Current estimates suggest that even in the most resourced settings, the aetiology of encephalitis is identified in less than half of clinical cases. It is acknowledged that filling this gap needs a ...combination of rigorous sampling and improved diagnostic technologies. Next generation sequencing (NGS) methods are powerful tools with the potential for comprehensive and unbiased detection of pathogens in clinical samples. We reviewed the use of this new technology for the diagnosis of suspected infectious encephalitis, and discuss the feasibility for introduction of NGS methods as a frontline diagnostic test.
A systematic literature review was performed, using MESH and text word searches for variants of “sequencing” and “encephalitis” in Medline and EMbase, and searching bibliographies and citations using the Web of Science database. Two authors independently reviewed, extracted and summarised data.
The review identified 25 articles reporting 44 case reports of patients with suspected encephalitis for whom NGS was used as a diagnostic tool. We present the data and highlight themes arising from these cases. There are no randomly controlled trials to assess the utility of NGS as a diagnostic tool.
There is increasing evidence of a role for NGS in the work-up of undiagnosed encephalitis. Lower costs and increasing accessibility of these technologies will facilitate larger studies of these patients. We recommend NGS should be considered as a front-line diagnostic test in chronic and recurring presentations and, given current sample-to-result turn-around times, as second-line in acute cases of encephalitis.
Noroviruses are a leading cause of human gastroenteritis worldwide. The norovirus genotype GII.4 is the most prevalent genotype in the human population and has caused six pandemics since 1995. A ...novel norovirus lineage containing the GII.P16 polymerase and pandemic GII.4 Sydney 2012 capsid was recently detected in Asia and Germany. We demonstrate that this lineage is also circulating within the UK and USA and has been circulating since October 2014 or earlier. While the lineage does not contain unique substitutions in the capsid, it does contain polymerase substitutions close to positions known to influence polymerase function and virus transmission. These polymerase substitutions are shared with a GII.P16-GII.2 virus that dominated outbreaks in Germany in Winter 2016. We suggest that the substitutions in the polymerase may have resulted in a more transmissible virus and the combination of this polymerase and the pandemic GII.4 capsid may result in a highly transmissible virus. Further surveillance efforts will be required to determine whether the GII.P16-GII.4 Sydney 2012 lineage increases in frequency over the coming months.
Highlights • Incidence of non-rotavirus infections not increased in vaccination era. • Norovirus infections have highest viral titre compared to other viral GI infections. • No seasonal trend in ...norovirus incidence; correlation with hospital admissions. • All GI viruses able to cause chronic infections, predominantly in immunocompromised patients. • Breadth of norovirus genotypes has implications for vaccine development.
Norovirus incidence was compared between severe combined immunodeficiency children with (n = 10) and without (n = 8) B cells. 60% of B+ and 63% of B–patients developed norovirus infections therefore ...norovirus replication in B lymphocytes is not essential for infection.
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.
Molecular techniques are used in the clinical microbiology laboratory to support culture-based diagnosis of infection and are particularly useful for detecting difficult to culture bacteria or ...following empirical antimicrobial treatment.
Broad-range 16S rRNA PCR is a valuable tool that detects a wide range of bacterial species. Diagnostic yield is low for some sample types but can be improved with the addition of qPCR panels targeting common bacterial pathogens.
To evaluate the performance of a broad-range 16S rRNA gene PCR and the additional diagnostic yield of targeted qPCR applied to specimens according to a local testing algorithm.
In total, 6130 primary clinical samples were collected as part of standard clinical practice from patients with suspected infection during a 17 month period. Overall, 5497 samples were tested by broad-range 16S rRNA gene PCR and a panel of targeted real-time qPCR assays were performed on selected samples according to a local testing algorithm. An additional 633 samples were tested by real-time qPCR only. The 16S rRNA gene PCR was performed using two assays targeting different regions of the 16S rRNA gene. Laboratory developed qPCR assays for seven common bacterial pathogens were also performed. Data was extracted retrospectively from Epic Beaker Laboratory Information Management System (LIMS).
Broad-range 16S rRNA gene PCR improves diagnostic yield in culture-negative samples and detects a large range of bacterial species.
spp.,
s spp. and the Enterobacteriaceae family are detected the most frequently in samples with a single causative organism, but mixed samples frequently contained anaerobic species. The highest diagnostic yield was obtained from abscess, pus and empyema samples; 44.9 % were positive by 16S and 61 % were positive by the combined 16S and targeted qPCR testing algorithm. Samples with a particularly low diagnostic yield were blood, with 3.3 % of samples positive by 16S and CSF with 4.8 % of samples positive by 16S. The increased diagnostic yield of adding targeted qPCR is largest (~threefold) in these two sample types.
