An end-point multiplex RT-PCR for SARS-CoV-2, Influenza A and B detection, including simultaneous RNAse P amplification: a timely tool for more accessible differential diagnosis.

The multiplex molecular diagnostic assays described for severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), influenza A (IAV) and B (IBV) viruses have been mainly based on real-time reaction, which limits their access to many laboratories or diagnostic institutions. To contribute to available strategies and expand access to differential diagnosis, we describe an end-point multiplex RT-PCR targeting SARS-CoV-2, IAV and IBV with simultaneous endogenous control amplification. Initially, we looked for well-established primers sets for SARS-CoV-2, IAV, IBV and RNAse P whose amplicons could be distinguished on agarose gel. The multiplex assay was then standardized by optimizing the reaction mix and cycle conditions. The limit of detection (LoD) was determined using titrated viruses (for SARS-CoV-2 and IAV) and by dilution from a pool of IBV-positive samples. The diagnostic performance of the multiplex was evaluated by testing samples with different RNAse P and viral loads, previously identified as positive or negative for the target viruses. The amplicons of IAV (146 bp), SARS-CoV-2 (113 bp), IBV (103 bp) and RNAse P (65 bp) were adequately distinguished in our multiplex. The LoD for SARS-CoV-2, IAV and IBV was 0.02 TCID50/ml, 0.07 TCID50/ml and 10-3 from a pool of positive samples, respectively. All samples positive for SARS-CoV-2 (n=70, Ct 17.2-36.9), IAV (n=53, Ct 14-34.9) and IBV (n=12, Ct 23.9-31.9) remained positive in our multiplex assay. RNAse P from negative samples (n=40, Ct 25.2-30.2) was also amplified in the multiplex. Overall, our assay is a timely and alternative tool for detecting SARS-CoV-2 and influenza viruses in laboratories with limited access to supplies/equipment.

Tracking SARS-CoV-2 in rivers as a tool for epidemiological surveillance.

The aim of this work was to evaluate if rivers could be used for SARS-CoV-2 surveillance. Five sampling points from three rivers (AR-1 and AR-2 in Arenales River, MR-1 and MR-2 in Mojotoro River, and CR in La Caldera River) from Salta (Argentina), two of them receiving discharges from wastewater plants (WWTP), were monitored from July to December 2020. Fifteen water samples from each point (75 in total) were collected and characterized physico-chemically and microbiologically and SARS-CoV-2 was quantified by RT-qPCR. Also, two targets linked to human contributions, human polyomavirus (HPyV) and RNase P, were quantified and used to normalize SARS-CoV-2 concentration, which was compared to reported COVID-19 cases. Statistical analyses allowed us to verify the correlation between SARS-CoV-2 and the concentration of fecal indicator bacteria (FIB), as well as to find similarities and differences between sampling points. La Caldera River showed the best water quality; FIBs were within acceptable limits for recreational activities. Mojotoro River's water quality was not affected by the northern WWTP of the city. Instead, Arenales River presented the poorest water quality; at AR-2 was negatively affected by the discharges of the southern WWTP, which contributed to significant increase of fecal contamination. SARS-CoV-2 was found in about half of samples in low concentrations in La Caldera and Mojotoro Rivers, while it was high and persistent in Arenales River. No human tracers were detected in CR, only HPyV was found in MR-1, MR-2 and AR-1, and both were quantified in AR-2. The experimental and normalized viral concentrations strongly correlated with reported COVID-19 cases; thus, Arenales River at AR-2 reflected the epidemiological situation of the city. This is the first study showing the dynamic of SARS-CoV-2 concentration in an urban river highly impacted by wastewater and proved that can be used for SARS-CoV-2 surveillance to support health authorities.

Validation of a duplex PCR technique using the gen E and RNase P for the diagnosis of SARS-CoV-2.

INTRODUCTION: Reverse transcriptase - polymerase chain reaction (RT-PCR) is the standard technique for SARS-CoV-2 diagnosis. The World Health Organization recommends the Charité-Berlin protocol for COVID-19 diagnosis, which requires triple PCR, limiting the process capability of laboratories and delaying the results. In order to reduce these limitations, a duplex PCR is validated for the detection of the E and ribonuclease P genes. METHODS: We compared the limit of detection, sensitivity and specificity of the duplex PCR technique (E gene and Rnasa P) against the monoplex standard (E gene) in RNA samples from a SARS-CoV-2 isolate and 88 clinical specimens with previously known results. The repeatability and reproducibility of the threshold cycle values ​​(Ct) were determined in two independent laboratories of the Faculty of Medicine of the Universidad de Antioquia, using different reagents and real time instruments. RESULTS: There were no significant differences in the Ct results between both techniques (P = .84). Using the monoplex PCR of E gene as a reference, the interrater reliability analysis showed similarity between the two techniques, with a kappa coefficient of 0.89, the sensitivity and the specificity of duplex PCR were 90% and 87%, respectively. CONCLUSIONS: Duplex PCR does not affect the sensitivity and specificity reported by the Charité, Berlin protocol, being a useful tool for SARS-CoV-2 screening in clinical samples.

Molecular investigation of haemotropic mycoplasmas and Coxiella burnetii in free-living Xenarthra mammals from Brazil, with evidence of new haemoplasma species.

