Astrovirus VA1/HMO-C: an increasingly recognized neurotropic pathogen in immunocompromised patients.

BACKGROUND: An 18-month-old boy developed encephalopathy, for which extensive investigation failed to identify an etiology, 6 weeks after stem cell transplant. To exclude a potential infectious cause, we performed high-throughput RNA sequencing on brain biopsy. METHODS: RNA-Seq was performed on an Illumina Miseq, generating 20 million paired-end reads. Nonhost data were checked for similarity to known organisms using BLASTx. The full viral genome was sequenced by primer walking. RESULTS: We identified an astrovirus, HAstV-VA1/HMO-C-UK1(a), which was highly divergent from human astrovirus (HAstV 1-8) genotypes, but closely related to VA1/HMO-C astroviruses, including one recovered from a case of fatal encephalitis in an immunosuppressed child. The virus was detected in stool and serum, with highest levels in brain and cerebrospinal fluid (CSF). Immunohistochemistry of the brain biopsy showed positive neuronal staining. A survey of 680 stool and 349 CSF samples identified a related virus in the stool of another immunosuppressed child. CONCLUSIONS: The discovery of HAstV-VA1/HMO-C-UK1(a) as the cause of encephalitis in this case provides further evidence that VA1/HMO-C viruses, unlike HAstV 1-8, are neuropathic, particularly in immunocompromised patients, and should be considered in the differential diagnosis of encephalopathy. With a turnaround from sample receipt to result of <1 week, we confirm that RNA-Seq presents a valuable diagnostic tool in unexplained encephalitis.

Whole-genome enrichment and sequencing of Chlamydia trachomatis directly from clinical samples.

BACKGROUND: Chlamydia trachomatis is a pathogen of worldwide importance, causing more than 100 million cases of sexually transmitted infections annually. Whole-genome sequencing is a powerful high resolution tool that can be used to generate accurate data on bacterial population structure, phylogeography and mutations associated with antimicrobial resistance. The objective of this study was to perform whole-genome enrichment and sequencing of C. trachomatis directly from clinical samples. METHODS: C. trachomatis positive samples comprising seven vaginal swabs and three urine samples were sequenced without prior in vitro culture in addition to nine cultured C. trachomatis samples, representing different serovars. A custom capture RNA bait set, that captures all known diversity amongst C. trachomatis genomes, was used in a whole-genome enrichment step during library preparation to enrich for C. trachomatis DNA. All samples were sequenced on the MiSeq platform. RESULTS: Full length C. trachomatis genomes (>95-100% coverage of a reference genome) were successfully generated for eight of ten clinical samples and for all cultured samples. The proportion of reads mapping to C. trachomatis and the mean read depth across each genome were strongly linked to the number of bacterial copies within the original sample. Phylogenetic analysis confirmed the known population structure and the data showed potential for identification of minority variants and mutations associated with antimicrobial resistance. The sensitivity of the method was >10-fold higher than other reported methodologies. CONCLUSIONS: The combination of whole-genome enrichment and deep sequencing has proven to be a non-mutagenic approach, capturing all known variation found within C. trachomatis genomes. The method is a consistent and sensitive tool that enables rapid whole-genome sequencing of C. trachomatis directly from clinical samples and has the potential to be adapted to other pathogens with a similar clonal nature.

ARHAI: antiviral resistance.

Development of antiviral resistance is a particular concern for the Advisory Committee on Antimicrobial Resistance and Healthcare-Associated Infections (ARHAI). Over the last 4 years, considerable time has been devoted to examining the ability of the UK to monitor the presence and transmission of antiviral resistance. Resistances to antiviral agents in influenza virus, HIV and hepatitis B and C viruses were identified as the main targets. The emphasis is on a network of laboratories that are able to perform diagnostic tests for resistance and to participate in surveillance programmes with co-ordination either through a central reference facility in the HPA or a collaborative study group.

Evolutionary genetics of human enterovirus 71: origin, population dynamics, natural selection, and seasonal periodicity of the VP1 gene.