Broad-range PCR is a powerful technique that can detect a very large range of bacterial pathogens but has limited diagnostic sensitivity. The data in this report supports a testing strategy that combines broad-range and targeted bacterial PCR assays for maximizing diagnosis of infection in culture-negative specimens. This is particularly justified for blood and CSF samples. Alternative approaches, such as metagenomic sequencing, are needed to provide the breadth of broad-range PCR and the sensitivity of targeted qPCR panels.
Norovirus full-genome sequencing is challenging due to sequence heterogeneity among genomes. Previous methods have relied on PCR amplification, which is problematic due to primer design, and ...transcriptome sequencing (RNA-Seq), which nonspecifically sequences all RNA, including host and bacterial RNA, in stool specimens. Target enrichment uses a panel of custom-designed 120-mer RNA baits that are complementary to all publicly available norovirus sequences, with multiple baits targeting each position of the genome, which overcomes the challenge of primer design. Norovirus genomes are enriched from stool RNA extracts to minimize the sequencing of nontarget RNA. SureSelect target enrichment and Illumina sequencing were used to sequence full genomes from 507 norovirus-positive stool samples with reverse transcription-real-time PCR cycle threshold (CT) values of 10 to 43. Sequencing on an Illumina MiSeq system in batches of 48 generated, on average, 81% on-target reads per sample and 100% genome coverage with >12,000-fold read depth. Samples included genotypes GI.1, GI.2, GI.3, GI.6, GI.7, GII.1, GII.2, GII.3, GII.4, GII.5, GII.6, GII.7, GII.13, GII.14, and GII.17. When outliers were accounted for, we generated >80% genome coverage for all positive samples, regardless of CT values. A total of 164 samples were tested in parallel with conventional PCR genotyping of the capsid shell domain; 164/164 samples were successfully sequenced, compared to 158/164 samples that were amplified by PCR. Four of the samples that failed capsid PCR analysis had low titers, which suggests that target enrichment is more sensitive than gel-based PCR. Two samples failed PCR due to primer mismatches; target enrichment uses multiple baits targeting each position, thus accommodating sequence heterogeneity among norovirus genomes.
Metagenomic next-generation sequencing (mNGS) is an untargeted technique for determination of microbial DNA/RNA sequences in a variety of sample types from patients with infectious syndromes. mNGS is ...still in its early stages of broader translation into clinical applications. To further support the development, implementation, optimization and standardization of mNGS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mNGS for viral diagnostics to share methodologies and experiences, and to develop application guidelines. Following the ENNGS publication Recommendations for the introduction of mNGS in clinical virology, part I: wet lab procedure in this journal, the current manuscript aims to provide practical recommendations for the bioinformatic analysis of mNGS data and reporting of results to clinicians.
The advent of PCR testing for the presence of viral genomes has led to the identification of parvovirus B19 (PVB19) as a causative agent of myocarditis.
The clinical presentation, course and outcome ...of children with PVB19 myocarditis was ascertained through a retrospective review. The PVB19 viral genome was detected by PCR from whole blood or endomyocardial biopsy specimens in patients presenting with new onset heart failure.
Seventeen patients presented at a median age of 1.3 years (range: 0.4-15.4 years) in cardiac failure with a mean fractional shortening of 15±3%. Eleven patients required mechanical ventilation and intravenous inotropes and seven required extra-corporeal mechanical oxygenation. Four of the five deaths occurred in patients who had a short prodromal illness of less than 48 hours. All patients with ST segment elevation died (n=4). All non-fulminant cases survived. Event-free survival occurred in 11/17 (65%) patients. Five (29%) patients died and one patient underwent heart transplantation. Complete recovery of cardiac function occurred within a median of 12 months (range: 1-48) in five patients. There was incomplete recovery in five patients and one patient had persistent dilated cardiomyopathy.
PVB19 can cause a devastating myocarditis in children. Children with fulminant myocarditis, ST segment changes or a short prodrome have the worst outcome. Transplantation may be considered, but is rarely required in the acute period if mechanical circulatory support is utilised. If the initial presentation is survived, recovery of the myocardium can occur even in those who had fulminant myocarditis.
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