Giuliano Gentili

World Health Organization collaborative study to establish a replacement WHO international standard for hepatitis C virus RNA nucleic acid amplification technology assays

Background and Objectives  A collaborative study was undertaken to establish a replacement for the current (1st) World Health Organization (WHO) hepatitis C virus (HCV) International Standard, 96/790.

Immunity to diphtheria in the 3–19 year age group in Italy

Diphtheria antibody level in serum samples obtained from 270 subjects aged 4-70 was measured by in vitro seroneutralization test on Vero cells. Of the studied population, 27.8% had an antibody titre below the protective level (< 0.01 IU/ml). The prevalence of susceptible subjects showed a significant age-related increase (p < 0.01), with the highest value (53.8%) in the 31-40 age group. Mean antibody titre was maximum in individuals aged 13-14, then decreased reaching the minimum level in the 41-50 age group. These data suggest that in individuals aged 30-50, diphtheria immunity is not satisfactory, both for prevalence of immune subjects and for antibody levels. Therefore, a revaccination of adults with reduced doses of diphtheria toxoid may be advisable.

The use of an immunoenzymatic assay for the estimation of tetanus antitoxin in human sera: a comparison with seroneutralization and indirect haemagglutination.

The ability of an enzyme-linked immunosorbent assay (ELISA) to detect tetanus antitoxin in human sera has been evaluated in comparison with the in vivo seroneutralization test. The results of this study, carried out on 171 serum samples, show that ELISA is a sensitive and specific in vitro test for immunity to tetanus in man; it reveals the minimum protective level of 0.01 IU/ml and is well correlated with seroneutralization. A comparison has also been made with indirect haemagglutination. Differences in specificity in low titered sera, although not statistically significant, have been observed. Reported data suggest that the ELISA may be used for the estimation of tetanus antitoxin in sero-epidemiological surveys and for clinical purposes with reliability equal--and perhaps superior--to that of IHA.

The outbreak of hepatitis A in Italian patients with hemophilia : facts and fancies

To determine whether an outbreak of hepatitis A that occured in 52 Italian hemophiliacs was acquired through the infusion of contaminated factor VIII or through environmental enteric transmission, a case‐control study of the first 29 infected patients was carried out. Case patients were neither more nor less likely than controls (hemophiliacs without HAV infection) to have traveled to high‐risk countries, consumed raw shellfish or had contact with persons with jaundice. The case patients, however, were more likely than controls to have received a factor VIII concentrate treated with solvent‐detergent and to have had larger infusions of the concentrate during the presumed HAV incubation period. In the PCR analysis of HAV sequences from implicated lots of factor VIII, HAV sequences were found in 5 of 12 lots of factor VIII implicated in the outbreak. Three different strains of HAV were recovered, suggesting that lots were not contaminated from the same plasmapheresis donor. To obtain molecular evidence that the HAV detected in the factor VIII preparations was responsible for transmission of HAV, serial serum samples from two patients were tested for HAV gene sequences and compared with those of the implicated lots. The genomic sequences of HAV obtained for two matched sets of factor VIII and recipient serum samples were identical within each set, but different for the two sets.

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.

PREVALENCE OF TT VIRUS IN HEALTHY CHILDREN AND THALASSEMIC PEDIATRIC AND YOUNG ADULT PATIENTS

Background The recently discovered TT virus (TTV) has been shown to be highly prevalent in patients with cryptogenetic chronic liver disease and fulminant hepatitis. To study the frequency of TTV and to evaluate the possible association with liver disease, 37 pediatric and young adult patients with thalassemia, and 36 healthy children were included in the study. The sera of 100 blood donors selected randomly in the same period were also tested for TTV DNA. Methods The TTV amplification by polymerase chain reaction (PCR) was performed using a first set of primers that recognize an internal sequence into N22 and a second set of primers amplifying a sequence within 5´NCR (5´ noncoding region). Results The first set of primers revealed TTV DNA in 73% of thalassemic patients, in 8% of healthy children, and in 5% of healthy blood donors. With the second set of primers, the prevalence of TTV DNA was, respectively, 100% in thalassemic patients, 44.5% in healthy pediatric patients, and 87% in healthy blood donors. All individuals who tested positive for TTV by the first set of primers were also positive by the second primer set. The TTV infection seemed not to be the cause of altered transaminase levels. Sequencing of TTV clones from thalassemic patients showed the presence of different TTV variants in the same serum. Conclusion The prevalence of TTV in polytransfused children is similar to that detected in blood donors. Moreover, TTV can be detected in healthy children of all ages. The presence of TTV seems to have no clinical significance.

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.