Monitoring Viral Genetic Variation as a Tool To Improve Molecular Diagnostics for Mumps Virus.

Here, we report how the analysis of viral genetic variation using next-generation sequencing (NGS) can be used as a tool to improve mumps virus diagnostics. Analysis of NGS data from recently circulating mumps virus isolates allowed optimization of the current mumps virus real-time reverse transcription-PCR (RT-PCR) by primer and probe modifications due to nucleotide variations. The modified assay showed a higher efficiency and sensitivity than the previously used CDC protocol for the detection of currently circulating mumps virus strains and could therefore offer better support for outbreak control. The NGS sequence data were also used to make predictions of changes in the hemagglutinin-neuraminidase protein structure that could explain possible immune escape mechanisms.

Comparison of the fast track diagnostics respiratory 21 and Seegene Allplex multiplex polymerase chain reaction assays for the detection of respiratory viruses.

BACKGROUND: Real-time multiplex PCR assays are increasingly used for respiratory virus detection, and offer automated analysis in a closed tube system, but they have the disadvantage of low-throughput due to multiplexing limitations. In this study, the established fast-track respiratory 21 assay (FTD) (fast-track diagnostics, Junglinster Luxembourg) was compared to the new Seegene Allplex assay (Seegene) (Seegene Inc. Seoul, Korea) which offers greater multiplexing as multiple targets can be detected in each fluorescence channel. The Seegene Allplex assay is quicker to perform than previous Seegene respiratory multiplex assays. MATERIALS AND METHODS: The assays were evaluated using 199 mostly upper respiratory tract samples. RESULTS: A respiratory pathogen was found in 127/199 (63.8%) of samples by the FTD assay and 123/199 (61.8%) using the Seegene assay. Kappa agreement was between 0.87 and 1 for all targets except human bocavirus and adenovirus. CONCLUSION: Although the performance of the assays were similar, the Seegene assay had the advantage of simultaneous detection of two gene targets for each of the common Influenza A subtypes, improved throughput of 30 samples per run and automated result analysis. The FTD assay could only test 17 samples per run but validation for use on several different real-time thermal cyclers made it easier to integrate into an existing laboratory system. Both assays were cost effective compared to in-house multiplex PCR respiratory virus screening.

Impact of routine systematic polymerase chain reaction testing on case finding for Legionnaires' disease: a pre-post comparison study.

BACKGROUND: Legionnaires' disease cannot be clinically or radiographically distinguished from other causes of pneumonia, and specific tests are required to make the diagnosis. Currently, testing occurs erratically and, instead, clinicians rely on empiric treatment strategies and ignore public health implications of the diagnosis. We aimed to measure the increase in case detection of Legionnaires' disease following the introduction of routine polymerase chain reaction (PCR) testing of respiratory specimens. PCR is the most sensitive diagnostic tool for Legionnaires' disease. METHODS: In a quasi-experimental study in Christchurch, New Zealand, we compared the number of cases of Legionnaires' disease requiring hospitalization diagnosed during a 2-year period before the introduction of a routine PCR testing strategy (November 2008-October 2010) with a similar period after the introduction (November 2010-October 2012). With this testing strategy, all respiratory specimens from hospitalized patients with pneumonia sent to the region's sole tertiary-level laboratory were tested for Legionella by PCR, whether requested or not. RESULTS: During November 2008 to October 2010, there were 22 cases of Legionnaires' disease compared with 92 during November 2010 to October 2012. Of 1834 samples tested since November 2010, 1 in 20 was positive, increasing to 1 in 9 during peak Legionella season (November to January). Increasing bacterial load was associated with increasing disease severity. CONCLUSIONS: In our region, the burden of Legionnaires' disease is much greater than was previously recognized. Routine PCR testing provides results within a clinically relevant time frame and enables improved characterization of the regional epidemiology of Legionnaires' disease.

The burden of Legionnaires' disease in New Zealand (LegiNZ): a national surveillance study.

