Identification of two novel subtypes of hepatitis C virus genotype 8 and a potential new genotype successfully treated with direct acting antivirals.

BACKGROUND: Hepatitis C virus (HCV) has a high genetic diversity and is classified into 8 genotypes and over 90 subtypes with some endemic to specific world regions. This could compromise direct-acting antiviral (DAA) efficacy and global HCV elimination. METHODS: We characterised HCV subtypes 'rare' to the UK (non-1a/1b/2b/3a/4d) by whole genome sequencing via a national surveillance programme. Genetic analyses to determine the genotype of samples with unresolved genotypes were undertaken by comparison with ICTV HCV reference sequences. RESULTS: Two HCV variants were characterised as being closely related to the recently identified genotype 8 (GT8), with >85% pairwise genetic distance similarity to GT8 sequences and within the typical inter-subtype genetic distance range. The individuals infected by the variants were UK residents originally from Pakistan and India. In contrast, a third variant was only confidently identified to be more similar to GT6 compared to other genotypes across 6% of the genome and was isolated from a UK resident originally from Guyana. All three were cured with pangenotypic DAAs (Sofosbuvir + Velpatasvir or Glecaprevir + Pibrentasvir) despite the presence of resistance polymorphisms in NS3 (80 K/168E), NS5A (28 V/30S/62L/92S/93S) and NS5B (159F). CONCLUSIONS: This study expands our knowledge of HCV diversity by identifying two new GT8 subtypes and potentially a new genotype.

Increased reports of severe myocarditis associated with enterovirus infection in neonates, United Kingdom, 27 June 2022 to 26 April 2023.

Enteroviruses are a common cause of seasonal childhood infections. The vast majority of enterovirus infections are mild and self-limiting, although neonates can sometimes develop severe disease. Myocarditis is a rare complication of enterovirus infection. Between June 2022 and April 2023, twenty cases of severe neonatal enteroviral myocarditis caused by coxsackie B viruses were reported in the United Kingdom. Sixteen required critical care support and two died. Enterovirus PCR on whole blood was the most sensitive diagnostic test. We describe the initial public health investigation into this cluster and aim to raise awareness among paediatricians, laboratories and public health specialists.

Clinical evaluation of a Hepatitis C Virus whole-genome sequencing pipeline for genotyping and resistance testing.

OBJECTIVES: We sought to evaluate clinically a hepatitis C virus (HCV) whole-genome, next-generation sequencing (NGS) pipeline that is agnostic to viral genotype. METHODS: Performance of the NGS pipeline was assessed through comparison of results with Sanger sequencing (SS) of partial HCV genomes. RESULTS: There was 98.7% (376/381) concordance for viral subtype between SS and NGS. The positive and negative per cent agreements for determination of resistance-associated substitutions were 97.8% (95% CI 92.5-99.4%) and 99.9% (95% CI 99.5-100.0%), respectively. The NGS pipeline was also able to detect novel subtypes, mixtures, recombinants, transiently occurring resistance mutations and distinguish re-infection with the same subtype from relapse. DISCUSSION: Particular scenarios where NGS may be used include settings without universal access to pan-genotypic antiviral regimens, those infected with a 'rare' subtype or who have been failed by first-line therapy, and in cases of suspected re-infection.

Real world SOF/VEL/VOX retreatment outcomes and viral resistance analysis for HCV patients with prior failure to DAA therapy.

