P L Yap

Detection of a novel DNA virus (TT virus) in blood donors and blood products

Summary Background A newly discovered DNA virus, transfusion- transmitted virus (TTV), has been implicated as a cause of post-transfusion hepatitis. We investigated the frequency of TTV viraemia in UK blood donors, and the extent to which TTV contaminates blood products such as factor VIII and IX clotting factors. We also investigated the possible aetiological role of TTV in cryptogenic fulminant hepatic failure (FHF). Methods We extracted DNA from plasma of blood donors and patients with FHF, and from blood products (factor VIII and IX clotting-factor concentrates, immunoglobulin preparations). We detected TTV by PCR using primers from a conserved region in the TTV genome. Findings TTV viraemia was detected in 19 (1·9%) of 1000 non-remunerated regular blood donors. Infection occurred more frequently in older donors (mean age 53 years), compared with the age prolife of donors infected with hepatitis C virus and other parenterally-transmitted viruses. TTV contamination was found in ten (56%) of 18 batches of factor VIII and IX concentrate manufactured from such non- remunerated donors, and in seven (44%) of 16 batches of commercially available products. Whereas solvent or detergent treatment had little effect on the detection of TTV in factor VIII and IX by PCR, this virucidal step seemed to inactivate TTV infectivity. TTV infection was detected in four (19%) of 21 patients with FHF; in three cases, infection was detected at the onset of disease and could thus not be excluded from its aetiology. Interpretation TTV viraemia is frequent in the blood-donor population, and transmission of TTV through transfusion of blood components may have occurred extensively. Clinical assessment of infected donors and recipients of blood and blood products, and assessment of TTVs aetiological role in hepatic and extra-hepatic disease, are urgently needed.

Detection of three types of hepatitis C virus in blood donors : investigation of type-specific differences in serologic reactivity and rate of alanine aminotransferase abnormalities

The serologic reactivity and epidemiology associated with different hepatitis C virus (HCV) variants were investigated in a cohort of 113 anti‐HCV‐positive donors. In Scotland, HCV type 1 accounted for one‐ half of all infections; 40 percent of subjects were infected with HCV type 3, and the remainder were infected with type 2. Reactivity with the NS‐4‐encoded antigens in the first‐generation anti‐c100 assay was absent in 68 percent of donors infected with types 2 and 3, as compared with 10 percent for those infected with type 1. Even when combined with surrogate marker testing, first‐generation tests would have failed to detect 12 percent of HCV‐infected blood donors. The age distribution, incidence of past infection with hepatitis B virus, and reported risk factors were similar in donors infected with types 1 and 3 (mean ages were 31.9 and 29.9; 18 and 17.5% were positive for antibody to hepatitis B core antigen; and 47 and 48% had past intravenous drug abuse). However, the distributions of alanine aminotransferase levels were significantly different in those infected with type 3 (abnormally raised in 83%) and those infected with type 1 (55% abnormal alanine aminotransferase; p < 0.05) or type 2 (60%; p < 0.01) and those who were nonviremic (8%; p < 0.0001). These data suggest that HCV type 1 is the most common HCV infection in blood donors and that infection with HCV type 3 may be associated with more severe liver disease, because of more recent infection or because of a greater inherent pathogenicity of type 3 variants.

Investigation of the pattern of hepatitis C virus sequence diversity in different geographical regions: implications for virus classification

Genotypes of hepatitis C virus (HCV) present within 104 samples from HCV-infected individuals from Africa, the Middle East, the Indian subcontinent and South-East Asia were identified by sequence comparisons in the core and NS-5 regions. Relatively short sequences (such as the 222 bp fragment of NS-5) provided effective discrimination of types, subtypes and isolates, and produced equivalent relationships between genotypes as were found upon comparison of longer sequences of NS-5, of the core region, and by comparison of the limited number of complete genomic sequences currently available. Measurement of evolutionary distances in the core and NS-5 regions allowed 79 of the 104 samples to be identified as examples of known genotypes, while 17 of the remainder could be provisionally classified as new subtypes of types 1 (Nigeria), 2 (Gambia), 3 (India, Pakistan and Bangladesh) and 4 (Middle East) on the basis of sequence comparison in core and NS-5 (n = 9) or provisionally using core alone (n = 8). The remaining sequences from Thailand made up two groups showing no close similarity to any of the six major genotypes classified to date, although one corresponded to an as yet unclassified variant of HCV also found in Thailand. However, phylogenetic analysis of the core and NS-5 regions indicated a distant relationship between these sequences with variants found in Vietnam and with type 6a, and collectively they formed a diverse single phylogenetic group. The existence of great diversity within a single genotype was also found amongst type 3 sequences in the Indian subcontinent, amongst type 4 variants in Central Africa and the Middle East, and amongst type variants in Nigeria. These findings may provide clues for understanding the origins and mechanisms of transmission of HCV.

