Global distribution of transfusion-transmitted virus.
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Global Health↗
Biomedical subjects
P Simmonds
Publications and source records attributed to P Simmonds.
Detection of a novel DNA virus (TTV) in blood donors and blood products.
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 TTV's aetiological role in hepatic and extra-hepatic disease, are urgently needed.
Adult↗
Sexual transmission of GB virus C/hepatitis G virus.
Although it is established that infection with GB virus C (GBV-C) or hepatitis G virus (HGV) can be transmitted parenterally, the prevalence of GBV-C/HGV viremia in the general population (2-5%) is relatively high compared with other parenterally borne viruses such as hepatitis C virus. To investigate the possibility of sexual transmission of GBV-C/HGV, we determined the frequency of viremia by the polymerase chain reaction and serological reactivity to the E2 protein by ELISA in samples collected from individuals at risk for sexually transmitted diseases attending a city genitourinary medicine clinic. GBV-C/HGV viremia was detected in 27 of 87 male homosexuals (31%) and 9 of 50 prostitutes (18%), frequencies significantly greater than those in matched controls (2/63) and local blood donors (2.3%). Among nonviremic individuals, a high frequency of serological reactivity to the E2 protein of GBV-C/HGV was also observed in the risk groups (male homosexuals: 14/60; prostitutes: 11/41), although these figures are likely to be underestimates of the frequency of past infection as detectable anti-E2 reactivity may attenuate rapidly over time following resolution of infection. Infection with GBV-C/HGV was more frequent among those coinfected with human immunodeficiency virus type 1. Among male homosexuals from whom retrospective samples were available, evidence for de novo infection was found in 9 of 22 individuals over a mean sampling time of 2.9 years, predicting an annualized incidence of GBV-C/HGV infection of approximately 11% in this group. The high prevalence and incidence of GBV-C/HGV infection in these individuals and prostitutes provides strong evidence for its spread by sexual contact. Further studies are required to investigate the mechanism of its transmission and the clinical significance of acute and persistent infection in these risk groups.
Adolescent↗
Classification, nomenclature, and database development for hepatitis C virus (HCV) and related viruses: proposals for standardization. International Committee on Virus Taxonomy.
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Databases, Factual↗
In situ polymerase chain reaction amplification of HIV-1 DNA in brain tissue.
A direct in situ polymerase chain reaction (IS-PCR) assay is described for the detection of HIV-1 proviral DNA in formalin fixed paraffin embedded brain tissue. Biotin-16-dUTP is incorporated during the PCR process and microwave pretreatment of tissue sections ensures that no non-specific incorporation into damaged or nicked genomic DNA occurs. Two methods are compared to detect the biotinylated amplified product, the use of an avidin-biotin-alkaline phosphatase complex (ABC) and the application of tyramide signal amplification (TSA) which allows both chromogenic and fluorescence detection. TSA detection enhances the sensitivity of IS-PCR, permitting fewer PCR cycles and preserving tissue morphology.
Brain↗
Detection of GB virus C RNA by GBV-C LCx and two PCR assays with primers from the 5' non-coding and NS5B region.
The objective of this study was to compare the sensitivity of three different reverse transcriptase-polymerase chain reaction (RT-PCR) based tests, for detection of GB virus C (GBV-C) RNA. One commercial and two 'in house' RT-PCR assays were employed in the testing of serum samples from 114 chronic hepatitis C infected individuals. A part of the 5' non-coding region (5'NCR) of the GBV-C genome was amplified by the GBV-C LCx assay (Abbott) and one of the 'in house' RT-PCR tests. In the other 'in house' RT-PCR a segment of the NS5B region was amplified. The 'in house' assays included the use of internal controls that were co-amplified with use of the same outer PCR primers as the virus targets. The GBV-C LCx from Abbott and 5'NCR 'in house' PCR tests detected 28 and 27 GBV-C positive individuals, respectively. The sample positive only in the LCx test was confirmed by the 'in house' 5'NCR RT PCR using an increased virus input. In comparison, the NS5B 'in house' PCR test detected 24 of the GBV-C positive samples. One sample showed no amplification of internal controls/virus target in the 5'NCR 'in house' PCR and another samples was amplification negative in the NS5B PCR. The PCR assays with primers from the 5'NCR of the virus genome e.g. the GBV-C LCx, were more sensitive compared with RT-PCR using primers from the NS5B region. The GBV-C LCx seemed to be the most sensitive and robust assay. Internal controls included in the 'in house' assays identified two samples with failure of the amplification.