Sustained viral response (SVR) rates for direct-acting antiviral (DAA) therapy for hepatitis C virus (HCV) infection routinely exceed 95%. However, a small number of patients require retreatment. Sofosbuvir, velpatasvir and voxilaprevir (SOF/VEL/VOX) is a potent DAA combination primarily used for the retreatment of patients who failed by DAA therapies. Here we evaluate retreatment outcomes and the effects of resistance-associated substitutions (RAS) in a real-world cohort, including a large number of genotype (GT)3 infected patients. 144 patients from the UK were retreated with SOF/VEL/VOX following virologic failure with first-line DAA treatment regimens. Full-length HCV genome sequencing was performed prior to retreatment with SOF/VEL/VOX. HCV subtypes were assigned and RAS relevant to each genotype were identified. GT1a and GT3a each made up 38% (GT1a n = 55, GT3a n = 54) of the cohort. 40% (n = 58) of patients had liver cirrhosis of whom 7% (n = 4) were decompensated, 10% (n = 14) had hepatocellular carcinoma (HCC) and 8% (n = 12) had received a liver transplant prior to retreatment. The overall retreatment SVR12 rate was 90% (129/144). On univariate analysis, GT3 infection (50/62; SVR = 81%, p = .009), cirrhosis (47/58; SVR = 81%, p = .01) and prior treatment with SOF/VEL (12/17; SVR = 71%, p = .02) or SOF+DCV (14/19; SVR = 74%, p = .012) were significantly associated with retreatment failure, but existence of pre-retreatment RAS was not when viral genotype was taken into account. Retreatment with SOF/VEL/VOX is very successful for non-GT3-infected patients. However, for GT3-infected patients, particularly those with cirrhosis and failed by initial SOF/VEL treatment, SVR rates were significantly lower and alternative retreatment regimens should be considered.

Technical Validation of a Hepatitis C Virus Whole Genome Sequencing Assay for Detection of Genotype and Antiviral Resistance in the Clinical Pathway.

Choice of direct acting antiviral (DAA) therapy for Hepatitis C Virus (HCV) in the United Kingdom and similar settings usually requires knowledge of the genotype and, in some cases, antiviral resistance (AVR) profile of the infecting virus. To determine these, most laboratories currently use Sanger technology, but next-generation sequencing (NGS) offers potential advantages in throughput and accuracy. However, NGS poses unique technical challenges, which require idiosyncratic development and technical validation approaches. This applies particularly to virology, where sequence diversity is high and the amount of starting genetic material is low, making it difficult to distinguish real data from artifacts. We describe the development and technical validation of a sequence capture-based HCV whole genome sequencing (WGS) assay to determine viral genotype and AVR profile. We use clinical samples of known subtypes and viral loads, and simulated FASTQ datasets to validate the analytical performances of both the wet laboratory and bioinformatic pipeline procedures. We show high concordance of the WGS assay compared to current "gold standard" Sanger assays. Specificity was 92.3 and 96.1% for AVR and genotyping, respectively. Discordances were due to the inability of Sanger assays to assign the correct subtype or accurately call mixed drug-resistant variants. We show high repeatability and reproducibility with >99.8% sequence similarity between sequence runs as well as high precision for variant frequency detection at >98.8% in the 95th percentile. Post-sequencing bioinformatics quality control workflows allow the accurate distinction between mixed infections, cross-contaminants and recombinant viruses at a threshold of >5% for the minority population. The sequence capture-based HCV WGS assay is more accurate than legacy AVR and genotyping assays. The assay has now been implemented in the clinical pathway of England's National Health Service HCV treatment programs, representing the first validated HCV WGS pipeline in clinical service. The data generated will additionally provide granular national-level genomic information for public health policy making and support the WHO HCV elimination strategy.

Enhanced Detection of DNA Viruses in the Cerebrospinal Fluid of Encephalitis Patients Using Metagenomic Next-Generation Sequencing.

The long and expanding list of viral pathogens associated with causing encephalitis confounds current diagnostic procedures, and in up to 50% of cases, the etiology remains undetermined. Sequence-agnostic metagenomic next-generation sequencing (mNGS) obviates the need to specify targets in advance and thus has great potential in encephalitis diagnostics. However, the low relative abundance of viral nucleic acids in clinical specimens poses a significant challenge. Our protocol employs two novel techniques to selectively remove human material at two stages, significantly increasing the representation of viral material. Our bioinformatic workflow using open source protein- and nucleotide sequence-matching software balances sensitivity and specificity in diagnosing and characterizing any DNA viruses present. A panel of 12 cerebrospinal fluid (CSFs) from encephalitis cases was retrospectively interrogated by mNGS, with concordant results in seven of nine samples with a definitive DNA virus diagnosis, and a different herpesvirus was identified in the other two. In two samples with an inconclusive diagnosis, DNA viruses were detected and in a virus-negative sample, no viruses were detected. This assay has the potential to detect DNA virus infections in cases of encephalitis of unknown etiology and to improve the current screening tests by identifying new and emerging agents.

