Showing papers by "Joanne E. Martin published in 2021"

Expression of p16 Within Myenteric Neurons of the Aged Colon: A Potential Marker of Declining Function.

Abstract: Human colonic neuromuscular functions decline among the elderly. The aim was to explore the involvement of senescence. A preliminary PCR study looked for age-dependent differences in expression of CDKN1A (encoding the senescence-related p21 protein) and CDKN2A (encoding p16 and p14) in human ascending and descending colon (without mucosa) from 39 (approximately 50: 50 male: female) adult (aged 27-60 years) and elderly donors (70-89 years). Other genes from different aging pathways (e.g., inflammation, oxidative stress, autophagy) and cell-types (e.g., neurons, neuron axonal transport) were also examined. Unlike CDKN1A, CDKN2A (using primers for p16 and p14 but not when using p14-specific primers) was upregulated in both regions of colon. Compared with the number of genes appearing to upregulate in association with temporal age, more genes positively associated with increased CDKN2A expression (respectively, 16 and five of 44 genes studied for ascending and descending colon). Confirmation of increased expression of CDKN2A was sought by immunostaining for p16 in the myenteric plexus of colon from 52 patients, using a semi-automated software protocol. The results showed increased staining not within the glial cells (S100 stained), but in the cytoplasm of myenteric nerve cell bodies (MAP2 stained, with identified nucleus) of ascending, but not descending colon of the elderly, and not in the cell nucleus of either region or age group (5,710 neurons analyzed: n = 12-14 for each group). It was concluded that increased p16 staining within the cytoplasm of myenteric nerve cell bodies of elderly ascending (but not descending) colon, suggests a region-dependent, post-mitotic cellular senescence-like activity, perhaps involved with aging of enteric neurons within the colon.

Megarectum: systematic histopathological evaluation of 35 patients and new common pathways in chronic rectal dilatation.

Abstract: Aims Megarectum is well described in the surgical literature but few contemporary pathological studies have been undertaken. There is uncertainty whether ‘idiopathic’ megarectum is a primary neuromuscular disorder or whether chronic dilatation leads to previously reported and unreported pathological changes. We sought to answer this question. Methods Systematic histopathological evaluation (in accord with international guidance) of 35 consecutive patients undergoing rectal excision surgery for megarectum (primary: n=24) or megarectum following surgical correction of anorectal malformation (secondary: n=11) in a UK university hospital with adult/paediatric surgical and gastrointestinal neuropathology expertise. Results We confirmed some previously reported observations, notably hypertrophy of the muscularis propria (27 of 35, 77.1% of patients) and extensive fibrosis (30 of 35, 85.7% of patients). We also observed unique and previously unreported features including elastosis (19 of 33, 57.6%) and the presence of polyglucosan bodies (15 of 32, 46.9% of patients). In contrast to previous literature, few patients had any strong evidence of specific forms of visceral neuropathy (5 of 35, including 3 plexus duplications) or myopathy (6 of 35, including 3 muscle duplications). All major pathological findings were common to both primary and secondary forms of the disease, implying that these may be a response to chronic rectal distension rather than of primary aetiology. Conclusions In the largest case series reported to date, we challenge the current perception of idiopathic megarectum as a primary neuromuscular disease and propose a cellular pathway model for the features present. The severe morphological changes account for some of the irreversibility of the condition and reinforce the need to prevent ongoing rectal distension when first identified.

Meta-analysis of rapid direct-to-PCR assays for the qualitative detection of SARS-CoV-2