Hepatitis C quantification and sequencing in blood products, haemophiliacs, and drug users

The polymerase chain reaction (PCR) detected specific hepatitis C viral (HCV) RNA sequences in plasma from 15 of 21 haemophiliacs (12 HCV-antibody positive) and 7 of 27 intravenous drug users (13 HCV-antibody positive). Quantification of RNA-positive samples showed high levels of HCV (10(5) to 10(6) copies of RNA/ml) in infected patients. HCV was more frequently found in haemophiliacs infected with human immunodeficiency virus (11/11 HIV-positive and 4/10 HIV-negative patients). HCV-RNA was detected in all batches of commercially available factor VIII tested and in low concentrations in some pools of plasma donations from volunteers. Factor VIII, manufactured from volunteer donations, was uniformly negative by PCR. Phylogenetic analysis of viral sequences showed two distinct groups: one was associated with intravenous drug users and the other with haemophiliacs infected with Scottish factor VIII preparations. Both were distinct from sequences found in commercially available factor VIII.

Molecular epidemiology of an outbreak of infection with hepatitis C virus in recipients of anti-D immunoglobulin

In a retrospective investigation of possible transmission of hepatitis C virus (HCV) by anti-rhesus D immunoglobulin (anti-D) in 1977, we compared variants infecting anti-D recipients in Ireland of one of the implicated batches with those of epidemiologically unrelated HCV-infected individuals. All 100 of the recipients of the batch investigated to date were infected with a single genotype (type 1), consistent with a single-source outbreak, whereas a wider range of genotypes (1, 2, and 3) were found in anti-HCV positive individuals from Ireland infected by different routes. Nucleotide sequences from a 222 base fragment from the NS-5 region of the genome amplified from stored aliquots of the implicated batch closely matched those detected in anti-D recipients 17 years after the transmission event. This study shows the value of molecular epidemiological techniques for identifying distant sources of infection, and for the epidemiological investigation of the current distribution and transmission of HCV in different populations.

Human parvovirus B19 and blood products

Background and objectives: Human B19 parvovirus (B19), identified in 1975, was only recognised as the causative agent of fifth disease in 1983. The incidence of viraemia is low, around 1 in 1,000, but is sufficient to ensure that most plasma pools for fractionation contain some virus. While infection usually occurs in childhood and is benign, chronic infection sometimes occurs and may be of concern in certain patient groups. Materials and methods: This review is based on a meeting held in March 1995, and addresses recent concerns regarding the potential transmission of B19 infection by pooled plasma products. Results: Recent data on the pathophysiology and assay of this virus are summarised along with possible approaches to donor screening, product screening, and virus removal. Only five cases of symptomatic infection have been reported in persons with haemophilia, but no technology for virus removal is established, and infection may be of concern in pregnant women, and in patients with enhanced red cell turnover or who are immunosuppressed, including those infected with human immunodeficiency virus, but only rarely in immunocompetent patients. Conclusions: For the future, well‐validated assays relevant to virus infectivity are required if blood donations, plasma pools, or plasma products are to be screened, and an in‐process virus inactivation step for B19 would be highly desirable. In the interim, non‐plasma or recombinant products or a selective transfusion policy might be used in patient groups in which B19 infection is of particular concern. Further clinical data on the prognosis and impact of B19 infection are needed to justify both such policies and the future adoption of new technologies designed to reduce any excess B19 infectivity arising from transfused products.

Serological responses to infection with three different types of hepatitis C virus

6. Scambler PJ, Carey AH, Wyse RKH, et al. Microdeletions within 22q1 1 associated with sporadic and familial DiGeorge syndrome. Genomics 1991, 10: 201-06 7. Driscoll DA, Budarf M, McDermid H, Emanuel BS. Molecular analysis of DiGeorge syndrome: 22q11 interstitial deletions Am J Hum Genet 1990, 47: A215. 8. Driscoll DA, Budarf ML, Emanuel BS. Mapping the critial region in DiGeorge syndrome. Am J Hum Genet 1991; 49: A86. 9. Kuwano A, Ledbetter SA, Dobyns WB, Emanuel BS, Ledbetter DH Detection of deletions and cryptic translocations in Miller-Dieker syndrome by in situ hybridization Am J Hum Genet 1991; 49: 707-14.