Surveillance of HIV-1 transmitted integrase strand transfer inhibitor resistance in the UK.

BACKGROUND: HIV treatment guidelines have traditionally recommended that all HIV-positive individuals are tested for evidence of drug resistance prior to starting ART. Testing for resistance to reverse transcriptase inhibitors and PIs is well established in routine care. However, testing for integrase strand transfer inhibitor (InSTI) resistance is less consistent. OBJECTIVES: To inform treatment guidelines by determining the prevalence of InSTI resistance in a national cohort of recently infected individuals. PATIENTS AND METHODS: Recent (within 4 months) HIV-1 infections were identified using a Recent Infection Testing Algorithm of new HIV-1 diagnoses in the UK. Resistance-associated mutations (RAMs) in integrase, protease and reverse transcriptase were detected by ultradeep sequencing, which allows for the sensitive estimation of the frequency of each resistant variant in a sample. RESULTS: The analysis included 655 randomly selected individuals (median age = 33 years, 95% male, 83% MSM, 78% white) sampled in the period 2014 to 2016 and determined to have a recent infection. These comprised 320, 138 and 197 samples from 2014, 2015 and 2016, respectively. None of the samples had major InSTI RAMs occurring at high variant frequency (≥20%). A subset (25/640, 3.9%) had major InSTI RAMs occurring only as low-frequency variants (2%-20%). In contrast, 47/588 (8.0%) had major reverse transcriptase inhibitor and PI RAMs at high frequency. CONCLUSIONS: Between 2014 and 2016, major InSTI RAMs were uncommon in adults with recent HIV-1 infection, only occurring as low-frequency variants of doubtful clinical significance. Continued surveillance of newly diagnosed patients for evidence of transmitted InSTI resistance is recommended to inform clinical practice.

Multi-Laboratory Comparison of Next-Generation to Sanger-Based Sequencing for HIV-1 Drug Resistance Genotyping.

Next-generation sequencing (NGS) is increasingly used for HIV-1 drug resistance genotyping. NGS methods have the potential for a more sensitive detection of low-abundance variants (LAV) compared to standard Sanger sequencing (SS) methods. A standardized threshold for reporting LAV that generates data comparable to those derived from SS is needed to allow for the comparability of data from laboratories using NGS and SS. Ten HIV-1 specimens were tested in ten laboratories using Illumina MiSeq-based methods. The consensus sequences for each specimen using LAV thresholds of 5%, 10%, 15%, and 20% were compared to each other and to the consensus of the SS sequences (protease 4-99; reverse transcriptase 38-247). The concordance among laboratories' sequences at different thresholds was evaluated by pairwise sequence comparisons. NGS sequences generated using the 20% threshold were the most similar to the SS consensus (average 99.6% identity, range 96.1-100%), compared to 15% (99.4%, 88.5-100%), 10% (99.2%, 87.4-100%), or 5% (98.5%, 86.4-100%). The average sequence identity between laboratories using thresholds of 20%, 15%, 10%, and 5% was 99.1%, 98.7%, 98.3%, and 97.3%, respectively. Using the 20% threshold, we observed an excellent agreement between NGS and SS, but significant differences at lower thresholds. Understanding how variation in NGS methods influences sequence quality is essential for NGS-based HIV-1 drug resistance genotyping.

Interpreting Viral Deep Sequencing Data with GLUE.

Using deep sequencing technologies such as Illumina's platform, it is possible to obtain reads from the viral RNA population revealing the viral genome diversity within a single host. A range of software tools and pipelines can transform raw deep sequencing reads into Sequence Alignment Mapping (SAM) files. We propose that interpretation tools should process these SAM files, directly translating individual reads to amino acids in order to extract statistics of interest such as the proportion of different amino acid residues at specific sites. This preserves per-read linkage between nucleotide variants at different positions within a codon location. The samReporter is a subsystem of the GLUE software toolkit which follows this direct read translation approach in its processing of SAM files. We test samReporter on a deep sequencing dataset obtained from a cohort of 241 UK HCV patients for whom prior treatment with direct-acting antivirals has failed; deep sequencing and resistance testing have been suggested to be of clinical use in this context. We compared the polymorphism interpretation results of the samReporter against an approach that does not preserve per-read linkage. We found that the samReporter was able to properly interpret the sequence data at resistance-associated locations in nine patients where the alternative approach was equivocal. In three cases, the samReporter confirmed that resistance or an atypical substitution was present at NS5A position 30. In three further cases, it confirmed that the sofosbuvir-resistant NS5B substitution S282T was absent. This suggests the direct read translation approach implemented is of value for interpreting viral deep sequencing data.

