Karen Cristiano

Prevalence of TT virus in plasma pools and blood products

A high prevalence of TT virus (TTV), a novel virus recently identified in the serum of a patient with post‐transfusion hepatitis of unknown aetiology, has been reported in blood donors worldwide. We investigated the presence of TTV DNA in several lots of blood products and in the corresponding plasma pools. In the process, we determined, from three sets of primers, the one which was most efficient in detecting the viral nucleic acid. This set amplifies the region closest to the 3′‐end of the TTV genome which was proved, by sequence analysis, to be more conserved than the other two regions. Whereas all 10 intravenous immunoglobulin and 21 albumin batches were TTV negative, 4/5 factor VIII concentrates and 4/10 intramuscular immunoglobulin batches were TTV positive. A high prevalence of TTV DNA (70%) was found in the plasma pools that were collected from four different countries. These results confirm the worldwide distribution of this virus and show that TTV is removed with a varying efficiency during the manufacture of blood products.

West Nile virus in the transfusion setting with a special focus on Italian preventive measures adopted in 2008-2012 and their impact on blood safety

. The majority of WNV infections in humans are asymptomatic while approximately 20% of infected individuals develop, after an incubation period of 3-14 days, a mild febrile illness for 3-6 days (West Nile fever, WNF) characterised by a variety of non-specific symptoms that do not allow WNF to be distinguished from other infectious illnesses on clinical examination. In less than 1% of infected individuals, particularly the elderly or immunocompromised subjects

Quantification of hepatitis C virus (HCV) RNA in a multicenter study: implications for management of HCV genotype 1-infected patients.

ABSTRACT Assessment of the viral load in hepatitis C virus (HCV) genotype 1-infected patients is critical before, during, and after antiviral therapy. In patients achieving a rapid virological response at week 4 of treatment, the viral load at the baseline is considered a predictive criterion of a sustained virological response 24 weeks after the discontinuation of treatment. A ≥2-log10 drop in the viral load at week 12 of treatment (early virological response) triggers the continuation of therapy. We organized a multicenter study (MS) for diagnostic laboratories involved in the quantification of HCV RNA. Commercial assays, including two based on real-time reverse transcription-PCR (TaqMan system), and in-house methods, were used by the 61 participants. The overall reproducibility of the commercial quantitative nucleic acid amplification techniques (qNAT) was acceptable. As the intermethod variability among commercial qNAT for HCV RNA was still present, the manufacturers of these test kits should join efforts to harmonize the means of quantification of HCV RNA. This study also shows that caution should be exercised when the baseline viral load is evaluated and when the 2-log10 reduction after 12 weeks of therapy is interpreted. Finally, this MS confirms the higher sensitivity of the commercial qNAT based on the TaqMan system, making them the elective assays for the monitoring of therapy.

External quality assessment for the detection of blood-borne viruses in plasma by nucleic acid amplification technology: the first human immunodeficiency virus and hepatitis B virus studies (HIV EQA/1 and HBV EQA/1) and the fifth hepatitis C virus study (HCV EQA/5)

Background and Objectives  This External Quality Assessment (EQA) study was aimed at assessing the proficiency of blood centres and blood product manufacturers in detecting, by nucleic acid amplification technology (NAT), the possible contamination of plasma with hepatitis C virus (HCV), human immunodeficiency virus (HIV) and hepatitis B virus (HBV).

External quality assessment for the detection of HCV RNA, HIV RNA and HBV DNA in plasma by nucleic acid amplification technology : a novel approach

Background and Objectives  In this EQA study a novel approach was used to assess the performance of blood centres and blood product manufacturers in detecting the possible contamination of plasma with HCV, HIV and HBV by NAT.

Hepatitis C virus testing of plasma pools by nucleic acid amplification technology: external quality assessment

Since 1 July 1999, in accordance with European regulations, only batches of blood products obtained from plasma pools tested and found to be non‐reactive for hepatitis C virus (HCV) RNA are being released. As monitoring the performance of manufacturers involved in plasma pool testing is important to ensure reliable amplification techniques, the Istituto Superiore di Sanità, as the Italian regulatory authority, organized an external quality assessment study.

