG) was also measured. Most lots made during 1993 to 1998 had detectable B19 DNA. The prevalence ranged from 56 to 100 percent and appeared to differ between manufacturers. The highest level of B19 DNA found was 10(6) genome equivalents (geq or international units [IU]) per mL. Forty percent of the lots tested contained 10(3) geq (IU) per mL. In comparison, both prevalence and levels in source plasma-derived AHF products made in 2001 to 2004 were lower. Both, however, remained unchanged in the recovered plasma-derived product because B19 NAT screening had not been implemented. Only an intermediate-purity AHF product was positive for the presence of anti-B19 IgG. The prevalence and levels of B19 DNA in AHF prepared from B19 NAT unscreened plasma were high but varied among products with different manufacturing procedures. B19 NAT screening of plasma effectively lowered the B19 DNA level in the final products and in the majority of cases rendered it undetectable and hence potentially reduced the risk of B19 transmission.

Cytokine responses in acute and persistent human parvovirus B19 infection

The aim of this study was to characterize the proinflammatory and T helper (Th)1/Th2 cytokine responses during acute parvovirus B19 (B19) infection and determine whether an imbalance of the Th1/Th2 cytokine pattern is related to persistent B19 infection. Cytokines were quantified by multiplex beads immunoassay in serum from B19-infected patients and controls. The cytokine responses were correlated with B19 serology, quantitative B19 DNA levels and clinical symptoms. In addition to a proinflammatory response, elevated levels of the Th1 type of cytokines interleukin (IL)-2, IL-12 and IL-15 were evident at time of the initial peak of B19 viral load in a few patients during acute infection. This pattern was seen in the absence of an interferon (IFN)-gamma response. During follow-up (20-130 weeks post-acute infection) some of these patients had a sustained Th1 cytokine response. The Th1 cytokine response correlated with the previously identified sustained CD8+ T cell response and viraemia. A cross-sectional study on patients with persistent B19 infection showed no apparent imbalance of their cytokine pattern, except for an elevated level of IFN-gamma response. No general immunodeficiency was diagnosed as an explanation for the viral persistence in this later group. Neither the acutely infected nor the persistently infected patients demonstrated a Th2 cytokine response. In conclusion, the acutely infected patients demonstrated a sustained Th1 cytokine response whereas the persistently infected patients did not exhibit an apparent imbalance of their cytokine pattern except for an elevated IFN-gamma response.

Human parvovirus B19 infection of monocytic cell line U937 and antibody-dependent enhancement

Human parvovirus B19 (B19) infects human erythroid lineage cells. Accumulating evidence also shows that B19 is detectable in nonerythroid lineage cells in vivo, but the mechanism of infection is still not clear. In this study, we explored the mode of B19 infection of human monocytic cell line U937. An in vitro infection study demonstrated B19 binding of U937 and slow replication of B19-DNA with B19-NS1 mRNA transcription. B19-DNA replication in U937 was accompanied by undetectable level of B19-VP1 mRNA transcription, indicating that B19 infection of U937 cells may be abortive. Levels of B19-DNA and B19-NS1 mRNA transcription increased in the presence of anti-B19 IgG antibodies, but this effect decreased in the presence of anti-Fc receptor antibodies, showing antibody-dependent enhancement by B19 infection. Antibody-dependent enhancement also caused the increased production of TNFα in U937. This study is the first to suggest B19 infection of nonerythroid lineage cells with antibody-dependent enhancement.

Persistent B19 infection in immunocompetent individuals: implications for transfusion safety

AbstractRecent reports suggested that parvovirus B19 (B19) might persist in immunocompetent individuals such as blood donors, but only cross-sectional data were available. Serial samples from a cohort of multitransfused patients with hemoglobinopathies and a cross-sectional population of pregnant women were tested for B19 markers. Of 76 red cell recipients, 6 (8%) had persistent viral DNA for 1 to 3 or more years, depending on the sensitivity of the genomic amplification assay. All patients also carried B19-specific immunoglobulin G (IgG). In contrast, 0.8% of 500 pregnant women carried both detectable B19 DNA and specific IgG. These results demonstrate that persistence of low levels of B19 DNA suggested by cross-sectional studies is frequent in multitransfused patients and that the virus may remain detectable several years after infection in nonimmunodeficient individuals. (Blood. 2005;106:2890-2895)

Parvovirus B19 viral loads in relation to VP1 and VP2 antibody responses in diagnostic blood samples

Background: Human parvovirus B19 infection is characterised by high peak viral load levels followed by episodes of prolonged viremia. The risk of transmission of parvovirus B19 by blood or blood products has been increasingly recognised and parameters that can predict the risk of transmission are subject of interest. Objectives: This study aimed to study correlations between B19 viral DNA loads and antibody responses to the viral antigens VP1 and VP2 in clinical serum samples. Study design: A panel of 1610 serum samples from patients clinically suspected from acute B19 infection were analysed. Antibodies were measured by the parvovirus anti-VP1 immuno-fluorescence assay (IFA) and the anti-VP2 enzyme immunoassay (EIA) from Biotrin. B19 viral loads were measured by a real-time PCR using the external WHO standard for DNA quantification. Results: Positive IgM responses were found in 154 (9.6%) of the 1610 sera tested. Based on the PCR results in a subset of 312 sera, the anti-VP2 EIA IgM showed a better combination of sensitivity/specificity (91%/94%) compared to the anti-VP1 IFA (66%/97%). B19 DNA levels in the sera strongly correlated with the levels of IgM antibodies, all sera with high viral loads (>10 6 IU/ml) having VP2 EIA IgM ratios above 3.0. Conclusions: The B19 VP2 IgM ELISA is superior to the B19 VP1 IgM IFA if verified by PCR. Anti-VP2 IgM antibodies in sera are indicative for the presence B19 DNA and can be used to predict high levels of B19 DNA in diagnostic sera.

