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Are plasma derivatives sources for parvovirus B19 transmission? Manal M. Baddour Associate Professor of Microbiology King Saud University السلام عليكم ورحمة الله وبركاته Ladies and gentlemen, today we will try to answer the query whether plasma products are sources of parvovirus B19 transmission as pertains to the Saudi society as this question has been addressed in other countries

Parvovirus B19 is an erythrovirus and was until recently the only known member of the Parvoviridae family of viruses causing disease in humans. Heegaard and Brown, 2002 But first lets remember together what is parvovirus B19? Parvovirus B19 is an erythrovirus and was until very recently the only known member of the Parvoviridae family of viruses causing disease in humans a small, nonenveloped virus containing a single-stranded DNA of 5600 nucleotides protein shell of approximately 280Å diameter diameter, 18–26 nm Parvoviruses are the smallest DNA-containing viruses that infect mammalian cells. Human Parvoviruses Human Erythrovirus (B19) (It is classified as an erythrovirus because it is highly tropic for erythroid progenitor cells and because complete replication of B19 has been found only in these cells) PARV4 (Jones et al., 2005) Acute infection syndrome Little known about epidemiology Human Bocavirus (Allander et al., 2005) HBoV may cause more severe respiratory illness in infants and children, similar to disease caused by RSV (4,5), HMPV (6,7), human coronavirus NL63 (8–14), and human coronavirus 229E Parvovirus B19 (B19V) – type member of Erythroviruses 3 different genotypes now recognized No evidence for different serotypes Highly erythrotropic Detection of DNA in blood and tissues for years following acute infection No evidence of reactivation/integration Parv4/5 is a new member of the Parvovidae Sequences are uncommon in blood and blood products No culture method available VLP can be expressed Do not resemble B19V Do not HA Parv4 Ab common in children/adults Human bocavirus in respiratory secretions of children < 5 Prevalence in other tissues/age groups Adeno-associated viruses – AAV2, 3, 5 are human viruses

The surface structures on the virus

Yellow boxes show human parvoviruses, B19, HBoV and Parv4/5, adenoassociated virus

Parvovirus B19 (B19) was discovered fortuitously in 1973 in the United Kingdom by Cossart, who was looking for the hepatitis B virus in blood from “healthy” donors. Cossart et al, 1975 Parvovirus B19 (B19) was discovered fortuitously in 1973 in the United Kingdom by the australian virologist, Yvonne Cossart, who was looking for the hepatitis B virus in blood from “healthy” donors and accidentally came across B19 It was thus given its name from the name of the blood bag where it was discovered (During electrophoresis, an abnormal band was noted in sample number 19, panel B.) Human parvovirus B19 Widespread in human populations 3 genotypes, limited genetic heterogeneity Acute, resolving infections, associated with intense viraemia Recent evidence for long term persistence

Electron Microscopy of B19 shows that The parvoviruses are small, non-enveloped viruses Thus they easily escape filtration (small) and solvent detergent treatment does not inactivate the virus (no lipids) and they are thermostable

Slapped Cheeks Usually infections are asymptomatic, but when symptomatic, The most common manifestation of infection with parvovirus B19 is erythema infectiosum (fifth disease) a mild illness common in children and young adults presenting with fever and characteristic “slapped cheek” rash and occasionally arthropathy. The rash could be somewhat intense. Due to the deposition of immune complexes. The typical rash (‘slapped cheek’ appearance) is immune mediated, since it coincides with the appearance of IgM and IgG specific antibodies At the age of 70 the seroprevalence exceeds 80% (Brown et al., Crit Rev Oncol Hemato 1994.)

Slapped Cheeks Or a very mild rash

Lacey Rash of the Dorsum of the Hands Sometimes, rash in the gloves and socks areas develops Treatment is IVIG (antibody to B19.)

Gloves and socks rash The gloves and socks rash These pictures were actually from 2 saudi cases (KFSHRC) reported by Abdullah alfadley et al.

Gloves and socks rash Again, the gloves and socks rash Bone marrow aspirates: giant proerythroblasts with eosinophilic nuclear inclusion bodies (lower).

