Alison Hardie

Epidemiology and Clinical Presentations of the Four Human Coronaviruses 229E, HKU1, NL63, and OC43 Detected over 3 Years Using a Novel Multiplex Real-Time PCR Method

ABSTRACT Four human coronaviruses (HCoV-229E, HCoV-HKU1, HCoV-NL63, and HCoV-OC43) are associated with a range of respiratory outcomes, including bronchiolitis and pneumonia. Their epidemiologies and clinical characteristics are poorly described and are often reliant on case reports. To address these problems, we conducted a large-scale comprehensive screening for all four coronaviruses by analysis of 11,661 diagnostic respiratory samples collected in Edinburgh, United Kingdom, over 3 years between July 2006 and June 2009 using a novel four-way multiplex real-time reverse transcription-PCR (RT-PCR) assay. Coronaviruses were detected in 0.3 to 0.85% of samples in all age groups. Generally, coronaviruses displayed marked winter seasonality between the months of December and April and were not detected in summer months, which is comparable to the pattern seen with influenza viruses. HCoV-229E was the exception; detection was confined to the winter of 2008 and was sporadic in the following year. There were additional longer-term differences in detection frequencies between seasons, with HCoV-OC43 predominant in the first and third seasons and HCoV-HKU1 dominating in the second (see Results for definitions of seasons). A total of 11 to 41% of coronaviruses detected were in samples testing positive for other respiratory viruses, although clinical presentations of coronavirus monoinfections were comparable to those of viruses which have an established role in respiratory disease, such as respiratory syncytial virus, influenza virus, and parainfluenza viruses. The novel multiplex assay for real-time pan-coronavirus detection enhances respiratory virus diagnosis, overcomes potential diagnostic problems arising through seasonal variation in coronavirus frequency, and provides novel insights into the epidemiology and clinical implications of coronaviruses.

Comparison of the Luminex Respiratory Virus Panel Fast Assay with In-House Real-Time PCR for Respiratory Viral Infection Diagnosis

ABSTRACT The Luminex xTAG Respiratory Virus Panel (RVP) assay has been shown to offer improved diagnostic sensitivity over traditional viral culture methods and to have a sensitivity comparable to those of individual real-time nucleic acid tests for respiratory viruses. The objective of this retrospective study was to test a new, streamlined version of this assay, the RVP Fast assay, which requires considerably less run time and operator involvement. The study compared the performance of the RVP Fast assay with those of viral culture, a direct fluorescent assay (DFA), and a panel of single and multiplex real-time PCRs in the testing of 286 respiratory specimens submitted to the Edinburgh Specialist Virology Centre for routine diagnosis of viral infection between December 2007 and February 2009. At least one respiratory viral infection was detected in 13.6% of specimens by culture and DFA combined, in 49.7% by real-time PCR, and in 46.2% by the RVP Fast assay. The sensitivity and specificity of the RVP Fast assay compared to the results of real-time PCR as the gold standard were 78.8% and 99.6%, respectively. Real-time PCR-positive specimens missed by the RVP Fast assay generally had low viral loads or were positive for adenovirus. Additionally, a small number of specimens were positive by the RVP Fast assay but were not detected by real-time PCR. For some viral targets, only a small number of positive results were found in our sample set using either method; therefore, the sensitivity of detection of the RVP Fast assay for individual targets could be investigated further with a greater number of virus-positive specimens.

A Genetic Analysis of Hepatitis C Virus Transmission between Injection Drug Users

Hepatitis C virus (HCV) genotype 1a and 3a partial NS5B gene segment sequences obtained from 154 HCV-infected injection drug users were studied to determine the extent to which HCV transmission occurs between injection drug user communities in London, Edinburgh, Glasgow (United Kingdom), Marseilles (France), and Melbourne. Phylogenetic relationships between sequences were analyzed by conventional methods and by a recently developed method that numerically scores the extent of sequence segregation between groups through calculation of association indices. The association indices revealed that none of the cities sampled support an HCV population that is completely isolated from that circulating in the other cities. Sequences from Melbourne were most isolated, whereas those from London were most dispersed. This suggests that HCV transmission between these cities occurs, with London playing a pivotal role. The degree of city-specific segregation of HCV subtype 1a sequences was linearly related to that of subtype 3a, indicating that these subtypes have spread through similar transmission networks.