5' Untranslated Regions↗
Transfusion virology: progress and challenges.
Discoveries of new human viruses and new technologies for their detection have made, and will continue to make, major contributions to the safety of blood transfusion. This article discusses the practical issues involved in the implementation of additional serological screening tests for viruses such as human T-lymphotropic virus, and reviews current information on the prevalence and pathogenicity of more recently discovered viruses, such as hepatitis G virus (HGV) or GB virus-C (GBV-C) and human herpes virus 8, a potential aetiological agent of Kaposi's sarcoma. Progress in the technology behind nucleic acid amplification techniques, such as the polymerase chain reaction (PCR), makes direct detection of viruses such as human immunodeficiency virus and hepatitis C virus possible. The use of such methods for screening will allow the earlier detection of acutely-infected individuals and the elimination of transmission from 'window' period donations before seroconversion for antibody. Establishing a framework for PCR-based screening would also enable the testing for others such as hepatitis A virus, parvovirus B19 and GBV-C/HGV for which serological detection methods cannot be or have not been developed.
Blood Transfusion↗
The effect of treatment with alpha-interferon on hepatitis G/GBV-C viraemia. The CONSTRUCT Group.
BACKGROUND: Hepatitis G virus (HGV) or GBV-C is frequently detected in patients co-infected with hepatitis C virus (HCV). This study investigated host and virologic factors influencing the response to HGV/GBV-C to alpha-interferon treatment. METHODS: HGV/GBV-C was detected and quantified by nested polymerase chain reaction. The influence of variables such as liver biopsy appearance, liver function abnormalities, and response of HCV to interferon treatment was monitored. RESULTS: Fourteen of the 25 HGV/GBV-C-infected patients treated with interferon (3-6 MIU three times a week for 6 months) became non-viraemic during treatment, although all relapsed after treatment withdrawal at 6 months, with no net change in virus load between 0 and 12 months. CONCLUSIONS: Predictive factors for clearance of HGV/GBV-C viraemia by interferon were pre-treatment severity of liver disease (median Knodell score of 4, compared with 7 for non-responders; P = 0.030) and alanine aminotransferase levels (median, 114, 182 for non-responders; P = 0.039). Clearance was associated with the treatment response of HCV. Nine of 13 who cleared HGV/GBV-C also cleared HCV, compared with 3 of 11 HGV/GBV-C non-responders; P = 0.05). The shared susceptibility of HGV/GBV-C and HCV to interferon treatment suggests a link between the mechanism of clearance of the two viruses.
Adult↗
Prevalence, incidence, and clinical characteristics of hepatitis G virus/GB virus C infection in Scottish blood donors.
The prevalence, incidence, clinical features, and natural history of hepatitis G virus (HGV) or GB virus C (GBV-C) were investigated in a non-remunerated blood donor population to determine its clinical significance and its impact on blood safety. Of 1020 regular blood donors, 23 (2.25%) were positive for plasma HGV/GBV-C RNA. Alanine aminotransferase levels were lower than in uninfected donors (median, 20 IU/mL; 32 IU/mL in controls; P=.015). Clinical examination produced no other evidence for hepatitis or for shared nonhepatic diseases. Fifteen of 17 donors excreted HGV/GBV-C in saliva (mean level, 8x103 copies of RNA/mL). Testing of previous donations indicated an incidence of 170-200 new infections with HGV/GBV-C per 100,000 donor-years. The absence of further clinicopathologic data and the limitations of current polymerase chain reaction-based methods for screening suggests that it is neither necessary nor practical to commence screening.
Adult↗
HIV encephalitis, proviral load and dementia in drug users and homosexuals with AIDS. Effect of neocortical involvement.
