A new TaqMan real-time PCR assay to detect Parachlamydia acanthamoebae and to monitor its co-existence with SARS-COV-2 among COVID-19 patients.

Human respiratory infections caused by a large variety of microbial pathogens are the most common diseases responsible for hospitalization, morbidity and mortality. Parachlamydia acanthamoebae, a Chlamydia-related bacterium, has been found to be potentially associated with these diseases. An early and accurate diagnosis of this pathogen could be useful to avoid the potential respiratory complications linked especially to COVID-19 patients and to set suitable outbreak control measures. A TaqMan-PCR assay was developed to detect and quantify Parachlamydia acanthamoebae in environmental and clinical samples from patients of all ages with COVID-19. The selected hydrolysis probe displayed no cross-reaction with the closely related Chlamydia or the other tested pathogens. This q-PCR achieved good reproducibility and repeatability with a detection limit of about 5 DNA copies per reaction. Using this q-PCR assay, Parachlamydia acanthamoebae was detected in 2/78 respiratory specimens and 9/47 water samples. Only one case (1.3%) of Parachlamydia acanthamoebae and SARS-COV-2 co-infection was noticed. To our knowledge, the combination of these two respiratory pathogens has not been described yet. This new TaqMan-PCR assay represents an efficient diagnostic tool to survey Parachlamydia acanthamoebae on a large-scale screening programs and also during outbreaks.

Development of a duplex q-PCR for the simultaneous detection of Parachlamydia acanthamoebae and Simkania negevensis in environmental and clinical samples.

Parachlamydia acanthamoebae and Simkania negevensis, two Chlamydia-like bacteria, have been recently recognized as emerging human respiratory pathogens. The prevalence and frequency of these bacteria in the environment and among atypical pneumonia patients are still underestimated by classical cultures, immunohistochemistry and serology which are non-specific, long and tedious methods. This study aims to develop a new duplex probe-based q-PCR assay for the simultaneous detection and quantification of P. acanthamoebae and S. negevensis. The selected hydrolysis probes displayed no cross-reaction with the closely related Chlamydia or the other tested waterborne pathogens. The assay achieved a large dynamic range for quantification (from 5 × 106 to 5 DNA copies/reaction). Efficiencies of FAM and JOE label probes weren't affected when they were combined. They were close to 100%, indicating the linear amplification. The application of this diagnostic tool resulted in 9/47 (19%) and 4/47 (8.5%) positive water samples for P. acanthamoebae and S. negevensis, respectively. P. acanthamoebae was also covered from 2/78 (2.5%) respiratory specimens and only one case (1/200 = 0.5%) of P. acanthamoebae and SARS-CoV-2 co-infection was noticed. While S. negevensis wasn't detected in clinical samples, the developed duplex q-PCR was shown to be an accurate, highly sensitive, and robust diagnostic tool for the detection and quantification of P. acanthamoebae and S. negevensis.

In silico evidence of antiviral activity against SARS-CoV-2 main protease of oligosaccharides from Porphyridium sp.

The coronavirus pandemic (COVID-19) has created an urgent need to develop effective strategies for prevention and treatment. In this context, therapies against protease Mpro, a conserved viral target, would be essential to contain the spread of the virus and reduce mortality. Using combined techniques of structure modelling, in silico docking and pharmacokinetics prediction, many compounds from algae were tested for their ability to inhibit the SARS-CoV-2 main protease and compared to the recent recognized drug Paxlovid. The screening of 27 algal molecules including 15 oligosaccharides derived from sulfated and non-sulphated polysaccharides, eight pigments and four poly unsaturated fatty acids showed high affinities to interact with the protein active site. Best candidates showing high docking scores in comparison with the reference molecule were sulfated tri-, tetra- and penta-saccharides from Porphyridium sp. exopolysaccharides (SEP). Structural and energetic analyses over 100 ns MD simulation demonstrated high SEP fragments-Mpro complex stability. Pharmacokinetics predictions revealed the prospects of the identified molecules as potential drug candidates.

Apigenin analogues as SARS-CoV-2 main protease inhibitors: In-silico screening approach.

The COVID-19 new variants spread rapidly all over the world, and until now scientists strive to find virus-specific antivirals for its treatment. The main protease of SARS-CoV-2 (Mpro) exhibits high structural and sequence homology to main protease of SARS-CoV (93.23% sequence identity), and their sequence alignment indicated 12 mutated/variant residues. The sequence alignment of SARS-CoV-2 main protease led to identification of only one mutated/variant residue with no significant role in its enzymatic process. Therefore, Mpro was considered as a high-profile drug target in anti-SARS-CoV-2 drug discovery. Apigenin analogues to COVID-19 main protease binding were evaluated. The detailed interactions between the analogues of Apigenin and SARS-CoV-2 Mpro inhibitors were determined as hydrogen bonds, electronic bonds and hydrophobic interactions. The binding energies obtained from the molecular docking of Mpro with Boceprevir, Apigenin, Apigenin 7-glucoside-4'-p-coumarate, Apigenin 7-glucoside-4'-trans-caffeate and Apigenin 7-O-beta-d-glucoside (Cosmosiin) were found to be -6.6, -7.2, -8.8, -8.7 and -8.0 kcal/mol, respectively. Pharmacokinetic parameters and toxicological characteristics obtained by computational techniques and Virtual ADME studies of the Apigenin analogues confirmed that the Apigenin 7-glucoside-4'-p-coumarate is the best candidate for SARS-CoV-2 Mpro inhibition.