Performance evaluation of fully automated cobas® 6800 CMV PCR for the detection and quantification of cytomegalovirus DNA in neonatal urine and saliva, and adult urine, saliva, and vaginal secretion.

Laboratory testing for cytomegalovirus (CMV) in bodily fluids is essential to manage congenital and prenatal CMV infection. The rapid and fully automated cobas® CMV PCR is approved only for the testing of plasma in transplant patients. To evaluate the performance of the cobas® CMV to detect and quantify CMV DNA in neonatal and adult female urine, saliva, and vaginal secretion, the limit of detection (LoD), limit of quantification (LoQ), imprecision, linearity, PCR efficiency, bias, analytical specificity, cross-reactivity, and cross-contamination of the cobas® CMV for urine, saliva, and vaginal secretion was determined. The performance of the assay was evaluated prospectively with two laboratory-developed PCR assays using neonatal and adult urine, saliva swabs, and vaginal swabs. The LoD and LoQ were 31 and 100 IU/mL, respectively, for urine, and 81 and 100 IU/mL, respectively, for vaginal secretion. The LoD and LoQ for saliva were the same (200 IU/mL). The cobas® CMV was precise (coefficient of variation ≤10%), linear (R2 ≥ 0.995), and efficient (1.07 and 1.09) between 100 and 250,000 IU/mL for the sample types. The bias and analytical specificity was <±0.30 log10 IU/mL and 100%, respectively. Cross-reactivity with non-CMV pathogens was not detected. Cross-contamination rate was 0.28%. The diagnostic accuracy, sensitivity, and specificity of the cobas® CMV for neonatal urine and saliva were ≥95.0%, ≥93.3%, and ≥90.4%, respectively. The overall percent agreement for adult urine, saliva, and vaginal secretion was 86.6%, 94.5%, and 89.4%, respectively. Taken together, the cobas® CMV demonstrated acceptable analytical and diagnostic performance, and is suitable for routine diagnostic laboratory investigation of CMV infection in neonates and adults.

Detecting human cytomegalovirus in urine, vagina and saliva: Impact of biological fluids and storage durations and temperatures on CMV DNA recovery.

Sample collection, transport and storage conditions vary in the human cytomegalovirus (CMV) shedding literature. Currently, limited data exist on the impact of biological fluids and pre-analytical sample handling on the detection of CMV DNA. To evaluate CMV DNA recovery from urine, vaginal fluid and saliva stored in different conditions, adult urine, vaginal and saliva fluids and swabs, stored with or without selected nucleic acid preservation media at various durations and temperatures, was compared by polymerase chain reaction (PCR) quantitation of spiked samples and self-collected urine (n = 45) and vaginal swabs (n = 58) from CMV seropositive pregnant women. There was a time-dependent reduction in CMV DNA recovery from urine, urine diluted in phosphate-buffered saline, and saliva stored at 2-8°C, but not from urine preserved in cobas® PCR transport media (CPM) (urine/CPM). For vaginal fluid, a reduction in recovery was evident after 7 days storage at 2-8°C. CMV DNA recovery over 91 days was similar between -80°C and -20°C storage for urine and vaginal swabs preserved in CPM, and saliva swabs preserved in eNAT® PCR transport media. A statistically significant change in CMV DNA recovery after 25 months storage (median) at -80°C was not observed for self-collected urine/CPM and vaginal swab/CPM from pregnant women. Taken together, recovery of CMV DNA is dependent on fluid type and storage conditions. To improve the validity and reliability of detection at different storage durations and temperatures, the use of nucleic acid preserving transport media at the point of collection for urine, vaginal fluid and saliva may be essential.

Performance evaluation of the Viasure PCR assay for the diagnosis of monkeypox: A multicentre study.