Presence, persistence and effects of pre-treatment HIV-1 drug resistance variants detected using next generation sequencing: A Retrospective longitudinal study from rural coastal Kenya.

BACKGROUND: The epidemiology of HIV-1 drug resistance (HIVDR) determined by Sanger capillary sequencing, has been widely studied. However, much less is known about HIVDR detected using next generation sequencing (NGS) methods. We aimed to determine the presence, persistence and effect of pre-treatment HIVDR variants detected using NGS in HIV-1 infected antiretroviral treatment (ART) naïve participants from rural Coastal Kenya. METHODS: In a retrospective longitudinal study, samples from HIV-1 infected participants collected prior [n = 2 time-points] and after [n = 1 time-point] ART initiation were considered. An ultra-deep amplicon-based NGS assay, calling for nucleotide variants at >2.0% frequency of viral population, was used. Suspected virologic failure (sVF) was defined as a one-off HIV-1 viral load of >1000 copies/ml whilst on ART. RESULTS: Of the 50 eligible participants, 12 (24.0% [95% CI: 13.1-38.2]) had at least one detectable pre-treatment HIVDR variant against Protease Inhibitors (PIs, n = 6 [12%]), Nucleoside Reverse Transcriptase Inhibitors (NRTIs, n = 4 [8.0%]) and Non-NRTIs (n = 3 [6.0%]). Overall, 15 pre-treatment resistance variants were detected (frequency, range: 2.3-92.0%). A positive correlation was observed between mutation frequency and absolute load for NRTI and/or NNRTI variants (r = 0.761 [p = 0.028]), but not for PI variants (r = -0.117 [p = 0.803]). Participants with pre-treatment NRTI and/or NNRTI resistance had increased odds of sVF (OR = 6.0; 95% CI = 1.0-36.9; p = 0.054). CONCLUSIONS: Using NGS, pre-treatment resistance variants were common, though observed PI variants were unlikely transmitted, but rather probably generated de novo. Even when detected from a low frequency, pre-treatment NRTI and/or NNRTI resistance variants may adversely affect treatment outcomes.

Determining the Origins of Human Immunodeficiency Virus Type 1 Drug-resistant Minority Variants in People Who Are Recently Infected Using Phylogenetic Reconstruction.

BACKGROUND: Drug-resistant minority variants (DRMinVs) detected in patients who recently acquired human immunodeficiency virus type 1 (HIV-1) can be transmitted, generated de novo through virus replication, or technical errors. The first form is likely to persist and result in treatment failure, while the latter two could be stochastic and transient. METHODS: Ultradeep sequencing of plasma samples from 835 individuals with recent HIV-1 infection in the United Kingdom was performed to detect DRMinVs at a mutation frequency between 2% and 20%. Sequence alignments including >110 000 HIV-1 partial pol consensus sequences from the UK HIV Drug Resistance Database (UK-HDRD), linked to epidemiological and clinical data from the HIV and AIDS Reporting System, were used for transmission cluster analysis. Transmission clusters were identified using Cluster Picker with a clade support of >90% and maximum genetic distances of 4.5% or 1.5%, the latter to limit detection to likely direct transmission events. RESULTS: Drug-resistant majority variants (DRMajVs) were detected in 66 (7.9%) and DRMinVs in 84 (10.1%) of the recently infected individuals. High levels of clustering to sequences in UK-HDRD were observed for both DRMajV (n = 48; 72.7%) and DRMinV (n = 63; 75.0%) sequences. Of these, 43 (65.2%) with DRMajVs were in a transmission cluster with sequences that harbored the same DR mutation compared to only 3 (3.6%) sequences with DRMinVs (P < .00001, Fisher exact test). Evidence of likely direct transmission of DRMajVs was observed for 25/66 (37.9%), whereas none were observed for the DRMinVs (P < .00001). CONCLUSIONS: Using a densely sampled HIV-infected population, we show no evidence of DRMinV transmission among recently infected individuals.

