Diagnosis of Multisystem Inflammatory Syndrome in Children by a Whole-Blood Transcriptional Signature.

BACKGROUND: To identify a diagnostic blood transcriptomic signature that distinguishes multisystem inflammatory syndrome in children (MIS-C) from Kawasaki disease (KD), bacterial infections, and viral infections. METHODS: Children presenting with MIS-C to participating hospitals in the United Kingdom and the European Union between April 2020 and April 2021 were prospectively recruited. Whole-blood RNA Sequencing was performed, contrasting the transcriptomes of children with MIS-C (n = 38) to those from children with KD (n = 136), definite bacterial (DB; n = 188) and viral infections (DV; n = 138). Genes significantly differentially expressed (SDE) between MIS-C and comparator groups were identified. Feature selection was used to identify genes that optimally distinguish MIS-C from other diseases, which were subsequently translated into RT-qPCR assays and evaluated in an independent validation set comprising MIS-C (n = 37), KD (n = 19), DB (n = 56), DV (n = 43), and COVID-19 (n = 39). RESULTS: In the discovery set, 5696 genes were SDE between MIS-C and combined comparator disease groups. Five genes were identified as potential MIS-C diagnostic biomarkers (HSPBAP1, VPS37C, TGFB1, MX2, and TRBV11-2), achieving an AUC of 96.8% (95% CI: 94.6%-98.9%) in the discovery set, and were translated into RT-qPCR assays. The RT-qPCR 5-gene signature achieved an AUC of 93.2% (95% CI: 88.3%-97.7%) in the independent validation set when distinguishing MIS-C from KD, DB, and DV. CONCLUSIONS: MIS-C can be distinguished from KD, DB, and DV groups using a 5-gene blood RNA expression signature. The small number of genes in the signature and good performance in both discovery and validation sets should enable the development of a diagnostic test for MIS-C.

Comparing lateral flow testing with a rapid RT‐PCR method for SARS‐CoV‐2 detection in the United Kingdom—A retrospective diagnostic accuracy study

Background and Aims In late 2019, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) emerged in Wuhan, China. Rapid global spread led to the coronavirus disease 2019 (Covid‐19) pandemic. Accurate detection of SARS‐CoV‐2 has become a vitally important tool in controlling the spread of the virus. Lateral flow devices (LFDs) offer the potential advantage of speed and on‐site testing. The sensitivity of these devices compared to reverse transcription‐polymerase chain reaction (RT‐PCR) has been questioned. Methods We compared the sensitivity of the Innova LFD, used widely in the United Kingdom, with our rapid RT‐PCR method using stored positive samples. Samples with a range of viral loads (original Ct values 18.9–36.5) were tested. Results The Innova LFD was found to be 6000–10,000 times less sensitive than RT‐PCR for SARS‐CoV‐2 detection. Overall, the LFD detected 46.2% of the positives detected by RT‐PCR, with 50% of these observed to be weak LFD positives. At lower viral loads, such as 10,000–100,000 RNA copies/ml, the LFD detected 22.2% of positives. In addition, two strong positives (3 and 1.5 million RNA copies/ml) were not detected by the LFD. Conclusion The argument for use of LFD kits is that they detect infectious virus and hence contagious individuals. However, there is a lack of conclusive evidence supporting this claim. The Innova LFD has been subject to a Class I recall by the US Food and Drug Administration, but is still approved and widely used in the United Kingdom.

Comparing lateral flow testing with a rapid RT-PCR method for SARS-CoV-2 detection in the United Kingdom-A retrospective diagnostic accuracy study.