BACKGROUND: Monkeypox virus (MPXV) is the causative agent of the 2022 monkeypox global outbreak. Rapid detection of MPXV infection is essential to inform patient management and public health response. Currently, there is a lack of established real-time PCR assays to support a rapid diagnosis of monkeypox. OBJECTIVES: To evaluate the performance characteristics of the Viasure MPXV PCR assay in three London teaching hospitals. STUDY DESIGN: Prospectively collected paired patient swabs from matched or unmatched anatomical sites were evaluated by the reference laboratory and Viasure MPXV PCR assays. A subset of samples were also tested for HSV, VZV, and/or Treponema pallidum DNA. RESULTS: 217 paired samples were evaluated. 91.2% of the paired swabs generated concordant results whilst 8.8% generated discordant results. The accuracy, diagnostic sensitivity, diagnostic specificity, positive predictive value, negative predictive value, likelihood ratio positive, and likelihood ratio negative of the Viasure PCR assay across the hospitals were 93.2 - 96.3%, 90.0 - 100%, 88.2 - 100%, 94.9 - 100%, 87.9 - 100%, 8.50 - 14.41, and 0.00 - 0.10 respectively. MPXV co-infections with HSV were detected in two patients. Five patients were negative for monkeypox but positive for herpes or chickenpox. CONCLUSIONS: The Viasure MPXV PCR assay demonstrated excellent performance characteristics, was easy to use, and is fit for routine diagnostic purpose. Where implemented, the assay would allow rapid and accurate laboratory diagnosis of MPXV infections and support a timely management of monkeypox. To reduce the risk of false negative detections, vesicular lesions from any anatomical site should be preferentially and optimally sampled.

Effectiveness of rapid SARS-CoV-2 genome sequencing in supporting infection control for hospital-onset COVID-19 infection: Multicentre, prospective study.

Background: Viral sequencing of SARS-CoV-2 has been used for outbreak investigation, but there is limited evidence supporting routine use for infection prevention and control (IPC) within hospital settings. Methods: We conducted a prospective non-randomised trial of sequencing at 14 acute UK hospital trusts. Sites each had a 4-week baseline data collection period, followed by intervention periods comprising 8 weeks of 'rapid' (<48 hr) and 4 weeks of 'longer-turnaround' (5-10 days) sequencing using a sequence reporting tool (SRT). Data were collected on all hospital-onset COVID-19 infections (HOCIs; detected ≥48 hr from admission). The impact of the sequencing intervention on IPC knowledge and actions, and on the incidence of probable/definite hospital-acquired infections (HAIs), was evaluated. Results: A total of 2170 HOCI cases were recorded from October 2020 to April 2021, corresponding to a period of extreme strain on the health service, with sequence reports returned for 650/1320 (49.2%) during intervention phases. We did not detect a statistically significant change in weekly incidence of HAIs in longer-turnaround (incidence rate ratio 1.60, 95% CI 0.85-3.01; p=0.14) or rapid (0.85, 0.48-1.50; p=0.54) intervention phases compared to baseline phase. However, IPC practice was changed in 7.8 and 7.4% of all HOCI cases in rapid and longer-turnaround phases, respectively, and 17.2 and 11.6% of cases where the report was returned. In a 'per-protocol' sensitivity analysis, there was an impact on IPC actions in 20.7% of HOCI cases when the SRT report was returned within 5 days. Capacity to respond effectively to insights from sequencing was breached in most sites by the volume of cases and limited resources. Conclusions: While we did not demonstrate a direct impact of sequencing on the incidence of nosocomial transmission, our results suggest that sequencing can inform IPC response to HOCIs, particularly when returned within 5 days. Funding: COG-UK is supported by funding from the Medical Research Council (MRC) part of UK Research & Innovation (UKRI), the National Institute of Health Research (NIHR) (grant code: MC_PC_19027), and Genome Research Limited, operating as the Wellcome Sanger Institute. Clinical trial number: NCT04405934.

Screening for cytomegalovirus shedding in vagina and saliva: Significant differences between biological fluids, swab types and storage durations in DNA recovery.

