Automation and standardisation of a quantitative multiplex PCR assay using PCR.Ai.

BACKGROUND: We previously undertook a prospective clinical study to evaluate PCR.Ai's (www.pcr.ai) accuracy and impact when automating the manual data-analysis and quality control steps associated with routine clinical pathogen testing using a non-quantitative multiplex quantitative real-time PCR (qPCR). In this study we demonstrated 100 % concurrence between our manual routine analysis method and PCR.Ai. This paper expands the evaluation of PCR.Ai's (www.pcr.ai) accuracy and impact using a multiplex quantitative real-time PCR (qPCR). OBJECTIVES: We evaluated the impact of PCR.Ai when used as the final interpretation/verification step for routine in-house multiplex quantitative qPCR tests for CMV, EBV and adenovirus from blood samples for a total of 1350 interpretations. STUDY DESIGN: We compared PCR.Ai to our existing manual interpretation, to determine accuracy and hands on time savings. RESULTS AND CONCLUSIONS: There was 100 % concurrence between validated CMV, EBV and adenovirus detection and quantitation by our manual routine analysis method and PCR.Ai. Furthermore, there were significant routine savings with PCR.Ai of 63 minutes/ run. Our conclusion is that for quantitative tests PCR.Ai is a highly accurate time-saving tool that reduces complexity of qPCR analysis and hence the need for specialists and hands-on time. It demonstrated capabilities to enable us to get results out more quickly with lower costs and less risk of errors.

Increasing drug-related mortality rates over the last decade in Scotland are not just due to an ageing cohort: A retrospective longitudinal cohort study.

BACKGROUND: In Europe, North America, and Australia, mortality due to drug-related (DR) causes amongst people who inject drugs (PWID) is a major issue. Our objective was to characterise temporal trends in DR mortality rates in a large cohort of PWID in Scotland over the past decade, all of whom had been diagnosed with hepatitis C virus (HCV) infection, and to investigate factors associated with DR mortality. METHODS: Retrospective longitudinal cohort study linking Scotland's national HCV Diagnosis Database and deaths registry. The study cohort consisted of all individuals with likely injection drug use-related route of HCV acquisition, who had been diagnosed with HCV between 1991 and 2018, and were alive and aged under 65 years on 1 January 2009. We used Lexis expansion to adjust for ageing cohort effects and calculated the mortality rate from an underlying/contributing DR cause over the period 2009-2018. We fitted Poisson regression models to estimate the temporal trend adjusting for attained age, sex, referral setting, region, and viraemic status at baseline. RESULTS: Amongst the study population (n = 35,065; 236,914 person-years), a total of 1900 DR deaths occurred; the DR mortality rate increased from 5.6/1000 [101 deaths] in 2009 to 12.4/1000 [342] person-years in 2018. Increasing trends were observed for all age-groups except 55-64 years. The overall DR mortality rate was highest for referrals for HCV testing from prison (11.0/1000) and hospital settings (10.0/1000). Mortality increased with calendar time period, with significantly raised adjusted rate ratios (RRs) from 2015 (RR=1.40, 95% CI:1.16-1.69) to 2018 (RR=2.23, 95% CI:1.88-2.64), compared with 2011-2012, for older age (35-44: RR=1.37, 95% CI:1.20-1.56; 45-54: RR=1.32, CI:1.14-1.53) compared with <35 years, for persons diagnosed with HCV since 2009 (RR=1.34, 95% CI:1.21-1.49), and for prison and hospital referrals (RRs of 1.30, 1.37) compared with GP referrals. CONCLUSION: Increasing DR mortality rates in Scotland over the past decade are not just due to an ageing cohort. Harm reduction services will likely need to expand and adapt to reverse the recent upward trends in DR mortality in PWID.

SARS-CoV-2 Positivity in Asymptomatic-Screened Dental Patients.

Enhanced community surveillance is a key pillar of the public health response to coronavirus disease 2019 (COVID-19). Asymptomatic carriage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a potentially significant source of transmission, yet remains relatively poorly understood. Disruption of dental services continues with significantly reduced capacity. Ongoing precautions include preappointment and/or at appointment COVID-19 symptom screening and use of enhanced personal protective equipment (PPE). This study aimed to investigate SARS-CoV-2 infection in dental patients to inform community surveillance and improve understanding of risks in the dental setting. Thirty-one dental care centers across Scotland invited asymptomatic-screened patients aged over 5 y to participate. Following verbal consent and completion of sociodemographic and symptom history questionnaire, trained dental teams took a combined oropharyngeal and nasal swab sample using standardized Viral Transport Medium-containing test kits. Samples were processed by the Lighthouse Lab and patients informed of their results by SMS/email with appropriate self-isolation guidance in the event of a positive test. All positive cases were successfully followed up by the national contact tracing program. Over a 13-wk period (from August 3, 2020, to October 31, 2020), 4,032 patients, largely representative of the population, were tested. Of these, 22 (0.5%; 95% CI, 0.5%-0.8%) tested positive for SARS-CoV-2. The positivity rate increased over the period, commensurate with uptick in community prevalence identified across all national testing monitoring data streams. To our knowledge, this is the first report of a COVID-19 testing survey in asymptomatic-screened patients presenting in a dental setting. The positivity rate in this patient group reflects the underlying prevalence in community at the time. These data are a salient reminder, particularly when community infection levels are rising, of the importance of appropriate ongoing infection prevention control and PPE vigilance, which is relevant as health care team fatigue increases as the pandemic continues. Dental settings are a valuable location for public health surveillance.