In this consecutive autopsy study, the pathological evidence of HIV encephalitis, which included the presence of giant cells and/or HIV p24 immunopositivity, was found more frequently in drug users (25 of 45; 56%) than in homosexual men (6 of 35; 17%) with AIDS (P < 0.01). Productive infection, as shown by HIV p24 positivity, was found in frontal lobe white matter in 29 of the 31 HIV encephalitis cases, but was also present in grey matter in 50% of the HIV encephalitis cases. Immunopositivity was confined to microglia, monocytes and most but not all giant cells. HIV-1 proviral load was determined by quantitative PCR in 65 of the 80 cases (separately in grey and white matter in 49 of these), and correlated well with the presence of HIV encephalitis (P < 0.001). Twenty-five patients with AIDS (13 male homosexuals, 12 drug users) showed no HIV encephalitis, opportunistic infection or cerebral lymphoma, while 18 (2 male homosexuals, 16 drug users) showed pure HIV encephalitis. Cognitive function had been assessed prospectively in this cohort and graded as normal or mildly, moderately or severely impaired. Because opportunistic infections and lymphomas of the brain may also lead to dementia, patients found to have these conditions at autopsy were excluded from the final analysis of the cases with dementia, so that the precise correlation between cognitive impairment and pure HIV encephalitis could be determined in this cohort without possible confounding variables. Fourteen of 18 patients with pure HIV encephalitis had shown cognitive impairment. Severe dementia correlated better with pure HIV encephalitis in cases in which grey matter involvement was present (7 out of 9) than in those in which only white matter was involved (2 out of 9) (P < 0.05), although milder degrees of cognitive impairment had been present in a further 5 HIV encephalitis cases. No correlation was found between zidovudine therapy and the degree of cognitive impairment. Systemic and cerebral opportunistic infections and lymphoma showed a negative association with HIV encephalitis, being more common in homosexuals than in drug users, despite comparable CD4 counts in the two groups. These findings suggest that neocortical productive HIV infection is a significant factor in AIDS-related dementia, although this may reflect merely a higher overall viral burden in the brain.
AIDS Dementia Complex↗
Neutralization of primary and T-cell line adapted isolates of human immunodeficiency virus type 1: role of V3-specific antibodies.
The role of the third variable domain (V3) of gp120 in the neutralization of primary and T-cell line adapted (TCLA) strains of human immunodeficiency virus type 1 (HIV-1) by serum from HIV-1-infected individuals was investigated. A primary virus isolate, M2424/4, when adapted to H9 cells, was more sensitive to neutralization on MT2 cells than the same stock passaged in PBMC. Neutralization of the PBMC-passaged stock by V3-specific MAbs was abrogated by addition of V3 (MN) peptide. However, exogenous V3 (MN) peptide failed to reduce the neutralization of this isolate on PBMC, or MT2 cells, by high titre anti-HIV-1 polyclonal human sera in contrast to the extensive reduction of neutralization by the same sera on MT2 cells using the prototype MN strain (4- to > or = 24-fold) and the TCLA M2424/H9 isolate (2- to 8-fold). These results indicate that the neutralization of primary virus isolates by serum from HIV-1-infected individuals is not significantly mediated by V3-specific antibodies.
Adaptation, Biological↗
Low level or absent in vivo replication of hepatitis C virus and hepatitis G virus/GB virus C in peripheral blood mononuclear cells.
To investigate which subsets of peripheral blood mononuclear cells (PBMCs) are susceptible to infection with hepatitis C virus (HCV) and hepatitis G virus (HGV) or GB virus C (GBV-C), a PCR-based assay using tagged primers in the core region (HCV) and NS3 region (HGV/GBV-C) for the specific detection of negative strand (replicating) viral RNA sequences was developed. In liver biopsy samples both positive and negative strands of HCV RNA were detected, at levels ranging from 3 to 11 x 10(6) RNA copies per 10(6) cells and 3.7-4.2 x 10(3) copies per 10(6) cells respectively, while lower frequencies of positive strands of GBV-C/HGV RNA were detected (from 13 biopsies, the highest frequency was 7.3 x 10(3) per 10(6) cells). In no samples were negative RNA strands detected. To investigate extra-hepatic replication of HCV and GBV-C/HGV, CD4+, CD8+ and B lymphocytes, monocytes and putative dendritic cell populations were separated from PBMCs from ten study subjects. Detection of positive strand HCV RNA was largely confined to B lymphocytes (at levels of up to 5 x 10(3) copies per 10(6) cells), while detection of negative strands was confined to a single subset (dendritic cells) of one of the study individuals. Similarly, GBV-C/HGV was detected at low levels in only twelve of twenty PBMC samples, while negative strands were uniformly absent. The low levels of HCV and GBV-C/HGV RNA in PBMCs suggest that these cells are at most a minor reservoir for virus replication. The absence of detectable replication of GBV-C/HGV suggests that the actual site of GBV-C/HGV replication remains to be discovered.