High proficiency in detecting the six major hepatitis C virus genotypes of laboratories involved in testing plasma by nucleic acid amplification technology

Summary Since the introduction of mandatory HCV RNA testing of plasma pools for fractionation by nucleic acid amplification technology, we have organised External Quality Assessment studies (EQAs) addressed to blood products manufacturers and blood centres. Here we report the results of a new EQA, the first one to include all six major HCV genotypes. The results, reported by laboratories worldwide, showed that genotypes 1, 2 and 3 were correctly identified in 100% of the tests, genotype 4 in 96·7% and genotypes 5 and 6 in 98·3% of the assays. As detection of all HCV genotypes is critical for laboratories involved in testing plasma for HCV, all six genotypes should continue to be included in the next EQA studies.

Overestimation of the Hepatitis C Virus RNA Content of Reference Preparations by the AMPLICOR HCV Monitor Test, Version 2.0

ABSTRACT An evaluation of the AMPLICOR hepatitis C virus (HCV) monitor test, version 2.0 (Roche Diagnostics), was carried out to investigate whether this test overestimates the HCV RNA content of reference preparations. Satisfactory accuracy was observed when the World Health Organization HCV international standard was included in the assay and a modified formula was used to calculate the viral content.

West Nile Virus in Europe and Safety of Blood Transfusion

West Nile virus (WNV) has become an increasing issue in the transfusion setting since 2002, when it was firstly shown in the USA that it can be transmitted through blood transfusion. Since then, several precautionary measures have been introduced in Europe in order to reduce the possible risk of transmission via transfusion/solid organ transplantation. In addition, the epidemiological surveillance has been tightened and the network for communication of human WNV cases strengthened. This review will focus on WNV circulation and the safety of blood in Europe.

Further evidence on the high proficiency of laboratories involved in plasma pool testing for HCV RNA by nucleic acid amplification technology.

We recently reported the results of an External Quality Assessment Study (EQA) on HCV RNA plasma pool testing addressed to manufacturers of blood products [1]. The study, carried out in two independent stages during the year 2000, demonstrated an excellent performance of participating laboratories. In fact, using validated nucleic acid amplification technology (NAT), manufacturers of blood products proved to fully comply with the relevant European guideline [2] by showing consistency with their stated detection limit. In order to continue providing laboratories involved in plasma pool testing for HCV RNA by NAT with an external quality measure of their proficiency, we organized a new EQA study for the year 2001. All laboratories, which took part in the previous study, participated in this EQA. In addition, two more manufacturers, one from Argentina and one from Switzerland, and three blood transfusion centres, one from Germany and two from Italy, were accepted as participants. This time the aim of the study was to confirm that participants comply with the European Pharmacopoeia requirement regarding the 100-IU/ml detection level and to verify the absence of cross contamination [3]. Consequently, the panel consisted of the following 20 coded samples: eight negative replicates, 10 replicates containing 100 IU/ml and two replicates containing 17 000 IU/ml. Two Italian reference preparations calibrated against the first WHO HCV RNA International Standard [4], ISS 0498 and ISS 0198, containing 1700 and 17 000 IU/ml, respectively, were used to prepare the positive samples. The former, recently characterized in an international collaborative study [5], was diluted to obtain the 100-IU/ml samples while the latter was used undiluted to prepare the high-titre samples. Before shipping it, the panel was tested in our laboratory by two operators in order to confirm the HCV positivity/negativity of the samples. Participants, who were assigned a random code, were asked to test the panel by their routine procedure. Seven out of the 15 participating laboratories used an in-house method while the rest used a commercial kit. As the samples were scheduled for shipment after 11 September 2001, serious problems with flight connections occurred in the aftermath of the terrorists’ attack in the USA. As a consequence, two laboratories received the panel thawed and only in one case was it possible to send a new panel. The deadline for sending the results was postponed from the end of November to mid-December 2001. All 15 participants sent their results including the laboratory that received the panel thawed but decided to test it anyway. As in this case degradation of HCV RNA during the long uncontrolled storage period could not be excluded, we did not evaluate the results of this laboratory. Still, we informed the laboratory of its results as these could be of some use for the participant. With respect to the other laboratories, all samples with a nominal concentration of 100 IU/ml and 17 000 IU/ml tested positive in 100% of the assays. Despite the presence in the panel of high-titre samples, crosscontamination was not observed as negative samples tested negative by all laboratories. In conclusion, the overall results could be considered satisfactory, confirming the high level of proficiency reached by laboratories involved in plasma pool testing for HCV RNA by NAT.