A European multi-centre External Quality Assessment (EQA) study on phenotypic and genotypic methods used for Herpes Simplex Virus (HSV) drug resistance testing.

BACKGROUND: Herpes Simplex Virus (HSV) drug resistance is a significant public health concern among immunocompromised individuals. Phenotypic assays are considered the gold standard method for detecting HSV drug resistance. However, plaque reduction assays (PRAs) are technically demanding, often with long turnaround times of up to four weeks. In contrast, genotypic tests can be performed within a few days. OBJECTIVES: The development and coordination of the first European External Quality Assessment (EQA) study to evaluate phenotypic and genotypic methods used for HSV drug resistance testing in specialised reference laboratories. STUDY DESIGN: Four HSV-1 or HSV-2 strains with different antiviral susceptibility profiles were isolated from clinical samples. Isolates were quantified by qPCR, and aliquoted in culture medium. One isolate was distributed at two dilutions to help assess assay sensitivity. The panel was distributed to five European centres with a six-week deadline for the return of phenotypic and genotypic results, together with clinical reports. RESULTS: Four out of five participating labs returned results by the deadline. Limited results were later available from the fifth lab. Phenotypic and genotypic data were largely, but not completely, concordant. An unusual resistance profile shown by one of the samples was explained by the detection of a mixed virus population after extensive further investigation by one of the centres. CONCLUSIONS: Discordant clinical outputs reflecting the diversity of phenotypic methodologies demonstrated the utility of this exercise. With emerging genotypic technologies looking to supplant phenotyping, there is a need for curated public databases, accessible interpretation tools and standardised control materials for quality management. By establishing a network of testing laboratories, we hope that this EQA scheme will facilitate ongoing progress in this area.

Efficient and unbiased metagenomic recovery of RNA virus genomes from human plasma samples.

RNA viruses cause significant human pathology and are responsible for the majority of emerging zoonoses. Mainstream diagnostic assays are challenged by their intrinsic diversity, leading to false negatives and incomplete characterisation. New sequencing techniques are expanding our ability to agnostically interrogate nucleic acids within diverse sample types, but in the clinical setting are limited by overwhelming host material and ultra-low target frequency. Through selective host RNA depletion and compensatory protocol adjustments for ultra-low RNA inputs, we are able to detect three major blood-borne RNA viruses - HIV, HCV and HEV. We recovered complete genomes and up to 43% of the genome from samples with viral loads of 104 and 103 IU/ml respectively. Additionally, we demonstrated the utility of this method in detecting and characterising members of diverse RNA virus families within a human plasma background, some present at very low levels. By applying this method to a patient sample series, we have simultaneously determined the full genome of both a novel subtype of HCV genotype 6, and a co-infecting human pegivirus. This method builds upon earlier RNA metagenomic techniques and can play an important role in the surveillance and diagnostics of blood-borne viruses.

Enhanced surveillance of HIV-1 drug resistance in recently infected MSM in the UK.

OBJECTIVES: To determine the prevalence of inferred low-frequency HIV-1 transmitted drug resistance (TDR) in MSM in the UK and its predicted effect on first-line therapy. METHODS: The HIV-1 pol gene was amplified from 442 newly diagnosed MSM identified as likely recently infected by serological avidity testing in 2011-13. The PCR products were sequenced by next-generation sequencing with a mutation frequency threshold of >2% and TDR mutations defined according to the 2009 WHO surveillance drug resistance mutations list. RESULTS: The majority (75.6%) were infected with subtype B and 6.6% with rare complex or unique recombinant forms. At a mutation frequency threshold of >20%, 7.2% (95% CI 5.0%-10.1%) of the sequences had TDR and this doubled to 15.8% (95% CI 12.6%-19.6%) at >2% mutation frequency (P < 0.0001). The majority (26/42, 62%) of low-frequency variants were against PIs. The most common mutations detected at >20% and 2%-20% mutation frequency differed for each drug class, these respectively being: L90M (n = 7) and M46IL (n = 10) for PIs; T215rev (n = 9) and D67GN (n = 4) for NRTIs; and K103N (n = 5) and G190E (n = 2) for NNRTIs. Combined TDR was more frequent in subtype B than non-B (OR = 0.38; 95% CI = 0.17-0.88; P = 0.024) and had minimal predicted effect on recommended first-line therapies. CONCLUSIONS: The data suggest differences in the types of low-frequency compared with majority TDR variants that require a better understanding of the origins and clinical significance of low-frequency variants. This will better inform diagnostic and treatment strategies.