Human enterovirus 71 (EV-71) is one of the major etiologic causes of hand, foot, and mouth disease (HFMD) among young children worldwide, with fatal instances of neurological complications becoming increasingly common. Global VP1 capsid sequences (n = 628) sampled over 4 decades were collected and subjected to comprehensive evolutionary analysis using a suite of phylogenetic and population genetic methods. We estimated that the common ancestor of human EV-71 likely emerged around 1941 (95% confidence interval [CI], 1929 to 1952), subsequently diverging into three genogroups: B, C, and the now extinct genogroup A. Genealogical analysis revealed that diverse lineages of genogroup B and C (subgenogroups B1 to B5 and C1 to C5) have each circulated cryptically in the human population for up to 5 years before causing large HFMD outbreaks, indicating the quiescent persistence of EV-71 in human populations. Estimated phylogenies showed a complex pattern of spatial structure within well-sampled subgenogroups, suggesting endemicity with occasional lineage migration among locations, such that past HFMD epidemics are unlikely to be linked to continuous transmission of a single strain of virus. In addition, rises in genetic diversity are correlated with the onset of epidemics, driven in part by the emergence of novel EV-71 subgenogroups. Using subgenogroup C1 as a model, we observe temporal strain replacement through time, and we investigate the evidence for positive selection at VP1 immunogenic sites. We discuss the consequences of the evolutionary dynamics of EV-71 for vaccine design and compare its phylodynamic behavior with that of influenza virus.

Molecular epidemiology of human enterovirus 71 in the United Kingdom from 1998 to 2006.

The last decade witnessed a significant increase in epidemic activity of human enterovirus 71 (EV71) in the Western Pacific Region (WPR). In most European countries, this risk is unrecognized despite occasional cases of severe disease and two severe outbreaks in Eastern Europe 30 years ago. In this study we report the first examination of the molecular epidemiology of EV71 in the United Kingdom from 1998 to 2006. Genomic regions encoding the 1D coat protein (VP1) and 3D polymerase (Pol) from 32 EV71 isolates associated with neurological or cutaneous manifestations were sequenced. Phylogenetic analyses of VP1 and 3D Pol sequences identified genotype C as the dominant strain. Several United Kingdom isolates had genetic linkages with predated C1 or C2 strains from Europe and the WPR. Recombination events were not detected between United Kingdom strains. However, a previously published Taiwanese strain was identified as an intergenotypic recombinant. EV71 genotype C appears to have continuous circulation in the United Kingdom from 1998 to 2006 with repeated introductions of new strains replacing previous strains. It is necessary to continuously monitor the molecular evolution and recombination events of EV71.

Antiviral therapy for polyomavirus-associated nephropathy after renal transplantation.

Polyomavirus-associated nephropathy (PVAN) has recently emerged as an important cause of allograft failure following renal transplantation. The BK virus is the most important polyomavirus associated with this condition. The mainstay of therapy for PVAN is a prompt immunosuppressive dose reduction in conjunction with careful monitoring for BK viraemia. A number of antiviral agents have been tried to help to reduce BK viral replication. So far, there has been only a single randomized controlled study on the use of one of these agents. Pooled data from various small case series did not show significant differences in outcome. Prospective randomized studies with a standardized protocol are urgently required.

A high HIV-1 strain variability in London, UK, revealed by full-genome analysis: Results from the ICONIC project.

