New methods for identifying infectious diseases.

BACKGROUND: The goal of clinical microbiology is to identify the cause of infection, aiding rapid treatment initiation or altering empirically chosen anti-microbial regimens. Automation and molecular techniques have brought about a revolution in the clinical laboratory, ensuring ever faster and more accurate diagnoses. In the last few years however, there have been a number of developments that radically alter the way that microbiology and other diagnostic laboratories are advancing. In particular, clinical microbiology will have the opportunity to intervene at the public health level as well as at the individual patient. SOURCES OF DATA, AREAS OF AGREEMENT AND CONTROVERSY: Experts in the new technologies discuss the advances and some of the key literature that has been published to-date. They touch upon both the potential benefits and some of the hurdles that must be overcome before the technologies are embraced fully into the clinical laboratory. GROWING POINTS: This review discusses a number of technologies that may alter the way in which clinical microbiology is used to investigate infectious disease. Diagnostic services in the UK are currently undergoing a process of rationalization, which involves a shift towards laboratory amalgamation, adoption of 24/7 working patterns and greater automation in order to reduce costs. This review explores technologies that are already or are expected to be important in this on-going transition because they simplify or accelerate the complex workflows that are required for pathogen identification.

Oseltamivir is protective for in-patient mortality in PCR confirmed influenza B and influenza A(H3N2) infections in an historic cohort of 1,048 patients hospitalised during the 2016-17 and 2017-18 influenza seasons.

Standard course oseltamivir 75mg two times daily for five days was associated with an 82% reduction of odds of in-patient death (OR 0.18 (0.07,0.51)) compared to no oseltamivir treatment (OR 1.0 Reference) in a final multivariable logistic regression model of a retrospective cohort of PCR confirmed influenza B and influenza A (H3N2) infected patients admitted to a large UK teaching hospital in influenza seasons 2016-17 and 2017-18. No difference of protective odds for standard course oseltamivir was observed between influenza B and influenza A (H3N2) nor between influenza seasons. These observations strongly support clinical guidelines for molecular testing for respiratory viruses on admission to hospital and prompt treatment of confirmed seasonal influenza B and A with oseltamivir 75mg twice daily for five days.

Streamlining SARS-CoV-2 confirmatory testing to reduce false positive results.

BACKGROUND: Confirmatory testing of SARS-CoV-2 results is essential to reduce false positives, but comes at a cost of significant extra workload for laboratories and increased turnaround time. A balance must be sought. We analysed our confirmatory testing pathway to produce a more refined approach in preparation for rising case numbers. METHODS: Over a 10-week low prevalence period we performed confirmatory testing on all newly positive results. Turnaround time was measured and results were analysed to identify a threshold that could be applied as a cut-off for future confirmatory testing and reduce overall workload for the laboratory. RESULTS: Between 22/06/20 and 31/08/20 confirmatory testing was performed on 108 newly positive samples, identifying 32 false positive results (30 %). Turnaround time doubled, increasing by an extra 17 h. There was a highly statistically significant difference between initial Relative Light Unit (RLU) of results that confirmed compared to those that did not, 1176 vs 721 (P < 0.00001). RLU = 1000 was identified as a suitable threshold for confirmatory testing in our laboratory: with RLU ≥ 1000, 55/56 (98 %) confirmed as positive, whereas with RLU < 1000 only 12/38 (32 %) confirmed. CONCLUSIONS: False positive SARS-CoV-2 tests can be identified by confirmatory testing, yet this may significantly delay results. Establishing a threshold for confirmatory testing streamlines this process to focus only on samples where it is most required. We advise all laboratories to follow a similar process to identify thresholds that trigger confirmatory testing for their own assays, increasing accuracy while maintaining efficiency for when case numbers are high.

Using Whole Genome Sequences to Investigate Adenovirus Outbreaks in a Hematopoietic Stem Cell Transplant Unit.

A recent surge in human mastadenovirus (HAdV) cases, including five deaths, amongst a haematopoietic stem cell transplant population led us to use whole genome sequencing (WGS) to investigate. We compared sequences from 37 patients collected over a 20-month period with sequences from GenBank and our own database of HAdVs. Maximum likelihood trees and pairwise differences were used to evaluate genotypic relationships, paired with the epidemiological data from routine infection prevention and control (IPC) records and hospital activity data. During this time period, two formal outbreaks had been declared by IPC, while WGS detected nine monophyletic clusters, seven were corroborated by epidemiological evidence and by comparison of single-nucleotide polymorphisms. One of the formal outbreaks was confirmed, and the other was not. Of the five HAdV-associated deaths, three were unlinked and the remaining two considered the source of transmission. Mixed infection was frequent (10%), providing a sentinel source of recombination and superinfection. Immunosuppressed patients harboring a high rate of HAdV positivity require comprehensive surveillance. As a consequence of these findings, HAdV WGS is being incorporated routinely into clinical practice to influence IPC policy contemporaneously.

A real-time Taqman method for hepatitis C virus genotyping and methods for further subtyping of isolates.

The HCV genome is highly heterogeneous; more and more genotypes, each with several distinct subtypes, are being identified around the world. Knowledge of genotype is important for planning of treatment regimes, whereas subtype identification is useful in epidemiological studies and outbreak investigation. We describe HCV genotyping and subtyping assays, based on real-time PCR, that are sensitive, specific, and reliable. These assays provide fast, accurate, and convenient methods for HCV genotyping/subtyping to support clinical practice.

A real-time Taqman method for hepatitis C virus genotyping.

BACKGROUND: There is the need for a rapid, inexpensive method for genotyping hepatitis C virus (HCV) to support clinical practice. OBJECTIVES: To develop a real-time (Rotor-Gene 3000) Taqman assay for HCV genotyping in a single tube. STUDY DESIGN: Seven type-specific probes, two for genotypes 1-3 and one for genotype 4 were designed around genotype-specific motifs in the 5' non-coding (NC) region to create two panels of probes. The first panel included two probes for genotype 1 detection and a single probe each for genotypes 2 and 3. The second panel had two probes for confirmation of genotypes 2 and 3 and a first line probe for genotype 4 detection. A comparative analysis of the Taqman assay against our in-house sequence-based method using 154 consecutive clinical samples, from HCV carriers in Cambridge, and four samples from the Quality Control for Molecular Diagnostics (QCMD) System was undertaken. RESULTS: 158 samples were analysed by conventional sequencing: 49% (n=78) were genotype 1, 11% (n=18) genotype 2, 30% (n=47) genotype 3 and 6% (n=10) genotype 4. For two samples, the sequence data was heterogeneous and difficult to analyse, suggesting mixed infection and for three samples, the viral load was insufficient for sequencing. Concordant results were obtained with the novel Taqman assay for 77/78 (99%) of genotype 1 isolates (positive with both genotype 1 probes), 17/18 (94%) of genotype 2 isolates, 43/47 (91%) of genotype 3 isolates and 10/10 (100%) genotype 4 isolates. One isolate, untypeable with sequencing was genotyped with the Taqman assay. CONCLUSIONS: The Taqman assay was sensitive, specific and reliable over a wide range of viral loads and could identify mixed infections. These results highlight the potential of the Taqman assay as a fast, accurate and convenient method for routine HCV genotyping.