Sometimes, Arthropathy develops in young adults especially females. Commonly develpos in the joints of the hand and feet joints, less often in the knees, elbows and shoulders

Ascites, hepatomegaly, cardiomegaly hydrops However, infection has been recognised as a potential risk for vulnerable patients such as pregnant women, immuno-compromised patients, or patients with increased haematopoiesis Infection during pregnancy may cause non immune fetal hydrops (with ascites, hepatomegaly and cardiomegaly) and spontaneous abortion or still-birth I do not claim that I know much about US but these are US pictures of fetuses with nonimmune hydrops fetalis due to B19 infection acquired during pregnancy (the earlier in pregnancy the 1ry infection occurs the more severe the outcome) Indeed, it has been estimated that upwards of 3,000 pregnancies per annum may be lost in the European Union and the United States due to B19 infection, based on an infection rate of 0.1% and a susceptible cohort of over 3 million pregnancies involving seronegative females. The virus is particularly threatening to the fetus since it has a unique affinity for the a cellular receptor (P-antigen) in fetal erythroid precursor cells in fetal liver

Other complications of parvovirus B19 infection are seen in individuals with underlying haematological disorders such as sickle cell disease and thalassemia which are particularly common in the eastern province of saudi arabia, resulting in aplastic crisis. These are pictures of the sickle cells, such unfortunate patients require frequent transfusions and some of them go into very painful crisis In patients whose hematocrit is dependent on brisk reticulocytosis, such as patients with hemolytic disease, anemia may occur due to parvovirus infection

Thalassemia Elliptocytosis This aplastic crisis occurs likewise in others hemolytic disease patients such as thalassemics and ovalocytosis patients.

The cellular receptor is the blood group P antigen The virus selectively targets early erythroid precursors, causing anemia. The cellular receptor is the blood group P antigen Brown et al., 1993 Parvovirus B19 exhibits an extraordinary high level of cell tropism as it selectively targets early erythroid precursors, causing anemia. The cellular receptor is the blood group P antigen globoside or globotetraosylceramide, Gb4), which is expressed on the erythrocytes and erythroid precursors of most individuals, as well as on other cells (megakaryocytes, endothelial cells, placental cells, the fetal liver, the heart, and other cell types) It occurs mainly in the red blood cell progenitor lineage (cfu-e) where it produces lytic infection by apoptosis. Entry into red cell progenitor cells involves specific receptors at the cell surface, such as globoside (P-blood group antigen) and/or KU80 and/or 53 integrin. In productive infection, two capsid proteins, the minor capsid protein VP1 and the major capsid protein VP2, are synthesized. The expression of VP2 alone or of VP1 and VP2 in combination leads to self-assembly of virus-like particles. The infection of susceptible cells and the expression of the nonstructural protein cause apoptosis, probably through induction of the caspase cascade. Nonstructural protein production is cytotoxic, even in cells in which the viral capsid proteins are not produced. Thus, cell death can occur even without virus replication. Parvovirus B19 can only replicate in erythroid precursor cells in human bone marrow, but it is known that infection of cells non-permissive for viral replication leads to an excess production of the viral non-structural protein (NS1) without production of capsid proteins. As the NS1 protein is cytotoxic and able to induce apoptosis, it probably plays a part in the pathogenetic process of the parvovirus B19 induced tissue damage. Parvovirus B19 exhibits an extraordinary high level of cell tropism; its replication is restricted to differentiating and proliferating erythroid precursors in human bone marrow or foetal liver.

After infection, the incubation period ranges from 6 to 18 days. Viremia in early infection occurs with extremely high titers (up to 1011 to 1013 genome equivalents/ml). In immunocompetent persons, viraemia is usually cleared a few weeks after infection. In immunocompromised patients, the virus can cause a persistent infection for several months and years mainly due to impairment of B cell function. Siegl and Cassinotti, 1998 After infection, the incubation period ranges from 6 to 18 d, although it can be longer. Viremia in early infection occurs with extremely high titers (up to 1011 to 1013 genome equivalents/ml). At this point most patients tend to be asymptomatic, some experience a flu-like syndrome and B19-infected blood donors can then easily go unrecognized In immunocompetent persons, viraemia is usually cleared a few weeks after infection as antibodies neutralise B19 infectivity. Although Donors can be infective even in presence of anti-B19. However, In immunocompromised patients (immunedeficiency diseases), the virus can cause a persistent infection for several months and years leading to chronic anemia as they are unable to clear the virus mainly due to impairment of B cell function. In subjects with immune dysfunction, e.g., patients with human immunodeficiency virus infection or congenital immunodeficiency, or recipients of organ transplants, parvovirus infection may become chronic

The frequency of B19-DNA blood donors has been estimated at rates of 1:167 to 1:35,000 Heegaard & Brown,2002 The frequency of B19-DNA blood donors has been estimated at rates of 1:167 to 1:35,000 Manufacturing pools may exceed one thousand litres and may comprise up to twenty thousand donations. No correlation between symptoms and levels of B19 DNA. A low B19 DNA titre can be detected for over one year. Antibodies neutralise B19 infectivity. Donors can be infective even in presence of anti-B19. Donors can be not infective despite a low B19 DNA level . For the manufacture of blood products plasma is pooled prior to fractionation.

