Alasdair Maclean

Development of a real-time RT-PCR for the detection of Swine-lineage Influenza A (H1N1) virus infections

Abstract Background A novel influenza A virus, subtype H1N1 of swine-lineage (H1N1 swl) has transmitted rapidly to many regions of the world with evidence of sustained transmission within some countries. Rapid detection and differentiation from seasonal influenza is essential to instigate appropriate patient and public health management and for disease surveillance. Objectives To develop a rapid and sensitive real-time reverse transcriptase polymerase chain reaction (rtRT-PCR) for confirmation of H1N1 swl. Study design A one-step rtRT-PCR approach was employed to target the matrix gene of the novel influenza A/H1N1 swl and validated against a panel of seasonal influenza A (H1N1 and H3N2), swine influenza A/H1N1 and avian influenza A/H5N1 viruses. The assay following validation was then used prospectively to detect H1N1 swl positive specimens from the recent outbreaks in the UK and the Republic of Ireland. Results The one-step H1N1 swl matrix rtRT-PCR successfully detected H1N1 swl clinical specimens and did not cross-react with seasonal influenza A, subtypes H1N1 and H3N2 viruses and swine influenza A (H1N1). The H1N1 swl matrix assay did cross react with H5N1. The H1N1 swl matrix assay was then compared to two other assays using a dilution series and a panel of untyped influenza A positive clinical samples. These experiments found the assay to have a comparable sensitivity to the established universal influenza A rtRT-PCR and was more sensitive than the H1N1 swl specific assay that targeted the H1 region. Conclusions The results demonstrate that the rtRT-PCR is sensitive and should be used alongside existing universal influenza A assays to rapidly detect the novel H1N1 swl virus.

Using multiplex real time PCR in order to streamline a routine diagnostic service.

Abstract An increasing number of virology laboratories are now utilising in house real time PCR assays as the frontline diagnostic tests. As the number of tests on offer increases the natural progression from this will be to rationalise their service via multiplexing. Since 2003 we have introduced a large number of qualitative and quantitative multiplex real time PCR assays into our routine testing service. This paper describes the development of the multiplex assays, the problems encountered and the resultant benefits to the routine service.

Development of a multiplex real-time RT-PCR that allows universal detection of influenza A viruses and simultaneous typing of influenza A/H1N1/2009 virus.

Abstract On June 11, 2009, the World Health Organization declared that the influenza A/H1N1/2009 virus had become the first influenza pandemic of the 21st century. Rapid detection and differentiation from seasonal and avian influenza would be beneficial for patient management and infection control. It was the aim of this study to develop a real-time RT-PCR that can detect all influenza A viruses and offer simultaneous typing for influenza A/H1N1/2009. This would be a useful addition to existing diagnostic protocols for influenza A. Its routine use would allow laboratories to screen out influenza A/H1N1/2009 positive samples rapidly and would reduce overall testing costs.

Prevalence of HCV NS3 pre-treatment resistance associated amino acid variants within a Scottish cohort

Highlights • Prevalence of HCV NS3 resistance-associated amino acid variants (RAV) was investigated.• Sanger sequencing was performed on a protease inhibitor treatment-naïve Scottish cohort (n = 146).• Overall 23.29% had a detectable RAV in the NS3 region; Q80K was the most prevalent.• Only 2.74% patients had more than one RAV.• The study highlighted the need for Q80K sequencing prior to simeprevir treatment.

The validation of a real-time RT-PCR assay which detects influenza A and types simultaneously for influenza A H1N1 (2009) and oseltamivir-resistant (H275Y) influenza A H1N1 (2009).

Abstract Influenza A H1N1 (2009) was declared by the World Health Organisation (WHO) as the first influenza pandemic of the 21st century. Rapid detection of influenza A and differentiation of influenza A H1N1 (2009) and seasonal influenza A is beneficial. In addition the rapid detection of antiviral resistant strains of influenza A H1N1 (2009) would be useful for clinicians to allow for change to an effective treatment at a much earlier stage if resistance is found. It was the aim of this study to develop a real-time RT-PCR that can detect all influenza A viruses and type simultaneously for influenza A H1N1 (2009) and oseltamivir resistant (H275Y) influenza A H1N1 (2009). This multiplex assay will allow laboratories to screen respiratory samples for all types of influenza A, influenza A H1N1 (2009) virus and oseltamivir resistant (H275Y) influenza A H1N1 (2009) virus in a rapid and cost effective format, ensuring that typing methods for seasonal and avian viruses are used on a smaller subset of samples. Since most virology laboratories already offer a molecular service for influenza A this assay could easily be implemented into most areas at little cost therefore increasing local access to resistance testing.

Letter to the editor: There is a need to consider all respiratory viruses in suspected mumps cases.

To the editor: The recent paper by Thompson et al. [1] highlighted the detection of influenza A (H3N2) virus in oral fluids from children with a clinical diagnosis of mumps. They concluded that influenza A(H3N2) virus should be considered as part of the differential diagnosis for mumps-like illness, particularly during influenza outbreaks caused by drifted strains. We report here similar findings in our Scottish cohort of patients during the 2014–15 winter season.

Prevalence of pre-treatment hepatitis C virus NS5A resistance associated amino-acid substitutions in genotype 1A infected patients in Scotland

BACKGROUND Hepatitis C (HCV) NS5A resistance associated amino-acid substitutions (RAS) can exist at baseline in treatment naïve individuals and have been shown to be associated with lower rates of sustained virological response (SVR) for patients infected with HCV genotype 1A (G1A) following treatment with NS5A inhibitors. OBJECTIVES The aim of this study was to measure the prevalence of baseline NS5A resistance in Scotland. STUDY DESIGN The study population consisted of 531 treatment naïve, G1A infected patients. The patient samples were collected between March and September 2017. The NS5A region was amplified and sequenced. RESULTS Baseline NS5A resistance in Scotland is high (16.8%) and is comparable to rates reported by a number of previously published studies. The high rate of baseline RAS, together with the high cost of direct-acting antivirals (DAAs), supports resistance testing to guide current patient treatment. However, given the rate at which new DAAs are currently being licensed with ever broader genotype efficacy and higher SVR rates, baseline resistance testing may not be required in the near future. CONCLUSIONS Baseline NS5A inhibitor resistance is high. The results of the present study support performing resistance testing at baseline for current regimens.

HIV-1 integrase inhibitor resistance among treatment naïve patients in the West of Scotland

BACKGROUND Transmitted integrase inhibitor resistance is rare, with only a small number of cases reported world-wide to date. OBJECTIVES The aim of this study was to assess whether transmitted integrase inhibitor resistance has occurred in Scotland and if so, could there be a case for performing genotypic integrase resistance testing at baseline. STUDY DESIGN The study population consisted of 106 treatment naïve, newly diagnosed, HIV positive patients. The patient samples were collected between October 2015 and March 2016 at the time of HIV diagnosis and prior to initiation of anti-retroviral therapy. The integrase region was amplified and sequenced. RESULTS We detected integrase inhibitor resistance (T66I/T) at baseline in one patient sample. This is a non-polymorphic mutation seen in patients receiving elvitegravir which confers high-level resistance to elvitegravir and intermediate resistance to raltegravir. A further 10 patients had accessory mutations which have minimal or no effect on susceptibility to integrase inhibitors. CONCLUSIONS Transmitted integrase inhibitor resistance remains rare. The results of the present study do not support performing integrase resistance testing at baseline.