B-Lymphocytes↗
Detection in chimpanzees of a novel flavivirus related to GB virus-C/hepatitis G virus.
Infection with hepatitis G virus (HGV) or GB virus-C (GBV-C) is widely distributed in human populations. Viruses related to GBV-C/HGV have been recovered from several New World primate species, including tamarins, owl monkeys and marmosets. To understand more about the relationship between GB viruses and their hosts, we used primers from the 5' non-coding (5'NC), non-structural 3 (NS3) and NS5 regions in nested polymerase chain reactions to screen for related viruses infecting non-captive chimpanzees (Pan troglodytes, troglodytes and verus subspecies). Sequences from the 5'NCR and NS5 regions were amplified from samples taken from 3 of 39 chimpanzees, and from one chimpanzee in the NS3 region. Sequence comparisons of each region revealed that the GB virus infecting chimpanzees was distinct from both GBV-C/HGV and from any of the known GBV-A sequences, but was more closely related to human viruses. GB viruses recovered from different chimpanzees were more diverse than variants of GBV-C/HGV found in humans, with 25% sequence divergence in the 5'NCR and 20% (9.5% amino acid) sequence divergence in NS5 between variants recovered from the troglodytes and verus subspecies, compared with 7.4% and 10.4% (1.9% amino acid) divergence amongst GBV-C/HGV variants infecting humans. Finding GBV-C/HGV-related viruses in an Old World monkey species suggests that GB-like viruses may be widely distributed in simians, and suggests a close evolutionary relationship with their natural hosts.
Amino Acid Sequence↗
High prevalence of hepatitis G virus infection in multiply transfused children with thalassaemia.
We investigated the prevalence of hepatitis G virus (HGV) RNA in relation to the frequency of blood transfusions in thalassaemic children and in volunteer blood donors in Thailand. Furthermore, we studied the frequency of coinfection with hepatitis B virus (HBV) and/or hepatitis C virus (HCV) as well as a possible relationship to the alanine aminotransferase (ALT) status of the blood samples, taken at random from thalassaemic children who have received multiple blood transfusions and from volunteer blood donors. The results show detectable HGV-RNA in 32.6% of transfusion patients and in 5% of blood donors. The prevalence of HGV-RNA peaked between the 11th and 50th transfusion. The relationship between HGV infection and ALT status was not statistically relevant.
Adolescent↗
Long-term evolution of the hypervariable region of hepatitis C virus in a common-source-infected cohort.
The long-term evolution of the hepatitis C virus hypervariable region (HVR) and flanking regions of the E1 and E2 envelope proteins have been studied in a cohort of women infected from a common source of anti-D immunoglobulin. Whereas virus sequences in the infectious source were relatively homogeneous, distinct HVR variants were observed in each anti-D recipient, indicating that this region can evolve in multiple directions from the same point. Where HVR variants with dissimilar sequences were present in a single individual, the frequency of synonymous substitution in the flanking regions suggested that the lineages diverged more than a decade previously. Even where a single major HVR variant was present in an infected individual, this lineage was usually several years old. Multiple lineages can therefore coexist during long periods of chronic infection without replacement. The characteristics of amino acid substitution in the HVR were not consistent with the random accumulation of mutations and imply that amino acid replacement in the HVR was strongly constrained. Another variable region of E2 centered on codon 60 shows similar constraints, while HVR2 was relatively unconstrained. Several of these features are difficult to explain if a neutralizing immune response against the HVR is the only selective force operating on E2. The impact of PCR artifacts such as nucleotide misincorporation and the shuffling of dissimilar templates is discussed.