献血血液のＲＨＡ検査による第ＶＩＩＩ因子製剤（クロスエイトＭ）原料血しょうからのパルボウイルスＢ１９除去効果

Since September 1997 the Japanese Red Cross has conducted a nationwide complete screening of human parvovirus B19 (B19) for all donated blood units by the receptor-mediated hemagglutination (RHA) method. RHA-positive units were excluded from source plasma for fractionation. The amounts of B19 DNA in pooled plasma and in factor VIII concentrates (Cross Eight M, plasma derived and monoclonal purified) were measured using a PCR method. All 112 batches of pooled plasma tested in 1996, before implementation of RHA screening, were B19 DNA-positive, with 83% of these contaminated with more than 3.8×105IU/ml of B19 DNA. In contrast, after implementing RHA screening, no detectable levels of B19 DNA were observed in 5% (1998), 16% (1999), 21% (2000) and 21% (2001) of batches, and batches contaminated with more than 3.8×105IU/ml of B19 DNA decreased to 18% (2001). B19 DNA content in the final products of factor VIII concentrate were reduced significantly when RHA-screened source plasma were used. Since September 1998, B19 DNA has not been detected in any of 78 lots of final products. Furthermore, no B19 DNA could be detected in any of 63 lots even in 1:10 concentrated solution. RHA screening for B19 has markedly reduced the viral load in source plasma for fractionation in Japan.

Large–Scale Screening for Human Parvovirus B19 DNA by PCR: Application to the Quality Control of Plasma for Fractionation

Background and Objectives: Because human parvovirus B19 (B19) has been transmitted by various plasma–derived medicinal products, the ‘Laboratoire français du Fractionnement et des Biotechnologies’ (LFB) implemented PCR screening of plasma pool samples for B19 DNA as part of the quality control of plasma source material. Materials and Methods: Plasma pool samples (average of 46.5 donations) were tested for B19 DNA by PCR and by immunological detection of PCR products. The viral DNA content was determined by means of a TaqMan™–based, quantitative PCR. Results: From plasma corresponding to 2–year collections in France, and representing 4.26 million donations from approximately 1.25 million voluntary unpaid donors, the average frequency of positive donations was 1/5,950 and reached 1/1,420 during an epidemic. Levels of B19 DNA in positive pools ranged from <10² to 10¹¹ copies/ml. Conclusion: A large–scale PCR plasma screening increased the safety of LFB’s wide range of products with respect to B19, a virus particularly resistant to physicochemical inactivation procedures.

Detection of human parvovirus B19 DNA in plasma pools and blood products derived from these pools: implications for efficiency and consistency of removal of B19 DNA during manufacture

The polymerase chain reaction (PCR) assay was used to detect human parvovirus B19 DNA in 38 blood products and start plasma pools from five different manufacturers. The products examined were albumin, factor VIII, intravenous (i.v.) and intramuscular (i.m) immunoglobulin batches. The majority of pools from all the manufacturers had detectable B19 DNA (64/75:85%; ranging from 60% to 100% for individual manufacturers). B19 DNA was found in 3/12 albumin samples, in 7/7 factor VIII samples, 3/15 IVIG samples and 3/4 IMIG samples. The levels of B19 DNA in pools varied from 10(2) to 10(9) genome equivalents/ml, whereas the levels in products varied from 10(2) to 10(6) genome equivalents/ml, but there was no clear relationship between the levels of B19 DNA in start pools and final products. The levels of B19 DNA varied between different batches of the same product from a single manufacturer, possibly due to small variations in the processing parameters. In addition, there was some indication from the study of IVIG samples that treatment at low pH may result in removal of PCR-detectable B19 DNA.

Manifestations and treatment of human parvovirus B19 infection in immunocompromised patients

We diagnosed infections from human parvovirus B19 in three patients by using dot-blot hybridization and a polymerase chain reaction to detect B19 DNA and using an enzyme immunoassay to detect IgG and IgM to B19. For 5 months a 5-year-old boy with acute lymphoblastic leukemia in remission had anemia without reticulocytes or bone marrow erythrocyte precursors. His serum lacked IgG and IgM to B19 but contained B19 DNA. He received gamma globulin intravenously (0.4 gm/kg/day for 5 days); his viremia promptly cleared and reticulocytosis developed. A 14-year-old boy with acute lymphoblastic leukemia in remission had fever, rash, neutropenia (less than 300 leukocytes/mm3), and a hemophagocytic syndrome lasting 3 weeks. His serum contained IgM to B19 and B19 DNA. Without therapy, IgG to B19 developed; although low levels of B19 DNA persisted, the leukocyte count returned to normal. In a 19-year-old patient with systemic lupus erythematosus and hemolytic anemia, an aplastic crisis lasted 2 weeks. Her serum lacked IgG and IgM to B19 but contained B19 DNA. Without therapy, IgG and IgM to B19 appeared, viremia diminished, and reticulocytosis occurred. These patients illustrate the varied manifestations of chronic B19 infections, the importance of DNA detection for diagnosis, and the possible efficacy of gamma globulin therapy.