BACKGROUND: Cytomegalovirus (CMV) shedding in genital and oral secretions during pregnancy is associated with adverse outcomes. Sample collection methods between studies are not uniform and currently there are limited data on the impact of biological fluids, swab types and storage durations on the detection of CMV DNA. OBJECTIVES: To evaluate the absorption efficiency and CMV DNA recovery of various commercially available swabs in vaginal and saliva fluids. STUDY DESIGN: The absorption volume of different swab types was evaluated. The recovery of CMV DNA over time from vaginal and saliva fluids, and vaginal and saliva swabs, was evaluated by PCR measurements of samples spiked with CMV standard. RESULTS: Absorption efficiency of swabs varied significantly. The duration of storage did not affect CMV DNA recovery from vaginal fluid or swabs, but did significantly affect CMV DNA recovery from neat saliva fluid and saliva swabs stored dry or in viral transport media (VTM). Flocked swab/eNAT media recovered the highest amount of CMV DNA from both fluids. In saliva, flocked swab/eNAT media and polyester swab/cobas media demonstrated a higher CMV DNA recovery than foam swab/VTM. 25% of dry saliva foam swabs were falsely negative for CMV DNA. CONCLUSIONS: Recovery of CMV DNA is dependent on sample type and swab type used. Flocked swab/eNAT media and polyester swab/cobas media appear acceptable, though flocked swab/eNAT media was superior demonstrating the best recovery of CMV DNA. For saliva, foam swabs stored dry or in VTM was shown to be inferior to flocked or polyester swabs.

The Alpha variant was not associated with excess nosocomial SARS-CoV-2 infection in a multi-centre UK hospital study.

OBJECTIVES: Recently emerging SARS-CoV-2 variants have been associated with an increased rate of transmission within the community. We sought to determine whether this also resulted in increased transmission within hospitals. METHODS: We collected viral sequences and epidemiological data of patients with community and healthcare associated SARS-CoV-2 infections, sampled from 16th November 2020 to 10th January 2021, from nine hospitals participating in the COG-UK HOCI study. Outbreaks were identified using ward information, lineage and pairwise genetic differences between viral sequences. RESULTS: Mixed effects logistic regression analysis of 4184 sequences showed healthcare-acquired infections were no more likely to be identified as the Alpha variant than community acquired infections. Nosocomial outbreaks were investigated based on overlapping ward stay and SARS-CoV-2 genome sequence similarity. There was no significant difference in the number of patients involved in outbreaks caused by the Alpha variant compared to outbreaks caused by other lineages. CONCLUSIONS: We find no evidence to support it causing more nosocomial transmission than previous lineages. This suggests that the stringent infection prevention measures already in place in UK hospitals contained the spread of the Alpha variant as effectively as other less transmissible lineages, providing reassurance of their efficacy against emerging variants of concern.

SARS-CoV-2 lineage B.1.1.7 is associated with greater disease severity among hospitalised women but not men: multicentre cohort study.

BACKGROUND: SARS-CoV-2 lineage B.1.1.7 has been associated with an increased rate of transmission and disease severity among subjects testing positive in the community. Its impact on hospitalised patients is less well documented. METHODS: We collected viral sequences and clinical data of patients admitted with SARS-CoV-2 and hospital-onset COVID-19 infections (HOCIs), sampled 16 November 2020 to 10 January 2021, from eight hospitals participating in the COG-UK-HOCI study. Associations between the variant and the outcomes of all-cause mortality and intensive therapy unit (ITU) admission were evaluated using mixed effects Cox models adjusted by age, sex, comorbidities, care home residence, pregnancy and ethnicity. FINDINGS: Sequences were obtained from 2341 inpatients (HOCI cases=786) and analysis of clinical outcomes was carried out in 2147 inpatients with all data available. The HR for mortality of B.1.1.7 compared with other lineages was 1.01 (95% CI 0.79 to 1.28, p=0.94) and for ITU admission was 1.01 (95% CI 0.75 to 1.37, p=0.96). Analysis of sex-specific effects of B.1.1.7 identified increased risk of mortality (HR 1.30, 95% CI 0.95 to 1.78, p=0.096) and ITU admission (HR 1.82, 95% CI 1.15 to 2.90, p=0.011) in females infected with the variant but not males (mortality HR 0.82, 95% CI 0.61 to 1.10, p=0.177; ITU HR 0.74, 95% CI 0.52 to 1.04, p=0.086). INTERPRETATION: In common with smaller studies of patients hospitalised with SARS-CoV-2, we did not find an overall increase in mortality or ITU admission associated with B.1.1.7 compared with other lineages. However, women with B.1.1.7 may be at an increased risk of admission to intensive care and at modestly increased risk of mortality.

COVID-19 antibody tests: statistical implications.