Amino Acid Sequence↗
Clinical significance of intrahepatic hepatitis C virus levels in patients with chronic HCV infection.
BACKGROUND: The clinical significance of a single assessment of circulating hepatitis C virus (HCV) RNA and its relation to the level of intrahepatic HCV RNA remains unclear. AIMS: To investigate the relation between intrahepatic HCV levels and clinicopathological characteristics of chronic HCV infection. PATIENTS: Ninety eight consecutive patients with chronic HCV infection were studied; none had received alpha interferon therapy. Of these, 12 patients were repeatedly negative for HCV RNA in serum by reverse transcriptase polymerase chain reaction (RT-PCR). METHODS: After diagnostic laparoscopy and liver biopsy, semiquantitative analysis of intrahepatic HCV RNA levels was carried out by limiting dilution of HCV cDNA. HCV genotypes were assessed in 96 patients by restriction fragment length polymorphism analysis of HCV cDNA. RESULTS: Ten out of 12 patients who were RT-PCR negative for HCV RNA in serum were RT-PCR positive in liver; however, this group had a significantly lower intrahepatic HCV level and serum aminotransferase level than the remaining 86 patients. Histological severity (cirrhosis: n = 10); histological activity index; HCV genotype (genotype 1: n = 41; genotype 2: n = 12; genotype 3: n = 36; genotype 4: n = 7); mode of infection (intravenous drug abuse: n = 58; post-transfusion: n = 10; haemophiliac: n = 4; sporadic: n = 26) and alcohol abuse did not affect the intrahepatic virus level. There was no correlation between patient age, duration of infection, and intrahepatic HCV level. CONCLUSIONS: Intrahepatic virus levels were not determined by host factors (age of patient, mode or duration of infection) or by virus factors (HCV genotype). Repeatedly negative RT-PCR for HCV RNA in serum does not indicate absence of HCV from the liver.
Adult↗
Detection of Chlamydia trachomatis in genital swabs: comparison of commercial and in house amplification methods with culture.
AIMS: To evaluate the sensitivity of the Roche Cobas, Roche Amplicor plate kit, ligase chain reaction (LCR), and an in house polymerase chain reaction (PCR) by titration of purified elementary bodies (EB) and also to test 245 urethral and endocervical specimens for Chlamydia trachomatis by the four assays as well as conventional culture. STUDY DESIGN: EB titrations were run in duplicate in each commercial assay and six times in the in house PCR. Clinical samples were aliquoted and tested by each assay and were considered positive if C trachomatis was detected by two or more separate tests or if the sample was either culture or immunofluorescence positive. Major outer membrane protein (MOMP) specific primers were used as a confirmatory assay for the in house PCR. RESULTS: The in house PCR, Roche Cobas Amplicor, LCR, and Amplicor plate kit gave detection limits of approximately 1, 1-2, 2, and 2-4 EBs respectively. By the criteria described above for definition of a C trachomatis positive result in clinical samples we identified 23 true positives among the 245 clinical specimens. The in house PCR detected all 23 giving a sensitivity of 100% and a specificity of 98%. The Roche Cobas Amplicor, Roche Amplicor plate kit, and LCR detected 21, 19, and 19 of these respectively giving sensitivities of 87.5%, 82%, and 82% respectively and specificities of 99.5%, 99%, and 100% respectively. The culture gave a sensitivity of 78% and specificity of 100%. CONCLUSION: All four amplification assays had a greater sensitivity than the culture used routinely in this laboratory. The in house plasmid PCR had the greatest sensitivity and when combined with confirmation by immunofluorescence detected the greatest number of positives. This increased sensitivity is likely to have been achieved by the use of a DNA purification step and of nested primers in the amplification stage and their combined use in routine diagnostic assays for chlamydia might increase the frequency of C trachomatis detections. However, this assay is much less user friendly than the two semiautomated commercial assays investigated in this study.
Bacterial Outer Membrane Proteins↗
Variability of the hepatitis C virus genome.
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Animals↗