BACKGROUND: Legionnaires' disease is under-diagnosed because of inconsistent use of diagnostic tests and uncertainty about whom to test. We assessed the increase in case detection following large-scale introduction of routine PCR testing of respiratory specimens in New Zealand. METHODS: LegiNZ was a national surveillance study done over 1-year in which active case-finding was used to maximise the identification of cases of Legionnaires' disease in hospitals. Respiratory specimens from patients of any age with pneumonia, who could provide an eligible lower respiratory specimen, admitted to one of 20 participating hospitals, covering a catchment area of 96% of New Zealand's population, were routinely tested for legionella by PCR. Additional cases of Legionnaires' disease in hospital were identified through mandatory notification. FINDINGS: Between May 21, 2015, and May 20, 2016, 5622 eligible specimens from 4862 patients were tested by PCR. From these, 197 cases of Legionnaires' disease were detected. An additional 41 cases were identified from notification data, giving 238 cases requiring hospitalisation. The overall incidence of Legionnaires' disease cases in hospital in the study area was 5·4 per 100 000 people per year, and Legionella longbeachae was the predominant cause, found in 150 (63%) of 238 cases. INTERPRETATION: The rate of notified disease during the study period was three-times the average over the preceding 3 years. Active case-finding through systematic PCR testing better clarified the regional epidemiology of Legionnaires' disease and uncovered an otherwise hidden burden of disease. These data inform local Legionnaires' disease testing strategies, allow targeted antibiotic therapy, and help identify outbreaks and effective prevention strategies. The same approach might have similar benefits if applied elsewhere in the world. FUNDING: Health Research Council of New Zealand.

Evidence that the gonococcal porA pseudogene is present in a broad range of Neisseria gonorrhoeae strains; suitability as a diagnostic target.

AIMS: The primary aim of the study was to determine if the gonococcal porA pseudogene is a stable sequence target for the detection of Neisseria gonorrhoeae by PCR. METHODS: A total of 240 gonococcal strains from various geographic locations were tested by porA pseudogene PCR. In addition, porA pseudogene PCR positivity rates were compared with established gonococcal assays in three Australian states. RESULTS: All N. gonorrhoeae isolates provided positive results in the porA pseudogene PCR. Positivity rates compared favourably with established gonococcal assays, with increased N. gonorrhoeae detection in the Northern Territory and Western Australia. CONCLUSIONS: The results of this multicentre study provide further evidence that the porA pseudogene is highly conserved across a diverse range N. gonorrhoeae strains and is a suitable PCR target for routine detection of N. gonorrhoeae.

Comparison of four multiplex PCR assays for the detection of viral pathogens in respiratory specimens.

Multiplex PCR has become the test of choice for the detection of multiple respiratory viruses in clinical specimens. However, there are few direct comparisons of different PCR assays. This study compares 4 different multiplex PCR assays for the recovery of common respiratory viruses. We tested 213 respiratory specimens using four different multiplex PCR assays: the xTAG respiratory viral panel fast (Abbott Molecular Laboratories), Fast-track Respiratory Pathogen assay (Fast-track Diagnostics), Easyplex respiratory pathogen 12 kit (Ausdiagnostics), and an in-house multiplex real-time PCR assay. The performance of the four assays was very similar, with 93-100% agreement for all comparisons. Other issues, such as through-put, technical requirements and cost, are likely to be as important for making a decision about which of these assays to use given their comparative performance.

Meningococcal disease deaths and the frequency of antibiotic administration delays.

OBJECTIVE: Meningococcal disease (MCD) remains a medical emergency and a frequent cause of death in previously healthy individuals. We aimed to determine the frequency and cause of delays in antibiotic administration in a cohort of deaths. METHODS: A retrospective chart review was undertaken on 140 hospitalised MCD deaths within New Zealand's serogroup B epidemic (1993-2004). RESULTS: Death after hospital presentation occurred rapidly (median 12 h) with 70% dying within 24 h. Delays of more than 2 h in time-to-antibiotic post hospital presentation occurred in 29%. The major contributors to these delays were the failure to include MCD within the differential diagnosis and prolonged assessment times. Multivariate logistic regression analysis, undertaken by combining study deaths which had meningococcal bacterial load results (n=9) with a survivor cohort (n=126) from a previous study, confirmed bacterial load as a major predictor of death (OR 7.5 per log10 cfu/mL increase; 95% CI 2.2-25.3; p=0.001). A non-significant increased risk of death per hour of antibiotic delay was seen (OR 1.18; 95% CI 0.90-1.55; p=0.22). CONCLUSIONS: Death from MCD occurred rapidly, with many patients not receiving antibiotics at the earliest opportunity. The introduction of recently developed rapid diagnostic markers into the identified delay-intervals could lead to a reduction in time-to-antibiotic and hopefully reduce case-fatality rates.

Direct detection and differentiation of Legionella spp. and Legionella pneumophila in clinical specimens by dual-color real-time PCR and melting curve analysis.

A dual-color LightCycler PCR assay targeting the 16S rDNA gene of Legionella spp. was established. By using two pairs of hybridization probes, Legionella spp. and Legionella pneumophila could be detected and differentiated simultaneously. With 26 culture-positive and 42 culture-negative respiratory specimens from patients with atypical pneumonia, 100% sensitivity and specificity was observed for L. pneumophila.