BACKGROUND & METHODS: The ICONIC project has developed an automated high-throughput pipeline to generate HIV nearly full-length genomes (NFLG, i.e. from gag to nef) from next-generation sequencing (NGS) data. The pipeline was applied to 420 HIV samples collected at University College London Hospitals NHS Trust and Barts Health NHS Trust (London) and sequenced using an Illumina MiSeq at the Wellcome Trust Sanger Institute (Cambridge). Consensus genomes were generated and subtyped using COMET, and unique recombinants were studied with jpHMM and SimPlot. Maximum-likelihood phylogenetic trees were constructed using RAxML to identify transmission networks using the Cluster Picker. RESULTS: The pipeline generated sequences of at least 1Kb of length (median = 7.46Kb, IQR = 4.01Kb) for 375 out of the 420 samples (89%), with 174 (46.4%) being NFLG. A total of 365 sequences (169 of them NFLG) corresponded to unique subjects and were included in the down-stream analyses. The most frequent HIV subtypes were B (n = 149, 40.8%) and C (n = 77, 21.1%) and the circulating recombinant form CRF02_AG (n = 32, 8.8%). We found 14 different CRFs (n = 66, 18.1%) and multiple URFs (n = 32, 8.8%) that involved recombination between 12 different subtypes/CRFs. The most frequent URFs were B/CRF01_AE (4 cases) and A1/D, B/C, and B/CRF02_AG (3 cases each). Most URFs (19/26, 73%) lacked breakpoints in the PR+RT pol region, rendering them undetectable if only that was sequenced. Twelve (37.5%) of the URFs could have emerged within the UK, whereas the rest were probably imported from sub-Saharan Africa, South East Asia and South America. For 2 URFs we found highly similar pol sequences circulating in the UK. We detected 31 phylogenetic clusters using the full dataset: 25 pairs (mostly subtypes B and C), 4 triplets and 2 quadruplets. Some of these were not consistent across different genes due to inter- and intra-subtype recombination. Clusters involved 70 sequences, 19.2% of the dataset. CONCLUSIONS: The initial analysis of genome sequences detected substantial hidden variability in the London HIV epidemic. Analysing full genome sequences, as opposed to only PR+RT, identified previously undetected recombinants. It provided a more reliable description of CRFs (that would be otherwise misclassified) and transmission clusters.

Retrospective hepatitis C seroprevalence screening in the antenatal setting-should we be screening antenatal women?

OBJECTIVES: An unlinked anonymous seroprevalence study was conducted to estimate the prevalence of hepatitis C virus (HCV) infection in samples derived from antenatal clinic attendees at 2 East London Hospitals. An unexpectedly high HCV seroprevalence of 2.6% (1.2% viraemic) had been revealed during an unlinked study of the emergency department at 1 of these hospitals. DESIGN: 1000 stored residual samples were tested for HCV antibody (anti-HCV) and reactive samples were further tested for HCV RNA. The study was reviewed by the East Midland NRES ethics committee project ID 181154, approval number 15/WS/0125. RESULTS: The anti-HCV reactivity rate was 0.5% (5/1000) with 0.1% (1/1000) confirmed viraemic. Prevalence for the other blood-borne viruses was higher: 1% (10/1000) were hepatitis B surface antigen positive and 0.3% were HIV antigen/antibody positive (3/1000). There were no co-infections. CONCLUSIONS: More data to establish the prevalence of HCV in the antenatal population is needed. The addition of anti-HCV testing to the well-established antenatal screening programme provides a unique opportunity to impact on the health of pregnant women, their children, partners and future pregnancies in this new era of treatment for hepatitis C.

2015 UK national guideline for the management of infection with Chlamydia trachomatis.

This guideline offers recommendations on the diagnostic tests, treatment regimens and health promotion principles needed for the effective management of Chlamydia trachomatis genital infection. It covers the management of the initial presentation, as well the prevention of transmission and future infection. The guideline is aimed at individuals aged 16 years and older presenting to healthcare professionals working in departments offering Level 3 care in sexually transmitted infections management within the UK. However, the principles of the recommendations should be adopted across all levels, using local care pathways where appropriate.

Genetic variations of hepatitis B virus.

The emergence of hepatitis B virus genetic variants occurs under the influence of host immunity, immunization, the use of immune globulin or antiviral chemotherapy. Most of these are probably the result of the 'immune escape' phenomenon. Some variants, in particular those in the precore and core promoter regions, have been associated with disease severity and progression. Surface antigen variants have implications for the accuracy of laboratory diagnosis and may reduce the effectiveness of vaccination. Polymerase variants are selected as a result of the use of antiviral chemotherapeutic agents. It is important to monitor the occurrence of these variants.