Increased case-finding and uptake of direct-acting antiviral treatment essential for micro-elimination of hepatitis C among people living with HIV: a national record linkage study.

OBJECTIVES: Micro-elimination of hepatitis C virus (HCV) in people living with HIV (PLHIV) and co-infected with HCV has been proposed as a key contribution to the overall goal of HCV elimination. While other studies have examined micro-elimination in HIV-treated cohorts, few have considered HCV micro-elimination among those not treated for HIV or at a national level. METHODS: Through data linkage of national and sentinel surveillance data, we examined the extent of HCV testing, diagnosis and treatment among a cohort of PLHIV in Scotland identified through the national database of HIV-diagnosed individuals, up to the end of 2017. RESULTS: Of 5018 PLHIV, an estimated 797 (15%) had never been tested for HCV and 70 (9%) of these had undiagnosed chronic HCV. The odds of never having been tested for HCV were the highest in those not on HIV treatment [adjusted odds ratio (aOR) = 7.21, 95% confidence interval (CI): 5.15-10.10). Overall HCV antibody positivity was 11%, and it was at its highest among people who inject drugs (49%). Most of those with chronic HCV (91%) had attended an HCV treatment clinic but only half had been successfully treated (54% for those on HIV treatment, 12% for those not) by the end of 2017. The odds of never having been treated for HCV were the highest in those not on HIV treatment (aOR = 3.60, 95% CI: 1.59-8.15). CONCLUSIONS: Our data demonstrate that micro-elimination of HCV in PLHIV is achievable but progress will require increased effort to engage and treat those co-infected, including those not being treated for their HIV.

The first case of HIV-2 in Scotland.

HIV-1 infects an estimated 37 million people worldwide, while the rarer HIV-2 infects 1-2 million worldwide. HIV-2 is mainly restricted to West African countries. The majority of patients in Scotland are diagnosed with HIV-1, but in 2013 the West of Scotland Specialist Virology Centre (WoSSVC) diagnosed Scotland's first HIV-2 positive case in a patient from Côte d'Ivoire. HIV-2 differs from HIV-1 in terms of structural viral proteins, viral transmissibility, prolonged period of latency, intrinsic resistance to certain antivirals and how to monitor the effectiveness of treatment. Over the course of 5 years the patient has required several changes in treatment due to both side effects and pill burden. This case highlights the complexity of HIV-2 patient management over time.

Impact of implementing respiratory point-of-care testing in a regional haemato-oncology unit.

Respiratory point-of-care testing (POCT) for the detection of influenza A, influenza B and respiratory syncytial virus (RSV) was implemented in response to recent RSV outbreaks at a regional haemato-oncology unit in Glasgow. This descriptive study, undertaken pre- and post-POCT implementation, suggests that POCT reduces the time taken to receive results and increases diagnostic rates in outpatients. It is likely that the reduction in turnaround time afforded by POCT also leads to a faster time to antiviral treatment, prompt isolation and a reduction in the number of hospital-acquired infections.

A European multicentre evaluation of detection and typing methods for human enteroviruses and parechoviruses using RNA transcripts.

Polymerase chain reaction (PCR) detection has become the gold standard for diagnosis and typing of enterovirus (EV) and human parechovirus (HPeV) infections. Its effectiveness depends critically on using the appropriate sample types and high assay sensitivity as viral loads in cerebrospinal fluid samples from meningitis and sepsis clinical presentation can be extremely low. This study evaluated the sensitivity and specificity of currently used commercial and in-house diagnostic and typing assays. Accurately quantified RNA transcript controls were distributed to 27 diagnostic and 12 reference laboratories in 17 European countries for blinded testing. Transcripts represented the four human EV species (EV-A71, echovirus 30, coxsackie A virus 21, and EV-D68), HPeV3, and specificity controls. Reported results from 48 in-house and 15 commercial assays showed 98% detection frequencies of high copy (1000 RNA copies/5 µL) transcripts. In-house assays showed significantly greater detection frequencies of the low copy (10 copies/5 µL) EV and HPeV transcripts (81% and 86%, respectively) compared with commercial assays (56%, 50%; P = 7 × 10-5 ). EV-specific PCRs showed low cross-reactivity with human rhinovirus C (3 of 42 tests) and infrequent positivity in the negative control (2 of 63 tests). Most or all high copy EV and HPeV controls were successfully typed (88%, 100%) by reference laboratories, but showed reduced effectiveness for low copy controls (41%, 67%). Stabilized RNA transcripts provide an effective, logistically simple and inexpensive reagent for evaluation of diagnostic assay performance. The study provides reassurance of the performance of the many in-house assay formats used across Europe. However, it identified often substantially reduced sensitivities of commercial assays often used as point-of-care tests.