Disease Progression in HIV-1-Infected Viremic Controllers.

BACKGROUND: The mechanism of CD4 T-cell decline in HIV-1 infection is unclear, but the association with plasma viral RNA load suggests viral replication is involved. Indeed, viremic controller patients with low viral RNA loads typically maintain high CD4 T-cell counts. Within a local cohort of 86 viremic controllers, we identify a subgroup (18 "discord controllers") with low CD4 T-cell counts that present clinical uncertainty. The underlying mechanism accounting for CD4 T-cell decline in the face of low or undetectable plasma (RNA) viral load remains unresolved. The objective of this study was to investigate the viral and host immune system dynamics in discord controllers by measuring cellular HIV-1 DNA load, T-cell populations, and T-cell activation markers. METHODS: We compared discord controllers (viral RNA load <2000 copies/mL, <450 CD4 T-cells/mm) with typical controllers (viral RNA load <2000 copies/mL, >450 CD4 T-cells/mm) and progressors (viral RNA load >10,000 copies/mL, <450 CD4 T-cells/mm). We quantified CD4/CD8 naive/central memory/effector memory subsets (CD45RA/RO ± CD62L), activation levels (CD38HLA-DR), and HIV-1 DNA load. RESULTS: Discord controllers resembled progressors showing high viral DNA load, depletion of naive CD4 T-cells, and higher activation in all CD4 T-cell subsets, compared with typical controllers. They were similar to typical controllers with lower CD8 T-cell activation compared with progressors. CONCLUSIONS: Our data are consistent with a relationship between CD4 T-cell activation and disease progression. HIV-1 DNA load may be a better marker of viral replication and disease progression than viral RNA load. Lower level CD8 T-cell activation correlates with low viral RNA load but not with disease progression or viral DNA load.

A MIQE-compliant real-time PCR assay for Aspergillus detection.

The polymerase chain reaction (PCR) is widely used as a diagnostic tool in clinical laboratories and is particularly effective for detecting and identifying infectious agents for which routine culture and microscopy methods are inadequate. Invasive fungal disease (IFD) is a major cause of morbidity and mortality in immunosuppressed patients, and optimal diagnostic criteria are contentious. Although PCR-based methods have long been used for the diagnosis of invasive aspergillosis (IA), variable performance in clinical practice has limited their value. This shortcoming is a consequence of differing sample selection, collection and preparation protocols coupled with a lack of standardisation of the PCR itself. Furthermore, it has become clear that the performance of PCR-based assays in general is compromised by the inadequacy of experimental controls, insufficient optimisation of assay performance as well as lack of transparency in reporting experimental details. The recently published "Minimum Information for the publication of real-time Quantitative PCR Experiments" (MIQE) guidelines provide a blueprint for good PCR assay design and unambiguous reporting of experimental detail and results. We report the first real-time quantitative PCR (qPCR) assay targeting Aspergillus species that has been designed, optimised and validated in strict compliance with the MIQE guidelines. The hydrolysis probe-based assay, designed to target the 18S rRNA DNA sequence of Aspergillus species, has an efficiency of 100% (range 95-107%), a dynamic range of at least six orders of magnitude and limits of quantification and detection of 6 and 0.6 Aspergillus fumigatus genomes, respectively. It does not amplify Candida, Scedosporium, Fusarium or Rhizopus species and its clinical sensitivity is demonstrated in histological material from proven IA cases, as well as concordant PCR and galactomannan data in matched broncho-alveolar lavage and blood samples. The robustness, specificity and sensitivity of this assay make it an ideal molecular diagnostic tool for clinical use.