The association of viral infection and chronic allograft nephropathy with graft dysfunction after renal transplantation.

BACKGROUND: The long-term effect of viral infections on graft dysfunction and rejection after renal transplantation is uncertain. METHODS: A cohort of 37 renal transplant recipients was followed prospectively for 3 years. Creatinine clearance rate at 6 months and 3 years and chronic allograft nephropathy were correlated with the detection of cytomegalovirus (CMV), human herpesvirus 6 and human herpesvirus 7 and BK virus DNA, CMV disease, and acute rejection. RESULTS: CMV disease was significantly associated with poor graft function at 6 months, whereas chronic allograft nephropathy was associated with graft dysfunction at 3 years. Both CMV disease and detection of human herpesvirus 6 DNA were associated with chronic allograft nephropathy. CONCLUSIONS: CMV disease was a significant cause of early graft dysfunction, whereas the presence of chronic allograft nephropathy was the main determinant of poor long-term graft function. The role of viral infections in chronic allograft nephropathy deserves further investigation.

The significance of 'anti-HBc only' in the clinical virology laboratory.

BACKGROUND: Isolated detection of hepatitis B core antibody (anti-HBc) in the absence of surface antigen (HBsAg) or antibody (anti-HBs) has been reported, particularly among individuals infected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV). The significance of this phenomenon is unknown and it is unclear whether all individuals with such serological pattern need further molecular investigations. OBJECTIVES: To determine the prevalence of 'anti-HBc only' in samples referred to a clinical virology laboratory and to evaluate its significance and possible mechanisms. STUDY DESIGN: Samples identified as anti-HBc positive (389/4359, 8.9%) during an 11-month period were investigated for HBsAg, anti-HBs, anti-HCV and anti-HIV. 'Anti-HBc only' samples were tested for HBV DNA using a nested qualitative PCR. Viral loads were measured in samples with detectable HBV DNA and the DNA sequences were analysed. RESULTS: Of 379 samples with detectable anti-HBc, 155 (40.9%) were 'anti-HBc only'. HBV DNA was detected in 6/151 (4%), all of which had a viral load <400 copies per ml. Anti-HIV was found in 50/151 (33.1%) and anti-HCV in 14/151 (9.3%). Of these, only one of the HIV infected patients had detectable HBV DNA. Phylogenetic analysis of the HBV surface gene from three patients showed a variety of genotypes (A, E and G). One sequence had a mutation in codon 144, which has previously been reported to give false negative HBsAg results. CONCLUSIONS: 'Anti-HBc only' is a common phenomenon in the clinical virology laboratory but only a small proportion of samples had detectable HBV DNA. The presence of HBsAg mutants with possible false negative HBsAg test result is of concern. Samples with 'anti-HBc only' could be used to monitor the emergence of these mutants.

Moving to nucleic acid-based detection of genital Chlamydia trachomatis.

Laboratory diagnosis of the bacterium Chlamydia trachomatis has gone through a complete phase of evolution since it was first identified as a significant cause of sexually transmitted infection. As a fragile, obligatory intracellular organism, it was initially only grown in eggs. Subsequently, diagnosis relied on culture in continuous cell lines. To address the limitations of culture, immunological methods were developed and direct antigen detection using enzyme immunoassay and immunofluorescence flourished. With the advent of molecular technologies, nucleic acid-based amplification techniques became the methods of choice, offering improved standard of care for diagnosis and opening up the possibility of screening using noninvasive, patient-acceptable specimens. In this article, the various currently available molecular methods are examined, some of the existing problems discussed and a view on what we think might happen in the next 5 years to the technology and requirement in diagnosis and screening is given.

Increase in susceptibility of young adults to hepatitis B infection in the Republic of Yemen.