Automation and standardisation of clinical molecular testing using PCR.Ai - A comparative performance study.

BACKGROUND: We undertook a prospective clinical study to evaluate PCR.Ai's (www.pcr.ai) accuracy and impact when automating the manual data-analysis and quality control steps associated with routine clinical pathogen testing using real-time PCR (qPCR). OBJECTIVES: We evaluated the impact of PCR.Ai when used as the final interpretation/verification step for routine in-house qPCR tests for respiratory pathogens and for norovirus for a total of 22,200 interpretations. STUDY DESIGN: We compared PCR.Ai to our existing manual interpretation, to determine accuracy and hands-on time savings. PCR.Ai was accurate. RESULTS AND CONCLUSIONS: There was 100% concurrence between validated respiratory virus and norovirus detection by our manual routine analysis method and PCR.Ai. Furthermore, there were significant routine savings with PCR.Ai of 45 min/respiratory run and 32 min/norovirus run. Our conclusion is that PCR.Ai is a highly accurate time-saving tool that reduces complexity of qPCR analysis and hence the need for specialists and hands-on time. It demonstrated capabilities to enable us to get results out more quickly with lower costs and less risk of errors.

Lessons learned from a pneumocystis pneumonia outbreak at a Scottish renal transplant centre.

BACKGROUND: Pneumocystis pneumonia (PCP) is an opportunistic infection occurring in renal transplant patients. Over a 14-month period an increase in PCP cases was identified among our renal transplant cohort. AIM: The outbreak population was studied to identify potential risk factors for the development of PCP. METHODS: A retrospective analysis of hospital records was carried out, with each case being matched with two case-linked controls. Information was collected on patient demographics, laboratory tests, and hospital visits pre and post development of infection. FINDINGS: No patients were receiving PCP prophylaxis at the time of infection and mean time from transplantation to developing PCP was 4.7 years (range: 0.51-14.5). The PCP group had a significantly lower mean estimated glomerular filtration rate than the control group (29.3 mL/min/1.73 m2 vs 70 mL/min-1 (P = 0.0007)). Three patients were treated for active cytomegalovirus (CMV) infection prior to PCP diagnosis and two had active CMV at the time of diagnosis compared to none in the control group (P = 0.001). Those who developed PCP were more likely to have shared a hospital visit with another patient who went on to develop PCP; 37% of clinic visits vs 19% (P = 0.014). CONCLUSION: This study highlights the ongoing risk of opportunistic infection several years after transplantation and adds weight to potential person-to-person Pneumocystis jirovecii transmission. Risk factors have been identified which may highlight those most at risk, enabling targeted rather than blanket long-term PCP prophylaxis.

Impact of previous hepatitis B infection on the clinical outcomes from chronic hepatitis C? A population-level analysis.

Chronic coinfection with hepatitis C virus (HCV) and hepatitis B virus (HBV) is associated with adverse liver outcomes. The clinical impact of previous HBV infection on liver disease in HCV infection is unknown. We aimed at determining any association of previous HBV infection with liver outcomes using antibodies to the hepatitis B core antigen (HBcAb) positivity as a marker of exposure. The Scottish Hepatitis C Clinical Database containing data for all patients attending HCV clinics in participating health boards was linked to the HBV diagnostic registry and mortality data from Information Services Division, Scotland. Survival analyses with competing risks were constructed for time from the first appointment to decompensated cirrhosis, hepatocellular carcinoma (HCC) and liver-related mortality. Records of 8513 chronic HCV patients were included in the analyses (87 HBcAb positive and HBV surface antigen [HBsAg] positive, 1577 HBcAb positive and HBsAg negative, and 6849 HBcAb negative). Multivariate cause-specific proportional hazards models showed previous HBV infection (HBcAb positive and HBsAg negative) significantly increased the risks of decompensated cirrhosis (hazard ratio [HR]: 1.29, 95% CI: 1.01-1.65) and HCC (HR: 1.64, 95% CI: 1.09-2.49), but not liver-related death (HR: 1.02, 95% CI: 0.80-1.30). This is the largest study to date showing an association between previous HBV infection and certain adverse liver outcomes in HCV infection. Our analyses add significantly to evidence which suggests that HBV infection adversely affects liver health despite apparent clearance. This has important implications for HBV vaccination policy and indications for prioritization of HCV therapy.