Background and Aims: In late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China. Rapid global spread led to the coronavirus disease 2019 (Covid-19) pandemic. Accurate detection of SARS-CoV-2 has become a vitally important tool in controlling the spread of the virus. Lateral flow devices (LFDs) offer the potential advantage of speed and on-site testing. The sensitivity of these devices compared to reverse transcription-polymerase chain reaction (RT-PCR) has been questioned. Methods: We compared the sensitivity of the Innova LFD, used widely in the United Kingdom, with our rapid RT-PCR method using stored positive samples. Samples with a range of viral loads (original Ct values 18.9-36.5) were tested. Results: The Innova LFD was found to be 6000-10,000 times less sensitive than RT-PCR for SARS-CoV-2 detection. Overall, the LFD detected 46.2% of the positives detected by RT-PCR, with 50% of these observed to be weak LFD positives. At lower viral loads, such as 10,000-100,000 RNA copies/ml, the LFD detected 22.2% of positives. In addition, two strong positives (3 and 1.5 million RNA copies/ml) were not detected by the LFD. Conclusion: The argument for use of LFD kits is that they detect infectious virus and hence contagious individuals. However, there is a lack of conclusive evidence supporting this claim. The Innova LFD has been subject to a Class I recall by the US Food and Drug Administration, but is still approved and widely used in the United Kingdom.

The temporal pattern and lifestyle associations of respiratory virus infection in a cohort study spanning the first two years of life.

BACKGROUND: Respiratory virus infection is common in early childhood, and children may be symptomatic or symptom-free. Little is known regarding the association between symptomatic/asymptomatic infection and particular clinical factors such as breastfeeding as well as the consequences of such infection. METHOD: We followed an unselected cohort of term neonates to two years of age (220 infants at recruitment, 159 who remained in the study to 24 months), taking oral swabs at birth and oropharyngeal swabs at intervals subsequently (at 1.5, 6, 9, 12, 18 and 24 months and in a subset at 3 and 4.5 months) while recording extensive metadata including the presence of respiratory symptoms and breastfeeding status. After 2 years medical notes from the general practitioner were inspected to ascertain whether doctor-diagnosed wheeze had occurred by this timepoint. Multiplex PCR was used to detect a range of respiratory viruses: influenza (A&B), parainfluenza (1-4), bocavirus, human metapneumovirus, rhinovirus, coronavirus (OC43, 229E, NL63, HKU1), adenovirus, respiratory syncytial virus (RSV), and polyomavirus (KI, WU). Logistic regression and generalised estimating equations were used to identify associations between clinical factors and virus detection. RESULTS: Overall respiratory viral incidence increased with age. Rhinovirus was the virus most frequently detected. The detection of a respiratory virus was positively associated with respiratory symptoms, male sex, season, childcare and living with another child. We did not observe breastfeeding (whether assessed as the number of completed months of breastfeeding or current feed status) to be associated with the detection of a respiratory virus. There was no association between early viral infection and doctor-diagnosed wheeze by age 2 years. CONCLUSION: Asymptomatic and symptomatic viral infection is common in the first 2 years of life with rhinovirus infection being the most common. Whilst there was no association between early respiratory viral infection and doctor-diagnosed wheeze, we have not ruled out an association of early viral infections with later asthma, and long-term follow-up of the cohort continues.

Molecular detection of SARS-CoV-2 using a reagent-free approach.

Shortage of reagents and consumables required for the extraction and molecular detection of SARS-CoV-2 RNA in respiratory samples has led many laboratories to investigate alternative approaches for sample preparation. Many groups recently presented results using heat processing method of respiratory samples prior to RT-qPCR as an economical method enabling an extremely fast streamlining of the processes at virtually no cost. Here, we present our results using this method and highlight some major pitfalls that diagnostics laboratories should be aware of before proceeding with this methodology. We first investigated various treatments using different temperatures, incubation times and sample volumes to optimise the heat treatment conditions. Although the initial data confirmed results published elsewhere, further investigations revealed unexpected inhibitory properties of some commonly used universal transport media (UTMs) on some commercially available RT-qPCR mixes, leading to a risk of reporting false-negative results. This emphasises the critical importance of a thorough validation process to determine the most suitable reagents to use depending on the sample types to be tested. In conclusion, a heat processing method is effective with very consistent Ct values and a sensitivity of 96.2% when compared to a conventional RNA extraction method. It is also critical to include an internal control to check each sample for potential inhibition.