Susceptibility to hepatitis B virus (HBV) infection among 987 young adult male blood donors in 2 major Yemeni cities was investigated. Hepatitis B surface antigen (HBsAg) was detected in 10.8% (107/987) of donors and 284 (28.8%) had evidence of ongoing or past HBV infection. Hepatitis B surface antibody (anti-HBs) only was detected in 34 (3.4%) donors. Thus, 67.8% (669/987) of donors had no detectable HBV-markers indicating susceptibility to infection. The proportion of HBV-susceptible donors decreased from 70.9% (249/351) in donors aged < 25 years to 69.4% (334/481) in those aged 25-34 years and 55.5% (86/155) in donors aged > 34 years (P = 0.002). The high proportion of susceptible young adults in a community with a high HBsAg carrier rate could be the result of changing epidemiology of hepatitis B in Yemen. Consideration should therefore be given to immunizing young adults as an adjunct to the current expanded infant immunization programme.

Is the addition of a standard HIV educational comment to virology laboratory reports effective in changing requesting behaviour?

BACKGROUND: It is not known whether the addition of general educational comments to virology laboratory reports can influence the requesting behaviour of practitioners. OBJECTIVES: To establish if there is any change in requesting behaviour after the addition of a standard comment to virology laboratory reports highlighting the need to include HIV testing when investigating patients presenting with a glandular fever (GF)-like illness. STUDY DESIGN: A standard comment to encourage inclusion of HIV testing was added to all GF screening reports from April 2010. The proportion of GF screening samples with concomitant HIV test requests before and after the introduction of the standard comment were compared over a 1 year period. RESULTS: A significant increase in concomitant HIV requests from 9.5% to 19.6% on GF screening samples from primary care practitioners was observed after the addition of the standard comment (p<0.0000001). This effect peaked at 5 months and although it waned, requests at one year were still higher than at baseline. CONCLUSIONS: Addition of a general HIV educational comment to virology laboratory reports is effective in changing requesting behaviour.

Missed opportunities for HIV testing--a costly oversight.

OBJECTIVES: In the UK, one-third of human immunodeficiency virus (HIV)-infected individuals are unaware of their diagnosis, and of those diagnosed a similar proportion have late stage disease. To address this National guidelines have been introduced promoting HIV testing across all medical specialities. We investigated HIV testing patterns in an inner London area with high local HIV prevalence, to identify missed opportunities for HIV testing and its consequences. METHODS: All human immunodeficiency virus (HIV) tests performed in 2008 at Guys and St Thomas' NHS Trust virology department were reviewed. Tests were stratified for location of request. Case-note review was carried out on all hospital HIV-positive diagnoses outside the genitourinary medicine (GUM) or screening settings to establish the circumstances surrounding the test, and missed opportunities for previous HIV testing. RESULTS: A total of 40,883 HIV tests were performed in 36,395 individuals. Three hundred and fifty-four (1%) tested positive. Excluding those from GUM or screening settings, 34 (2.8%) of the 1225 inpatients, 17 (0.3%) of the 5303 outpatients and 68 (1.12%) of the 5746 from primary care tested positive. Nineteen (41%) of 46 evaluable hospital diagnoses had presented to local healthcare services within the previous 12 months, 17 (37%) with an HIV indicator condition, but had not been tested. Of the 5303 outpatient tests conducted, 3148 (59%) were performed by either fertility or renal specialist teams. Other specialties conducted relatively few tests. The mean cost of admission for those diagnosed as an inpatient was £36,625 (range £331-223,000). The total cost for the 12 inpatients, who had presented to services in the preceding year but had not been tested was £439,500. CONCLUSION: Despite large numbers of HIV tests as screening tests in GUM and antenatal settings, relatively few tests occurred elsewhere with profound costs. Missed opportunities to access this high-prevalence HIV population is concerning and urgent engagement of primary, secondary and tertiary healthcare systems to increase HIV testing and prevent late-stage diagnoses is underway.