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K Cristiano

Publications and source records attributed to K Cristiano.

At least 19 recordsLinked to original sources
PubMed2004

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). MATERIALS AND METHODS: Three independent panels, one for each virus, were prepared at the Istituto Superiore di Sanità (ISS) by diluting the respective reference preparations. NAT methods used by the EQA participants included polymerase chain reaction (PCR) assays by Roche, transcription-mediated amplification (TMA) assays by Chiron and in-house PCR assays. RESULTS: Forty-three of the 45 participants (95.6%) in the HCV EQA/5 who used a validated method were consistently able to detect a nominal concentration of 100 IU/ml for all six major genotypes. In the case of the HIV EQA/1, all 35 participants detected the samples containing 1000 IU/ml HIV, while five (14.3%) did not identify the samples containing 100 IU/ml HIV. With respect to the HBV EQA/1, all 16 participants correctly identified the positive samples containing either 1000 IU/ml or 100 IU/ml HBV. No false-positive results were observed with any of the three panels. CONCLUSIONS: The HCV EQA/5 showed an improved proficiency of laboratories as compared with the HCV EQA/4. In fact, HCV genotypes 1, 2, 3 and 5 were correctly identified in 100% of the assays and genotypes 4 and 6 in 97.8% of the assays. While most of the participants in the HIV EQA/1 showed a good level of proficiency, an excellent performance was shown by all participants in the HBV EQA/1.

False Positive Reactions↗
PubMed2003

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

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.

Blood Transfusion↗
PubMed2001

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

BACKGROUND AND OBJECTIVES: 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. MATERIALS AND METHODS: A reference HCV RNA panel calibrated in international units (IU) was sent to each participant to be tested according to the validated procedure they routinely used in plasma pool testing. The panel consisted of 20 coded samples, four of which were obtained from a negative plasma pool. The remaining 16 samples, prepared by diluting the national reference preparation (ISS HCV RNA 0498), represented four half-log dilution series, each consisting of four samples containing 100, 32, 10 and 3.2 IU/ml of HCV RNA. RESULTS: The overall performance of the laboratories was very satisfactory. All laboratories correctly identified the negative samples. The 100- and 32-IU/ml samples were both detected in 98.4% of the assays, while the 10- and 3.2-IU/ml samples were detected in 73.4 and 50.0% of the assays, respectively. No substantial differences were observed between in-house procedures and commercial kits. CONCLUSION: This external quality assessment study showed that manufacturers of blood products have reached a high level of proficiency that fully complies with the European Pharmacopoeia requirements. This finding is reassuring in the context of the safety of blood products.

Blood Specimen Collection↗
PubMed1999

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.

Blood↗
PubMed1993

Hepatitis C infection in children and adolescents on haemodialysis and after renal transplant.

Serum antibodies to hepatitis C virus (HCV) were measured in children and adolescents on haemodialysis (HD, n = 20) and after renal transplant (RT, n = 33). Seropositivity was observed in 3 HD patients (15%) and in 7 RT patients (21.2%) with an enzyme-linked immunosorbent assay (2nd generation) and a recombinant immunoblotting assay (2nd generation). HCV RNA was detected by the polymerase chain reaction in the 10 patients with anti-HCV antibodies. Anti-HCV positivity was significantly correlated (P < 0.05) with the number of blood transfusions and the time on HD. Transaminase levels were not useful for screening. This study confirms that there is a high risk of HCV infection in children and adolescents on HD or after RT. Moreover, HCV infection is closely related to the number of blood transfusions as well as the time on HD.

Adolescent↗
PubMed1992

Prevalence of hepatitis C virus antibodies and hepatitis C virus-RNA in an urban population.

Several studies had been carried out on anti-hepatitis C virus (HCV) prevalence in populations with blood exposure risks and in blood donors. New tests are now available which allow the investigation to extend to other parameters such as antibody type and HCV-RNA. In this study the prevalence of anti-HCV c100-3 and the associated epidemiological, clinical, and virological markers were evaluated in subjects from an urban population located in central Italy. In positive cases the time persistence of HCV-RNA and anti-HCV antibody pattern was studied. For this purpose, sera from 1,484 randomly sampled individuals, aged 30-69 years, collected in 1985 and stored at -80 degrees C were retrospectively tested. The prevalence was 0.87% (i.e., 13 anti-HCV c100-3 positive cases). A significant association was observed with raised alanine transaminase (ALT) levels (P less than 0.001). Paired serum samples from 11 out of the 13 subjects collected in 1985 and 1991 were tested by nested polymerase chain reaction (PCR) using primers from the 5' non-coding region and by 4-RIBA. Concordant RIBA patterns between 1985 and 1991 were observed in the majority of positive paired sera (7/9) as well as for HCV-RNA (6/9). HCV-RNA was present in sera simultaneously positive to both types of antibody or to anti-c100-3 or anti-c22 alone. A wide spectrum of viral and antibody patterns in anti-HCV c100-3 positive sera was observed in this urban population and persisted for at least 6 years.

Adult↗
PubMed1992

Hepatitis C viral RNA in serum of patients with chronic non-A, non-B hepatitis: detection by the polymerase chain reaction using multiple primer sets.