Comparative evaluation of the Seegene Seeplex RV15 and real-time PCR for respiratory virus detection.

Respiratory virus infections contribute substantially both to hospitalizations of young children, and to morbidity in immunocompromised patients such as those with hematological malignancies. Their rapid and accurate diagnosis is essential to patient management. To evaluate the prospective utility of Seeplex® DPO technology in respiratory virus diagnosis, a panel of 99 respiratory samples positive by real-time RT-PCR for one or more viruses was assayed by the Seegene Seeplex® RV12 system. As well as being able to detect all 10 viruses in the real-time RT-PCR system with the exception of enteroviruses, RV12 can also distinguish between the two subgroups of RSV and detect two subgroups of coronaviruses. Seven of the nine viruses in common with the RT-PCR were detected reliably by RV12. Eleven samples RT-PCR-positive for Metapneumovirus and five samples positive for influenza B were not detected by RV12. Seegene developed a second-generation system, RV15, which not only allowed detection of three additional viruses, but also addressed the potential problems with RV12 specificity. To address these concerns, 84 respiratory samples positive for a range of viruses by real-time PCR were assayed with RV15. The results of this evaluation improved significantly upon those seen with RV12. The high throughput capabilities and potential lower technical requirements afforded by the Seeplex® system may offer an alternative to real-time RT-PCR systems.

Application of a novel in vitro selection technique to isolate and characterise high affinity DNA aptamers binding mammalian prion proteins.

Clinical diagnosis and research into transmissible spongiform encephalopathies are hampered by the lack of sufficiently sensitive and specific reagents able to adequately detect the normal cellular form of the prion protein, PrP(C), and the pathological isoform, PrP(Sc). In order to provide such reagents, we applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) against a recombinant murine prion protein, to select single-stranded DNA ligands (aptamers) of high affinity. The SELEX protocol and subsequent aptamer characterisation employed protein immobilisation/partitioning using nickel-complexed magnetic particles and a novel SYBR Green-mediated quantitative real-time PCR technique. Following eight rounds of selection, the enriched aptamer pool was cloned and 24 clones sequenced. Seven of these were 'orphan' clones and the remainder were grouped into three separate T-rich families. All but four of the aptamer clones exhibited specific binding to the murine prion protein and the majority also bound to human and ovine prion proteins. Dissociation constants (K(d)) ranged from 18 to 79 nM. Flow cytometry with fluorescein-labelled aptamers confirmed that binding to cells was dependent on the expression of PrP(C). Preliminary studies also indicate that a trivalent aptamer pool is capable of binding the pathological isoform PrP(Sc) following guanidinium denaturation.

Molecular investigations of an outbreak of parainfluenza virus type 3 and respiratory syncytial virus infections in a hematology unit.

A large simultaneous outbreak of respiratory syncytial virus (RSV) and parainfluenza type 3 (PIV-3) infections occurred on an adult hematology unit. Implementation of enhanced infection control was complicated by cocirculation of the two different viruses, with prolonged viral shedding from infected patients, and placed great pressure on health care staff; of 27 infected hematopoietic stem cell transplant patients, 9 died, and the unit was closed for 2 months. Retrospective molecular investigation of the virus strains involved in the outbreak was performed by analyzing part of the fusion gene of PIV-3 and part of the glycoprotein gene of RSV. Reverse transcription-PCR on nasopharyngeal aspirates from patients infected before and during the simultaneous outbreak generated amplicons for sequence analysis. A single strain of RSV and a single strain of PIV-3 had spread from person to person within the unit; 7 patients were infected with RSV, 22 were infected with PIV-3, and 4 were infected with both viruses. The PIV-3 outbreak had started at the beginning of August 3 months before the RSV outbreak; it had arisen when PIV-3 was introduced from the community by a patient and passed to another patient, who became chronically infected with the identical strain and, in spite of being nursed in isolation, was most likely the source from which widespread infection occurred in November. Had these early cases been linked to a common PIV-3 strain at the time of diagnosis, enhanced infection control precautions might have prevented the eventual extensive spread of PIV-3, making it much easier to deal with the later RSV outbreak.