Diagnosis of SARS-CoV-2 Infection with LamPORE, a High-Throughput Platform Combining Loop-Mediated Isothermal Amplification and Nanopore Sequencing.

LamPORE is a novel diagnostic platform for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA combining loop-mediated isothermal amplification with nanopore sequencing, which could potentially be used to analyze thousands of samples per day on a single instrument. We evaluated the performance of LamPORE against reverse transcriptase PCR (RT-PCR) using RNA extracted from spiked respiratory samples and stored nose and throat swabs collected at two UK hospitals. The limit of detection of LamPORE was 10 genome copies/µl of extracted RNA, which is above the limit achievable by RT-PCR, but was not associated with a significant reduction of sensitivity in clinical samples. Positive clinical specimens came mostly from patients with acute symptomatic infection, and among them, LamPORE had a diagnostic sensitivity of 99.1% (226/228; 95% confidence interval [CI], 96.9% to 99.9%). Among negative clinical specimens, including 153 with other respiratory pathogens detected, LamPORE had a diagnostic specificity of 99.6% (278/279; 98.0% to 100.0%). Overall, 1.4% (7/514; 0.5% to 2.9%) of samples produced an indeterminate result on first testing, and repeat LamPORE testing on the same RNA extract had a reproducibility of 96.8% (478/494; 94.8% to 98.1%). LamPORE has a similar performance as RT-PCR for the diagnosis of SARS-CoV-2 infection in symptomatic patients and offers a promising approach to high-throughput testing.

Transcriptomic analysis of the human placenta reveals trophoblast dysfunction and augmented Wnt signalling associated with spontaneous preterm birth.

Preterm birth (PTB) is the leading cause of death in under-five children. Worldwide, annually, over 15 million babies are born preterm and 1 million of them die. The triggers and mechanisms of spontaneous PTB remain largely unknown. Most current therapies are ineffective and there is a paucity of reliable predictive biomarkers. Understanding the molecular mechanisms of spontaneous PTB is crucial for developing better diagnostics and therapeutics. To address this need, we conducted RNA-seq transcriptomic analysis, qRT-PCR and ELISA on fresh placental villous tissue from 20 spontaneous preterm and 20 spontaneous term deliveries, to identify genes and signalling pathways involved in the pathogenesis of PTB. Our differential gene expression, gene ontology and pathway analysis revealed several dysregulated genes (including OCLN, OPTN, KRT7, WNT7A, RSPO4, BAMBI, NFATC4, SLC6A13, SLC6A17, SLC26A8 and KLF8) associated with altered trophoblast functions. We identified dysregulated Wnt, oxytocin and cellular senescence signalling pathways in preterm placentas, where augmented Wnt signalling could play a pivotal role in the pathogenesis of PTB due to its diverse biological functions. We also reported two novel targets (ITPR2 and MYLK2) in the oxytocin signalling pathways for further study. Through bioinformatics analysis on DEGs, we identified four key miRNAs, - miR-524-5p, miR-520d-5p, miR-15a-5p and miR-424-5p - which were significantly downregulated in preterm placentas. These miRNAs may have regulatory roles in the aberrant gene expressions that we have observed in preterm placentas. We provide fresh molecular insight into the pathogenesis of spontaneous PTB which may drive further studies to develop new predictive biomarkers and therapeutics.