Abstract: Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the ensuing COVID-19 pandemic present significant challenges to current diagnostic and therapeutic patient care pathways including whether new in vitro diagnostic tests can accurately identify and rule out current SARS-CoV-2 infection. The gold standard diagnostic test to identify a current SARS-CoV-2 infection is a central laboratory-based molecular assay using reverse transcription polymerase chain reaction (RT-PCR) with very high accuracy of detection but which typically requires 1-2 days turn-around for results. Therefore, rapid RTPCR assays and systems have been developed which can be deployed locally (near-patient or point of care (POC), provide faster results and not impact on already stressed central laboratory capacity. Rapid test results can be returned within the same clinical encounter, facilitating timely decisions that optimise the patient care pathway and support more rapid COVID-19 diagnosis, isolation and contract tracing activities1. Direct-to-PCR is an evolution of RT-PCR in which the patient sample is added directly to an amplification reaction without being subjected to prior nucleic acid extraction, purification, or quantification to reduce the time and monetary resources required to process samples.1 Rapid, direct-to-PCR systems further increase the speed of testing by combining rapid PCR instruments with direct-to-PCR assays, to generate results in less than two hours. This appears to be the first meta-analysis assessing the accuracy of rapid direct-to-PCR in the detection of SARS-CoV-2. In total, 10,957 unique records were identified and screened using a search string evaluation, 420 full-text reports and/or supplemental materials were assessed for inclusion. This resulted in 14 studies reporting 20 datasets with 4593 patient samples (1391 positive) included in the analysis. The overall agreement between the rapid direct RT-PCR and gold standard centralised laboratory RT-PCR was 97.1% with 93.60% positive percent agreement and 98.63% negative percent agreement. The Cohen’s kappa statistical coefficient k = 0.93, indicating an almost perfect agreement and Youden Index = 0.92. These results indicate that direct-to-PCR assays can perform equivalently to the standard centralised laboratory PCR systems for the detection of SARS-CoV-2. Objectives To assess the efficacy of rapid direct-to-PCR assays and systems for the detection of SARS-CoV-2 in the hospital, care home and medical research population in England from November 2020 to April 2021. Search methods Electronic searches of the Cochrane COVID-19 Study Register (which includes daily updates from PubMed and Embase and preprints from medRxiv and bioRxiv) were undertaken on the 30th of April 2020. Please see the PRISMA flow diagram below (figure 2). Selection criteria Studies of subjects with either suspected SARS-CoV-2 infection, known SARS-CoV-2 infection or known absence of infection, or those who were being screened for infection were included. Commercially available and research use rapid direct-to-PCR assays (without RNA extraction and purification reporting results within two hours) were included in the study. Data collection, extraction and analysis Studies were screened independently, in duplicate with any disagreements resolved by discussion with a third author. Study characteristics were extracted by one author and checked by a second; extraction of study results and assessments of risk of bias and applicability were undertaken independently in duplicate. Where studies were not publicly available, sites that undertook in-service evaluations of rapid direct-to-PCR system were contacted and asked to supply anonymised datasets. Both reviewers independently performed data extraction and verification and calculated 2×2 contingency tables of the number of true positives, false positives, false negatives and true negatives. They resolved any disagreements by discussion and by review with the third reviewer. Main results Twenty study cohorts were included (described in 14 study reports, including 4 unpublished reports), reporting results for 4593 samples (1391 with confirmed SARS-CoV-2). Studies were mainly from Europe and North America, and evaluated nine direct-to-PCR assays. Conclusions This appears to be the first meta-analysis assessing the accuracy of rapid direct-to-PCR in the detection of SARS-CoV-2. In total, 10,957 unique records were identified and screened using a search string evaluation, 420 full-text reports and/or supplemental materials were assessed for inclusion. This resulted in 14 studies reporting 20 datasets with 4593 patient samples (1391 positive) included in the analysis. The overall agreement between the rapid direct RT-PCR and gold standard centralised laboratory RT-PCR was 97.10% with 93.60% positive percent agreement and 98.63% negative percent agreement. The Cohen’s kappa statistical coefficient k = 0.93, indicating an almost perfect agreement and Youden Index = 0.92. These results show that direct-to-PCR assay perform equivalently to the gold standard centralised laboratory RT-PCR systems for the detection of SARS-CoV-2.

A prospective diagnostic study to measure the accuracy of detection of SARS-CoV-2 Variants Of Concern (VOC) utilising a novel RT-PCR GENotyping algorithm in an In silico Evaluation (VOC-GENIE)

Abstract: BackgroundSARS-CoV-2 variants of concern (VOCs) have been associated with higher rate of transmission, and evasion of immunisation and antibody therapeutics. Variant sequencing is widely utilized in the UK. However, only 0.5% (~650k) of the 133 million cumulative positive cases worldwide were sequenced (in GISAID) on 08 April 2021 with 97% from Europe and North America and only ~0.25% (~320k) were variant sequences. This may be due to the lack of availability, high cost, infrastructure and expert staff required for sequencing. Public health decisions based on a non-randomised sample of 0.5% of the population may be insufficiently powered, and subject to sampling bias and systematic error. In addition, sequencing is rarely available in situ in a clinically relevant timeframe and thus, is not currently compatible with diagnosis and treatment patient care pathways. Therefore, we investigated an alternative approach using polymerase chain reaction (PCR) genotyping to detect the key single nucleotide polymorphisms (SNPs) associated with increased transmission and immune evasion in SARS-CoV-2 variants. MethodsWe investigated the utility of SARS-CoV-2 SNP detection with a panel of PCR-genotyping assays in a large data set of 640,482 SARS-CoV-2 high quality, full length sequences using a prospective in silico trial design and explored the potential impact of rapid in situ variant testing on the COVID-19 diagnosis and treatment patient pathway. ResultsFive SNPs were selected by screening the published literature for a reported association with increased transmission and / or immune evasion. 344881 sequences contained one or more of the five SNPs. This algorithm of SNPs was found to be able to identify the four variants of concern (VOCs) and sequences containing the E484K and L452R escape mutations. InterpretationThe in silico analysis suggest that the key mutations and variants of SARS-CoV-2 may be reliably detected using a focused algorithm of biologically relevant SNPs. This highlights the potential for rapid in situ PCR genotyping to compliment or replace sequencing or to be utilized instead of sequences in settings where sequencing is not feasible, accessible or affordable. Rapid detection of variants with in situ PCR genotyping may facilitate a more effective COVID-19 diagnosis and treatment patient pathway. FundingThe study was funded by Primer Design (UK), with kind contributions from all academic partners.