Clotting factors Globulin Albumin Although parvovirus B19 is transmitted normally via the respiratory route or transplacental Administration of contaminated blood and plasma-derived medicinal products, particularly clotting factors can result in transmission

High seroconversion rates and some cases of symptomatic illness have been due to blood products (eg immune globulin, clotting factors) prepared from B19-containing plasma pools with reports of prominent B19 infection and worsening the patient’s condition as a result Hayakawa et al, 2002; Yee et al, 1995; Zanella et al 1995; Mortimer et al, 1983 Actually, Blood and marrow donors are not routinely screened for parvovirus and remain potential sources of disease transmission; even autologous donors can transmit the infection Thus many countries are now advocating screening of plasma pools to remove samples containing high B19 viral titres

Factor 8 Blood clot Hemophiliacs were found to be infected with B19 after receiving dry heat-treated or serum treated factors VIII and IX or solvent- or detergent-treated clotting factors Azzi et al, 1992; Yee et al 1996

Filtration column Why does transmission occur via blood products? This is because: Many countries do not mandate routine screening of blood donations for B19 DNA The virus is small and escapes filtration filtration does not remove the virus and solvent detergent treatment doesn’t also Filtration column

Aim of This Study To determine the frequency of parvovirus B19 DNA contamination in a range of plasma derivatives commercially available in the local market for clinical use. Very little is known about parvovirus in saudi arabia and none of the few published articles dealt with blood products or pcr Originally, the study aimed at testing plasma pools also as they are the source of plasma products but it was found that plasma products are all imported and none are manufactured locally in Saudi Arabia. Such data can be of extreme importance both for the manufacturing companies as well as the medical community as it can alert them to faulty techniques and can delineate the more effective means of eliminating B19 from such products. By doing so, needless suffering of already unfortunate patients can be avoided.

Material Samples from three batches of each of the following types of products from 2 companies were tested for parvovirus DNA by nested PCR: 1- human albumin 2- human coagulation factor VIII 3- human coagulation factor IX (one company) 4- human normal IV immunoglobulins Serum from a patient previously diagnosed to be positive for parvovirus B19 DNA by PCR was used as a control. Since the B19 genome is very stable several oligonucleotide pairs, amplifying various regions of the genome, have been successfully Used

Methods DNA from each of these products was extracted in triplicates from 200µl aliquots of the tested products using the QIAamp DNA Blood Minikit Nested PCR using primers A1 5´-AATGAAAACTTTCCATTTAATGA-3´ B1 5´-TCCTGAACTGGTCCCGGGGATGG-3´ 590 bp A2 5´-GCGTGGAAGTGTAGCTGTGCC-3´ B2 5´-CAGAGCTTTCACCACCACTGC-3´ 355 bp Frickhofen and Young, 1989 Since B19 parvovirus cannot be routinely grown in stable cell cultures, the diagnosis of B19 parvovirus infection is generally attempted either by direct detection of B19 antigens and B19 genome in clinical specimens or by detection of a specific antibody response in serum samples. Genome amplification assays are the most sensitive diagnostic tool to detect B19 DNA. Nested PCR is a modified PCR technique which involves 2 consecutive rounds of amplifications each involving a set of primers one amplifying a region internal to the other It is more sensitive than regular PCR (it can disclose between 10-100 copies of B19 DNA in a sample) so it was adopted as the study technique A representative sample of each type of product was spiked with parvovirus B19 DNA (extracted from serum of a previously determined B19 positive patient) and subjected to the same procedures to test for the efficiency of the technique. Nested PCR uses two sets of primers, one internal to the other, in two consecutive amplification rounds and the second round PCR product is identified by electrophoresis in agarose gel and ethidium bromide staining. The nested PCR can disclose between 10–100 copies of B19 DNA in a sample, with a sensitivity at least 10–100 times higher than that achieved by amplification with a single primer set. Nested PCR is reportedly more sensitive and more specific than traditional PCR although more demanding Carriere et al, 1993

Carriere et al, 1993

Results Results of the nested PCR amplification of the sampled plasma products showed that none of the studied products contained detectable levels of B19 DNA. For each assay, the positive controls were all positive and the negative controls were all negative.

Results, cont. A representative sample of each type of product was spiked with parvovirus B19 DNA (extracted from serum of a previously determined B19 positive patient) and subjected to the same procedures to test for the efficiency of the technique. Nevertheless, to guard against false negative results and to make sure that the negative results were not due to inhibitors in the samples

Results, cont. Lanes 1 2 3 4 5 6 7 8 9 Results were reassuring as gel electrophoresis of the amplicons (amplification products) showed that nested PCR was able to amplify the B19 DNA lane 1:molecular weight marker, lanes 2 to 5: spiked samples, lane 6: negative control (water), lane 7,8: positive control (B19 DNA), lane 9: molecular weight marker.