Prevalence of mixed genotype hepatitis C virus infections in the UK as determined by genotype-specific PCR and deep sequencing.

The incidence of mixed genotype hepatitis C virus (HCV) infections in the UK is largely unknown. As the efficacy of direct-acting antivirals is variable across different genotypes, treatment regimens are tailored to the infecting genotype, which may pose issues for the treatment of underlying genotypes within undiagnosed mixed genotype HCV infections. There is therefore a need to accurately diagnose mixed genotype infections prior to treatment. PCR-based diagnostic tools were developed to screen for the occurrence of mixed genotype infections caused by the most common UK genotypes, 1a and 3, in a cohort of 506 individuals diagnosed with either of these genotypes. The overall prevalence rate of mixed infection was 3.8%; however, this rate was unevenly distributed, with 6.7% of individuals diagnosed with genotype 3 harbouring genotype 1a strains and only 0.8% of samples from genotype 1a patients harbouring genotype 3 (P < .05). Mixed infection samples consisted of a major and a minor genotype, with the latter constituting less than 21% of the total viral load and, in 67% of cases, less than 1% of the viral load. Analysis of a subset of the cohort by Illumina PCR next-generation sequencing resulted in a much greater incidence rate than obtained by PCR. This may have occurred due to the nonquantitative nature of the technique and despite the designation of false-positive thresholds based on negative controls.

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.

Consecutive yearly outbreaks of respiratory syncytial virus in a haemato-oncology ward and efficacy of infection control measures.

BACKGROUND: Respiratory syncytial virus (RSV) causes significant respiratory tract infection in immunosuppressed patients. AIM: To describe two consecutive yearly outbreaks of RSV in our haemato-oncology ward. METHODS: Haematology patients presenting with respiratory symptoms were screened by polymerase chain reaction for viral respiratory pathogens using a saline gargle. FINDINGS: None of our patients had undergone bone marrow transplant but all had underlying haematological malignancies. Eight patients were affected in the first outbreak (mortality rate: 37.5%) and 12 patients were affected in the second (mortality rate: 8.3%). Extensive infection control measures were implemented in both outbreaks and were successful in preventing further cross-transmission. CONCLUSION: There was significant learning from both outbreaks and actions implemented with the aim of reducing the likelihood and impact of future outbreaks.

Investigation of outbreaks of Pneumocystis jirovecii pneumonia in two Scottish renal units.

Pneumocystis jirovecii is recognized as an opportunistic pathogen. In recent years, human-to-human transmission of P. jirovecii has been demonstrated. However, outbreaks of P. jirovecii infections are not well defined because the epidemiological setting that facilitates transmission is not fully understood. This article describes two outbreaks of P. jirovecii pneumonia (PCP) in renal transplant patients in the West of Scotland. In total, 25 patients in two geographically contiguous locations were affected. Allele B was identified as the dominant type, along with allele A3. It was not possible to determine the exact reason for clustering of cases, although the outpatient clinic setting featured in one of the outbreaks. The outbreaks ceased with the use of trimethoprim-sulphamethoxazole prophylaxis; the target populations that received prophylaxis were different in the two outbreaks. Infection control teams should be alert to the possibility of outbreaks of PCP.

Extensive multiplex PCR diagnostics reveal new insights into the epidemiology of viral respiratory infections.

Viral respiratory infections continue to pose a major global healthcare burden. At the community level, the co-circulation of respiratory viruses is common and yet studies generally focus on single aetiologies. We conducted the first comprehensive epidemiological analysis to encompass all major respiratory viruses in a single population. Using extensive multiplex PCR diagnostic data generated by the largest NHS board in Scotland, we analysed 44230 patient episodes of respiratory illness that were simultaneously tested for 11 virus groups between 2005 and 2013, spanning the 2009 influenza A pandemic. We measured viral infection prevalence, described co-infections, and identified factors independently associated with viral infection using multivariable logistic regression. Our study provides baseline measures and reveals new insights that will direct future research into the epidemiological consequences of virus co-circulation. In particular, our study shows that (i) human coronavirus infections are more common during influenza seasons and in co-infections than previously recognized, (ii) factors associated with co-infection differ from those associated with viral infection overall, (iii) virus prevalence has increased over time especially in infants aged <1 year, and (iv) viral infection risk is greater in the post-2009 pandemic era, likely reflecting a widespread change in the viral population that warrants further investigation.