The coronavirus disease 2019 (COVID-19) pandemic has had significant implications for society, with the introduction of restrictive social measures. Antibody tests provide a way of identifying patients who have been previously exposed to the virus and thus may have a degree of immunity. This is important in the development of public health policy, as local and national bodies seek to relax social restrictions in an attempt to mitigate the socioeconomic impact of the pandemic. This article explores the essential statistical concepts used to interpret the findings of diagnostic investigations, with examples illustrated using COVID-19 antibody tests.

Verification of the Roche cobas® 6800 PCR 200 µl and 500 µl protocols for the quantification of HIV-1 RNA, HBV DNA and HCV RNA and evaluation with COBAS® Ampliprep/COBAS® TaqMan® assays.

PURPOSE: The analytical performance of the cobas 6800 HIV-1, HBV and HCV assays was verified and evaluated to the COBAS Ampliprep/COBAS TaqMan assays. METHODOLOGY: The precision, limit of detection (LoD), limit of quantification (LoQ) and linearity were verified using pooled residual clinical samples. The analytical specificity was verified with negative plasma. HIV-1 analytical reactivity was verified with WHO reference preparations. Accuracy was verified using EQA plasma panels. Evaluation of the equipment was performed using prospectively collected clinical whole blood samples. RESULTS: Excellent precision was demonstrated using both testing protocols (coefficient of variation ≤15 %). The LoDs using the 500 and 200 µl protocols were 20 and 50 cp ml-1 for HIV-1, 10 and 20 IU ml-1 for HBV and 15 and 40 IU ml-1 for HCV, respectively. The LoQs were 40 and 100 cp ml-1 for HIV-1, 20 and 25 IU ml-1 for HBV and 30 and 80 IU ml-1 for HCV, respectively. Assays demonstrated good linearity (R2 ≥0.96). The analytical specificity of the assays was 100 %. There was excellent agreement between the cobas 6800 and CAP/CTM assays (kappa>0.94). The sensitivity, specificity, positive predictive value and negative predictive value for each of the assays were ≥99 %. The cobas HIV-1 and HCV mean quantifications were 0.03 log10 cp ml-1 and 0.17 log10 IU ml-1 higher than the CAP/CTM. The cobas HBV mean quantification was 0.17 log10 IU ml-1 lower than the CAP/CTM. Subtype/genotype specific differences were not observed. CONCLUSION: Cobas 6800 equipment and assays demonstrated excellent performance and correlated well with CAP/CTM assay results.

Clinical application of whole-genome sequencing to inform treatment for multidrug-resistant tuberculosis cases.

The treatment of drug-resistant tuberculosis cases is challenging, as drug options are limited, and the existing diagnostics are inadequate. Whole-genome sequencing (WGS) has been used in a clinical setting to investigate six cases of suspected extensively drug-resistant Mycobacterium tuberculosis (XDR-TB) encountered at a London teaching hospital between 2008 and 2014. Sixteen isolates from six suspected XDR-TB cases were sequenced; five cases were analyzed in a clinically relevant time frame, with one case sequenced retrospectively. WGS identified mutations in the M. tuberculosis genes associated with antibiotic resistance that are likely to be responsible for the phenotypic resistance. Thus, an evidence base was developed to inform the clinical decisions made around antibiotic treatment over prolonged periods. All strains in this study belonged to the East Asian (Beijing) lineage, and the strain relatedness was consistent with the expectations from the case histories, confirming one contact transmission event. We demonstrate that WGS data can be produced in a clinically relevant time scale some weeks before drug sensitivity testing (DST) data are available, and they actively help clinical decision-making through the assessment of whether an isolate (i) has a particular resistance mutation where there are absent or contradictory DST results, (ii) has no further resistance markers and therefore is unlikely to be XDR, or (iii) is identical to an isolate of known resistance (i.e., a likely transmission event). A small number of discrepancies between the genotypic predictions and phenotypic DST results are discussed in the wider context of the interpretation and reporting of WGS results.

Differences in outcome according to Clostridium difficile testing method: a prospective multicentre diagnostic validation study of C difficile infection.