The recently introduced antibody test for hepatitis C virus (HCV) infection has proven to have certain limitations. Since HCV itself is usually present in clinical specimens at very low titers, a useful assay for the virus must have very high sensitivity. We have developed a simple, highly sensitive assay for HCV RNA based on the polymerase chain reaction (PCR). In this test, RNA extracted from HCV infected serum or plasma is used as the template for double PCR with nested primers. Sensitivity studies demonstrate that this assay is able to detect HCV at or beyond the sensitivity level of chimpanzee infectivity. We tested, with several sets of nested primers, 40 patients with chronic non-A, non-B hepatitis (36 seropositive and 4 seronegative) and found that 35/40 were PCR positive including all 4 seronegative patients. Normal human plasma and plasma from hepatitis B infected patients did not react in this test. This assay has proven to be valuable for determining the presence of HCV in various samples; furthermore, it offers the possibility of diagnosis of HCV infection in seronegative patients.

Adult↗
PubMed1991

Decrease in serum hepatitis C viral RNA during alpha-interferon therapy for chronic hepatitis C.

OBJECTIVE: To assess the effect of alpha-interferon therapy on hepatitis C viral RNA in serum of patients with chronic hepatitis C. DESIGN: Retrospective testing for hepatitis C viral (HCV) RNA and antibody to the hepatitis C virus (anti-HCV) of stored serum samples from a randomized, double-blind, placebo-controlled trial of alpha-interferon therapy. SETTING: Warren Grant Magnuson Clinical Center of the National Institutes of Health, a tertiary referral center. PATIENTS: Forty-one patients with chronic non-A, non-B hepatitis were entered in this trial. INTERVENTIONS: Twenty-one patients were treated with alpha-interferon, and 20 patients were treated with placebo for 6 months. Seventeen placebo recipients were then treated with alpha-interferon for up to 1 year. METHODS: Samples were tested for anti-HCV by enzyme-linked immunosorbent assay. Hepatitis C viral RNA was detected in serum using the polymerase chain reaction. Titers of both antibody and RNA were determined by serial end-point dilution. MAIN RESULTS: At entry into the trial, 37 (90%) of 41 patients had anti-HCV and 39 (95%) had HCV RNA in serum. Anti-HCV titers decreased slightly with treatment. Serum levels of HCV RNA decreased in all patients who responded to alpha-interferon therapy with improvements in serum aminotransferases; in 17 of 21 responders (81%; 95% Cl, 58% to 95%) HCV RNA became undetectable. In contrast, in only 2 of 16 (12%; Cl, 2% to 38%) patients who did not respond to treatment did HCV RNA become undetectable. In 19 patients treated during the preliminary 6-month period with placebo, HCV RNA remained detectable. Finally, in the 11 patients who relapsed when treatment was stopped, HCV RNA reappeared in the serum, but in 4 of 7 patients with a sustained improvement in serum aminotransferases, HCV RNA remained undetectable. CONCLUSIONS: These results indicate that the clinical and serum biochemical response to alpha-interferon in chronic hepatitis C is associated with a loss of detectable HCV genome from serum.

Base Sequence↗
PubMed1991

Hepatitis C viral RNA in serum of patients with chronic non-A, non-B hepatitis: detection by the polymerase chain reaction using multiple primer sets.

The recently introduced antibody test for hepatitis C virus infection has already proved to be valuable in many situations such as screening blood donors and diagnosing chronically infected patients, but this antibody assay has certain limitations. Hepatitis C virus itself is usually present in clinical specimens at very low titers; therefore a useful assay for the virus must have very high sensitivity. We have developed a simple, highly sensitive assay for hepatitis C virus RNA based on the polymerase chain reaction. In this test RNA extracted from hepatitis C virus-infected serum or plasma is used as the template for double polymerase chain reaction with nested primers. Sensitivity studies demonstrate that this assay is able to detect hepatitis C virus at or beyond the sensitivity level of chimpanzee infectivity. Preliminary studies in chronic non-A, non-B hepatitis showed that 16 of 36 patients positive for antibody to hepatitis C virus and 2 of 4 patients negative for antibody to hepatitis C virus were positive in the polymerase chain reaction test. By retesting the polymerase chain reaction-negative patients with several sets of polymerase chain reaction primers, we found hepatitis C virus RNA in 35 of the 40 patients including all 4 seronegative patients. Normal human plasma and plasma from patients with hepatitis B infection did not react in this test. This assay has proved to be valuable for determining the presence of hepatitis C virus RNA in various samples. Furthermore, it offers the possibility of diagnosis of hepatitis C virus infection in patients who do not react in the presently available antibody tests.

Animals↗
PubMed1989

Co-amplification of multiple regions of the HIV-1 genome by the polymerase chain reaction: potential use in multiple diagnosis.

We have used the polymerase chain reaction (PCR) to detect by co-amplification, multiple regions of the HIV-1 genome in infected cells. Genomic RNA and DNA from productively infected H9 cells were independently extracted and amplified in reactions with and without reverse transcriptase respectively using primer pairs to the gag, env, tat and nef regions of the viral genome in the same reaction mixture. PCR-products were analysed by liquid hybridization with end labelled oligonucleotide probes followed by gel-electrophoresis (oligomer hybridization). The primer pairs were capable of detecting as few as 10 copies of RNA and 10-20 copies of integrated proviral DNA. The ability to co-amplify multiple target regions in the same incubation mixture provides a method for detecting and confirming the presence of HIV-1 in samples for which limited nucleic acid is available. In addition, in reconstitution experiments, the same method was used to detect HIV-1 and HTLV-I simultaneously with comparable sensitivity (20-40 gene copies each). This offers the possibility of simultaneous diagnosis of multiple viral infections, such as those that occur in AIDS, on the same sample preparation.

DNA, Viral↗