Results, cont. Another run with a clearer picture

The explanation of this is that: As of 2004, the European pharmacopoeia set an upper limit of B19 DNA in plasma pools to be < 104 IU/ml (Hokynar et al, J Clin MIcrobiol, 2004), the same was done by the FDA in USA. > 104 geq/ml leads to infection (European Pharmacopoeia. 2004.) It has been suggested that viruses in the presence of certain kinds of a.a. could be aggregated and effectively removed by a filter with a pore size larger than the size of the virus (Yokoyama et al, Vox Sang 2004)

Manufacturing Safeguards Pathogen Safety is a comprehensive approach with effective redundant measures that provide a high margin of safety Donor Plasma Donation Center Safety Step 2: Testing Donation Safety Step 1: Donor Screening Safety Step 3: Inventory Hold and Lookback Safety Step 9: Post-Marketing Surveillance Safety Step 4: Plasma Pool Testing Patient Safety Step 5 & 6: Quality Assurance GMP Begin Manufacturing The comprehensive approach to pathogen safety used as manufacturing safeguard against B19 transmission as implied by the plasma protein therapeutics association goes through the following steps Complete Manufacturing Safety Step 8: GCP Packaging Guidance Safety Step 7: Virus Inactivation Virus/TSE Removal The Plasma Protein Therapeutics Association

Reduction in Parvovirus B19 Viral Loads Pre-implementation PROCESS VIRAL REDUCTION 101- 109 IU/mL Production Pool No Testing Unknown Margin of Safety Implementation of Parvovirus B19 NAT Testing Mini-pool Testing 96-1200 donation samples High Titer Plasma How has this affected reduction in B19 loads? If plasma donations were not tested, then the production pool is likely to contain anywhere between 10(1) to 10(9) IU/ml of B19, processing does decrease the viral load but to an unknown level. On the other hand, if the plasma donations were tested (even as minipools) with elimination of high titered plasma, then the production will likely have a low viral titre and thus after processing we will be within the safety margin. As B19 has no envelope, it is resistant to detergent- or organic-solvent treatment. In addition, B19 is a small virus (diameter, 18–26 nm), which means that it is not easy to eliminate by filtration through virus removal membranes. As an In process control Inactivation of the virus has proved difficult and as a consequence: manufacturers of blood products have implemented screening measures to reduce the load of parvovirus B19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (NAT). Liquid-heat treatment, which is considered to be a highly reliable method for virus inactivation, has been introduced in the manufacturing processes of various plasma derivatives. Recent reports have shown that under liquid-heating conditions, B19, unlike other animal parvoviruses, was heat sensitive (Blu¨mel et al, 2002a; Yunoki et al, 2003). The reports suggested that, under the heating conditions employed in manufacturing process of albumin preparations, B19 should rapidly be inactivated and that its heat sensitivity should differ depending on the specific stabilizer used in the inactivation treatment (Yunoki et al, 2003). Further, this study confirmed that the liquid-heating step (60C for 10 h) rapidly inactivated B19. Based on these findings, we propose that heat/PEG-treated IVIG could be safe for B19 for the following reasons: (i) robustness against virus contamination offered by its manufacturing process demonstrated in virus clearance studies; (ii) B19 inactivation process at 60C for 10 h; and (iii) presence of neutralization antibodies in the final preparation.(Yunoki et al, bjh, 2004) <105 IU/mL Production Pool PROCESS VIRAL REDUCTION Increased Margin of Safety

Conclusions It is true that results obtained from the present study seem reassuring, probably due to implementation of strict screening of blood donors or plasma pools for parvovirus B19 DNA by the plasma products' manufacturing companies. Nevertheless, continuous monitoring of blood products should be a routine procedure to ensure safety of such products for recipients especially those at risk to avoid the hazardous possibilities of serious infections. However, we should not be disalluded by the negative results as the small sample size (only 3 batches of each product) could have attributed to these results. In fact in 1995, a report from dhahran health centre (where there is a high incidence of sickle cell disease), this report describes a localized outbreak of aplastic crisis and recent B19 infection in these patients (91% of 46 patients) without being able to give a reason for it. Another report by the same author in 1993 described aplastic crises in 3 sickle cell anemia patients. That reason could very well be from plasma products given to those patients. Primary prevention is not feasible as no vaccines against parvovirus B19 infection are currently approved

Conclusions, cont. Human bocavirus HBoV B 19 genotypes 2 (A6) & 3 (V9) Additionally, more recently, Human bocavirus B19 genotypes 2 (A6) & 3 (V9) have been discovered and have been linked to disease and need to be screened for likewise.

Thank You

Human Bocavirus Almost invariably non-persistent, short period of excretion Generally confined to infants and young children Three adults with immunosuppression (transplant) showed persistent infections (2 from 3 with multiple samples), high titres Peak incidence December/January Infections largely confined to < 2 years of age Strongly associated with lower respiratory tract infections