Development and Assay of RNA Transcripts of Enterovirus Species A to D, Rhinovirus Species A to C, and Human Parechovirus: Assessment of Assay Sensitivity and Specificity of Real-Time Screening and Typing Methods

ABSTRACT Nucleic acid amplification methods such as the PCR have had a major impact on the diagnosis of viral infections, often achieving greater sensitivities and shorter turnaround times than conventional assays and an ability to detect viruses refractory to conventional isolation methods. Their effectiveness is, however, significantly influenced by assay target sequence variability due to natural diversity and rapid sequence changes in viruses that prevent effective binding of primers and probes. This was investigated for a diverse range of enteroviruses (EVs; species A to D), human rhinoviruses (HRVs; species A to C), and human parechovirus (HPeV) in a multicenter assay evaluation using a series of full-length prequantified RNA transcripts. RNA concentrations were quantified by absorption (NanoDrop) and fluorescence methods (RiboGreen) prior to dilution in buffer supplemented with RNase inhibitors and carrier RNA. RNA transcripts were extremely stable, showing minimal degradation after prolonged storage at temperatures between ambient and −20°C and after multiple freeze-thaw cycles. Transcript dilutions distributed to six referral laboratories were screened by real-time reverse transcriptase PCR assays using different primers and probes. All of the laboratories reported high assay sensitivities for EV and HPeV transcripts approaching single copies and similar amplification kinetics for all four EV species. HRV detection sensitivities were more variable, often with substantially impaired detection of HRV species C. This could be accounted for in part by the placement of primers and probes to genetically variable target regions. Transcripts developed in this study provide reagents for the ongoing development of effective diagnostics that accommodate increasing knowledge of genetic heterogeneity of diagnostic targets.

A prospective study of community-associated Clostridium difficile infections: The role of antibiotics and co-infections

OBJECTIVES This prospective study was performed to determine the incidence, risk factors, severity and outcomes of community-associated Clostridium difficile infection (CA-CDI) in the SE of Scotland. METHODS All patients (335) diagnosed with laboratory confirmed CDI in the city of Edinburgh, East Lothian and Midlothian regions of Scotland between August 2010 and July 2011 were followed up for one year after diagnosis. Clinical details and laboratory markers were recorded. Stool samples were tested for C. difficile, other bacterial pathogens and norovirus. Molecular epidemiology of C. difficile isolates was studied by PCR-ribotyping. RESULTS Of the total 335 confirmed CDI cases, PCR-ribotype 001 was the commonest (14.1%), followed by PCR-ribotypes 078 (12.9%) and 015 (11.7%), respectively. CA-CDI represented 12.5% of the cases. In these, PCR-ribotype 078 was the commonest (19.0%), followed by PCR-ribotypes 014/020 (16.7%), PCR-ribotype 015 (14.3% and PCR-ribotype 001 (11.9%). A lower Charlson co-morbidity index and a lower age was observed in the CA-CDI group as was total number of different antibiotic classes whereas age >75 was more common in the HA-CDI group. On multivariable analysis presence of PCR-ribotype 078 was significantly associated with community acquisition (p = 0.006) whereas a greater proportion of immunosuppressed patients and those on antibiotics 8 weeks preceding diagnosis (p = 0.035 and p = 0.005 respectively) were found among HA-CDI cases. Charlson co-morbidity index, number of different antibiotics given in the eight weeks preceding onset, severity of infection and rural residence were not significantly different between the two groups. CONCLUSION This study demonstrates that patients with CA-CDI may also present with severe infection, are less likely to receive antibiotics prior to CDI, more likely to be younger in age and have a greater proportion of PCR-ribotype 078 compared with CDI acquired in a hospital setting. Hence a high level of vigilance must be maintained to detect CDI cases which present in the community without the traditional predisposing factors.

Development of a PCR-free electrochemical point of care test for clinical detection of methicillin resistant Staphylococcus aureus (MRSA)

An MRSA assay requiring neither labeling nor amplification of target DNA has been developed. Sequence specific binding of fragments of bacterial genomic DNA is detected at femtomolar concentrations using electrochemical impedance spectroscopy (EIS). This has been achieved using systematic optimisation of probe chemistry (PNA self-assembled monolayer film on gold electrode), electrode film structure (the size and nature of the chemical spacer) and DNA fragmentation, as these are found to play an important role in assay performance. These sensitivity improvements allow the elimination of the PCR step and DNA labeling and facilitate the development of a simple and rapid point of care test for MRSA. Assay performance is then evaluated and specific direct detection of the MRSA diagnostic mecA gene from genomic DNA, extracted directly from bacteria without further treatment is demonstrated for bacteria spiked into saline (10(6) cells per mL) on gold macrodisc electrodes and into human wound fluid (10(4) cells per mL) on screen printed gold electrodes. The latter detection level is particularly relevant to clinical requirements and point of care testing where the general threshold for considering a wound to be infected is 10(5) cells per mL. By eliminating the PCR step typically employed in nucleic acid assays, using screen printed electrodes and achieving sequence specific discrimination under ambient conditions, the test is extremely simple to design and engineer. In combination with a time to result of a few minutes this means the assay is well placed for use in point of care testing.