The distribution of hepatitis C virus genotypes in Turkish patients

Summary. The distribution of hepatitis C virus (HCV) genotypes was investigated in 89 HCV‐infected Turkish patients. Blood samples were collected from haemodialysis patients (n= 45), chronic liver disease (CLD) patients (n= 38), acute non‐A, non‐B (NANB) hepatitis patients (n= 2) and blood donors (n= 4). HCV RNA sequences were amplified in the 5″ non‐coding region and were typed by restriction fragment length polymorphism analysis. The predominant genotype was 1b (75.3%), followed by 1a (19.1%), 2 (3.4%) and 4 (2.2%). While there was no significant difference in the distribution of HCV genotypes with respect to age, sex, transfusion history, alanine aminotransferase levels or liver histology (in the CLD group), type 1a‐infected patients were younger than type 1b‐infected patients (P < 0.05) in the haemodialysis group. Serological reactivity to recombinant HCV proteins was assessed in 58 samples using the Chiron RIBA‐2 assay. The reactivity of samples from patients infected with type 1b with 5–1–1 and c100 antigens was significantly lower (P < 0.05) than the reactivity of samples from those infected with type 1a. These results, together with the results of two previous studies, indicate that HCV genotypes 1, 2, 3 and 4 are prevalent in different frequencies in the Turkish population. Determination of the genotype distribution of HCV in a geographical area may provide important clues for studying the epidemiology, transmission and pathogenesis of HCV‐related diseases and may also aid in improving serological assays to detect HCV infection.

Transmission of non-A, non-B hepatitis by pH4-treated intravenous immunoglobulin.

Abstract. Four patients (2 with X‐linked, one with common variable hypogammaglobulinaemia, and 1 with ulcerative colitis) developed non‐A, non‐B hepatitis (NANBH) following administration of a specific batch of intravenous immunoglobulin (IV IgG) manufactured by the Scottish National Blood Transfusion Service using the pH4/mild pepsin method. Each patient had normal serum ALT levels over a preceding period of 12–67 months, with raised values developing within 4–18 weeks of first administration of the implicated batch. Two patients had very mild symptoms of hepatitis, the other 2 being asymptomatic. Over a follow‐up period of 8–12 months, ALT levels returned to normal in 3 patients, but biopsy‐proven chronic NANBH developed in the fourth. The level of NANBH virus in the starting plasma used to manufacture this batch may have exceeded the capacity of the process to inactivate the virus. The transmission of NANBH by one of approximately 110 batches administered demonstrates the importance of continued close surveillance of recipients of IV IgG, even if asymptomatic, by regular monitoring of liver function tests and recording of all batches received.

Relevance of RIBA-3 supplementary test to HCV PCR positivity and genotypes for HCV confirmation of blood donors

HCV antibody screening of 624,910 blood donations resulted in 3,832 samples being referred for confirmation. All were tested by RIBA‐3 with 2,710 negative, 945 indeterminate and 177 positive results. HCV RNA was detected by PCR in an average of 69.5% of RIBA‐3 positives (4 bands 84.1%; 3 bands 74.1%; 2 bands 34.1%) and only 0.53% of RIBA‐3 indeterminates. Eighty‐four percent of samples with a total RIBA‐3 band intensity score (maximum 16) of ≥8 were PCR positive compared with only 22% of those with a score of <8. Total mean band intensities for HCV genotype 1 samples (n = 65) were 13.2, genotype 2 (n = 17) 11.4 and genotype 3 (n = 65) 11.2 with type 1 samples showing greater reactivity with c100 and c33 antibodies. No PCR positive type 1 samples were found with RIBA‐3 total band scores less than 8, no PCR positive type 2 samples less than 6, whilst PCR positive type 3 samples were found with scores as low as 2. NS5 indeterminates were the most common (40.2%) single band pattern but yielded no PCR positive samples, followed by c33 (23.3%) with one PCR positive and c100 (20.2%) with one PCR positive whilst c22 indeterminates were least common (16.3%) but included three PCR positive donors. All five RIBA‐3 indeterminate PCR positive donors were type 3.