BACKGROUND: Diagnosis of Clostridium difficile infection is controversial because of many laboratory methods, compounded by two reference methods. Cytotoxigenic culture detects toxigenic C difficile and gives a positive result more frequently (eg, because of colonisation, which means that individuals can have the bacterium but no free toxin) than does the cytotoxin assay, which detects preformed toxin in faeces. We aimed to validate the reference methods according to clinical outcomes and to derive an optimum laboratory diagnostic algorithm for C difficile infection. METHODS: In this prospective, multicentre study, we did cytotoxigenic culture and cytotoxin assays on 12,420 faecal samples in four UK laboratories. We also performed tests that represent the three main targets for C difficile detection: bacterium (glutamate dehydrogenase), toxins, or toxin genes. We used routine blood test results, length of hospital stay, and 30-day mortality to clinically validate the reference methods. Data were categorised by reference method result: group 1, cytotoxin assay positive; group 2, cytotoxigenic culture positive and cytotoxin assay negative; and group 3, both reference methods negative. FINDINGS: Clinical and reference assay data were available for 6522 inpatient episodes. On univariate analysis, mortality was significantly higher in group 1 than in group 2 (72/435 [16·6%] vs 20/207 [9·7%], p=0·044) and in group 3 (503/5880 [8·6%], p<0·001), but not in group 2 compared with group 3 (p=0·4). A multivariate analysis accounting for potential confounders confirmed the mortality differences between groups 1 and 3 (OR 1·61, 95% CI 1·12-2·31). Multistage algorithms performed better than did standalone assays. INTERPRETATION: We noted no increase in mortality when toxigenic C difficile alone was present. Toxin (cytotoxin assay) positivity correlated with clinical outcome, and so this reference method best defines true cases of C difficile infection. A new diagnostic category of potential C difficile excretor (cytotoxigenic culture positive but cytotoxin assay negative) could be used to characterise patients with diarrhoea that is probably not due to C difficile infection, but who can cause cross-infection.

Positive predictive value of the Becton Dickinson VIPER system and the ProbeTec GC Q x assay, in extracted mode, for detection of Neisseria gonorrhoeae.

OBJECTIVES: Performance of the new Becton Dickinson ProbeTec GC Q(x) assay on the BD VIPER platform was evaluated to ascertain whether confirmatory testing is required in our clinical setting. METHODS: Positive predictive value (PPV) was determined by comparison with culture and a confirmatory nucleic acid amplification test (NAAT)-based Neisseria gonorrhoeae assay from genital and extragenital samples (rectal and pharyngeal) collected from a genitourinary medicine (GUM) clinic. RESULTS: Among 14,223 clinical genital samples, 149 (1.0%) specimens were positive using the ProbeTec GC Q(x) assay, automated on the VIPER platform; 141 of these were confirmed by either culture or a real-time PCR targeting two gonococcal-specific targets (PPV 94.6%; 95% CI 90% to 98%). Among 840 pharyngeal samples, 26 (3.1%) were positive by the ProbeTec GC Q(x) assay; 13 were confirmed (PPV 50%; 95% CI 30% to 70%). Among 593 rectal samples, 17 tested positive by the ProbeTec GC Q(x) assay; all were confirmed (PPV 100%; 95% CI 80% to 100%). CONCLUSIONS: The lower 95% CI of the PPV for the ProbeTec GC Q(x) assay for genital specimens was >90% in this GUM clinic population, and therefore confirmatory testing for genital specimens is not required. Confirmatory testing of pharyngeal and rectal samples should continue in line with national guidelines.

Methods to determine fitness in bacteria.

Acquisition of antibiotic resistance may be associated with a physiological cost for the bacterium. Determination of growth rate and generation time is often used to measure fitness costs associated with antibiotic resistance. However, fitness costs may be small and difficult to quantify and multiple models are required. Available in vitro models that can be used to measure fitness include quantification of biofilm growth, survival in water, resistance to drying, and measurement of planktonic growth rates. The use of growth curve techniques to determine generation time is laborious, time-consuming, and can introduce sampling error. We have described the use of a semi-automated liquid culture system to estimate generation time in Burkholderia cepacia complex bacteria. We have also used the BacT/ALERT system to determine generation time and enumerate bacterial numbers in Mycobacterium tuberculosis. We describe methods for measuring biofilm growth and environmental survival in Burkholderia cepacia complex bacteria. These methods can be adapted for use with other organisms.