Clinical outcomes and macrolide resistance in Mycoplasma pneumoniae infection in Scotland, UK

Mycoplasma pneumoniae has a cyclical, epidemic pattern of infection and the most recent epidemic occurred in Europe in 2011. Macrolides are recommended for the treatment of M. pneumoniae respiratory tract infection, but macrolide resistance has been reported at low levels in Europe. The aim of the study was to examine the clinical impact of the recent M. pneumoniae epidemic in a hospital setting in Scotland and to determine whether macrolide-resistant strains are present. Data were analysed retrospectively for 307 patients with M. pneumoniae respiratory infection diagnosed in 2010 and 2011 in Edinburgh, UK. Genotypic macrolide resistance testing was also carried out in 32 patients in whom resistance was considered most likely, based on their clinical picture. We found that 175 patients (59 %) were admitted to hospital, 20 (7 %) were admitted to critical care and 97 (38 %) required oxygen. All 48 adult patients (100 %) were admitted to hospital, compared with 127 children (51 %). Adults were also more likely to require oxygen [odds ratio (OR) 4.964, P<0.001, 95 % confidence interval (CI) 2.129-11.803] and to be admitted to critical care (OR 4.909, P = 0.001, 95 % CI 1.735-13.829), compared with children. Macrolide resistance conferred by the 23S rRNA gene mutation was found in samples from 6 out of 32 patients (19 %) in the subset tested. The results suggest that the recent M. pneumoniae epidemic was associated with a significant burden of hospital admission locally. The study also describes the first case series of macrolide-resistant M. pneumoniae in the UK, indicating that macrolide resistance surveillance is warranted in preparation for the next epidemic.

Distribution of HPV types associated with cervical cancers in Scotland and implications for the impact of HPV vaccines

Background/metho:This study evaluated human papillomavirus (HPV) type prevalence in 370 Scottish invasive cervical cancers (ICCs) using HPV genotyping and HPV mRNA detection.Results:HPV 16 and/or 18 was detected in 72% of cancers overall and in 82% of HPV-positive cancers. HPV 45 and 16 were the most frequently transcribed types.Conclusion:A significant reduction in ICC in Scotland should be achieved through the HPV immunisation programme.

High-risk HPV detection in specimens collected in SurePath preservative fluid: comparison of ambient and refrigerated storage

Objective:  With moves to introduce human papillomavirus (HPV) triage at sentinel sites in England, it is essential that optimal storage and transport conditions are determined for efficient HPV detection using residual liquid‐based cytology specimens.

Clinical performance of RNA and DNA based HPV testing in a colposcopy setting: Influence of assay target, cut off and age.

BACKGROUND As HPV testing is used increasingly for cervical disease management, there is a demand to optimise the performance of HPV tests, particularly with respect to specificity. OBJECTIVES To compare the clinical performance of an HPV DNA and a RNA based test in women with cytological abnormalities. The influence of age and assay cut off on test performance was also assessed. STUDY DESIGN A prospective comparison of the Hybrid Capture 2 test (HC2) and the Aptima HPV assay (AHPV) was performed within a colposcopy setting. Clinical sensitivity and specificity were determined for the detection of cervical intraepithelial neoplasia (CIN) grade 2 or worse. RESULTS Both assays were >90% sensitive for the detection of CIN2+. AHPV was slightly more specific than HC2 [49.9% (46.8-53.1) vs 45.9% (42.8, 49.1), p<0.0001]. Raising HC2 cut off to 2 RLU did not improve specificity. A cut-off of 10 RLU increased specificity by approximately 10% - although this led to a reduction in sensitivity of 6.3% which equated to 24 missed cases of CIN2+. Both assays were more specific in women over 30 years of age, compared to women under 30 (p<0.001). CONCLUSION Although AHPV was more specific than HC2 in the total cohort (p<0.001), we found this difference to be smaller than other studies. This could be attributed to different indications for colposcopic referral across different settings. This study also confirms the relatively poor specificity of commercial HPV assays in women under 30.