Although mammals of the superorder Xenarthra are considered hosts of a wide range of zoonotic agents, works aiming at investigating the role of these animals as hosts for bacteria with zoonotic potential are rare. The present study aimed to investigate the occurrence and molecularly characterize Coxiella burnetii and haemoplasma (haemotropic mycoplasmas) DNA in blood and spleen samples from 397 free-living Xenarthra mammals (233 sloths, 107 anteaters and 57 armadillos) in five Brazilian states (Mato Grosso do Sul, São Paulo, Pará, Rondônia and Rio Grande do Sul). All biological samples from Xenarthra were negative in the qPCR for Coxiella burnetii based on the IS1111 gene. The absence of C. burnetii DNA in blood and spleen samples from Xenarthra suggests that these mammals may not act as possible hosts for this agent in the locations studied. When performed conventional PCR assays for the endogenous (gapdh) mammalian gene, 386 samples were positive. When screened by molecular assays based on the 16S rRNA gene of haemoplasmas, 81 samples were positive, of which 15.54% (60/386) were positive by conventional PCR and 5.44% (21/386) were positive by real-time PCR; three samples were positive in both assays. Of these, 39.74% (31/78) were also positive for the 23S rRNA gene and 7.69% (6/78) for the haemoplasma RNAse P gene. Among the samples positive for haemoplasmas, 25.64% (20/78) were obtained from anteaters (Tamandua tetradactyla and Myrmecophaga tridactyla), 39.74% (31/78) from sloths (Bradypus tridactylus, Bradypus sp. and Choloepus sp.) 34.61% (27/78) from armadillos (Priodontes maximus, Euphractus sexcinctus and Dasypus novemcinctus). A haemoplasma 16S rRNA sequence closely related and showing high identity (99.7%) to Mycoplasma wenyonii was detected, for the first time, in B. tridactylus. Based on the low identity and phylogenetic positioning of 16S rRNA and 23S rRNA sequences of haemoplasmas detected in anteaters and armadillos, the present study showed, for the first time, the occurrence of putative new Candidatus haemotropic Mycoplasma spp. ("Candidatus Mycoplasma haematotetradactyla" and "Candidatus Mycoplasma haematomaximus") in Xenarthra mammals from Brazil.

High sensitivity-low cost detection of SARS-CoV-2 by two steps end point RT-PCR with agarose gel electrophoresis visualization.

More than one year since Coronavirus disease 2019 (COVID-19) pandemic outbreak, the gold standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection is still the RT-qPCR. This is a limitation to increase testing capacities, particularly at developing countries, as expensive reagents and equipment are required. We developed a two steps end point RT-PCR reaction with SARS-CoV-2 Nucleocapsid (N) gene and Ribonuclease P (RNase P) specific primers where viral amplicons were verified by agarose gel electrophoresis. We carried out a clinical performance and analytical sensitivity evaluation for this two-steps end point RT-PCR method with 242 nasopharyngeal samples using the CDC RT-qPCR protocol as a gold standard technique. With a specificity of 95.8%, a sensitivity of 95.1%, and a limit of detection of 20 viral RNA copies/uL, this two steps end point RT-PCR assay is an affordable and reliable method for SARS-CoV-2 detection. This protocol would allow to extend COVID-19 diagnosis to basic molecular biology laboratories with a potential positive impact in surveillance programs at developing countries.

Quantitative detection of SARS-CoV-2 RNA in nasopharyngeal samples from infected patients with mild disease.

Diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) cases is based on the count of real-time reverse transcription-plymerase chain reaction (RT-PCR) positive people. Viral load by real-time RT-PCR has been suggested as a biomarker of the SARS-CoV-2 infection. However, the association of viral load and severity of the disease is not yet resolved. Nasopharyngeal samples from 458 patients were tested by RT-PCR for SARS-CoV-2 diagnosis. Relative quantitation was made by the comparative threshold cycle (ΔΔCt ) formula between ORF1ab viral and RNase P housekeeping genes. Absolute viral load was calculate using a reference positive control. Most prevalent clinical signs were cough (75.8%), myalgia (66.7%), and fever (48.5%). Hypertension (18.2%), neurological diseases (15.1%), and asthma and hypothyroidism (12.1%) were most frequent comorbidities. Fever, either as an exclusive symptom or combined with others, was associated with high viral loads ( 2-∆∆Ct range, 35.65-155.16; 4.25-4.89 log10 RNA copies/test]). During the first week after onset of symptoms in mild patients up to 60 years-old was detected the peak of viral load. Children under 10 years old have a high viral load (313.84; 2.50) in the first 2 days postinfection with a sharp decline thereafter. Cases between 10 and 49 years old mostly showed low and moderate viral load during the first 2 days postinfection (range, 0.03 to 17.24; -1.50 to 1.24). Patients over 60 years old have high viral load up to the second week after the onset of symptoms (range, 25.32-155.42; 1.40-2.19), indicating the longer presence of the virus in them. These findings suggest the viral load in nasopharyngeal swabs would help to monitor the SARS-CoV-2 infection in mild coronavirus disease 2019 cases.

A newborn screening pilot study using methylation-sensitive high resolution melting on dried blood spots to detect Prader-Willi and Angelman syndromes.

Prader-Willi (PWS) and Angelman (AS) syndromes are two clinically distinct imprinted disorders characterized by genetic abnormalities at 15q11-q13. Early diagnosis of both syndromes provides improved treatment and accurate genetic counseling. Whole blood (WB) is the most common DNA source of many methodologies to detect PWS and AS, however, the need of WB makes a massive screening difficult in newborns due to economic and technical limitations. The aim of this study was to adapt a Methylation-sensitive High-Resolution Melting (MS-HRM) approach from dried blood spot (DBS) samples, assessing the different DNA isolation techniques and diagnostic performance. Over a 1-year period, we collected 125 DBS cards, of which 45 had already been diagnosed by MS-HRM (20 PWS, 1 AS, and 24 healthy individuals). We tested three different DBS-DNA extraction techniques assessing the DNA concentration and quality, followed by MS-HRM and statistical comparison. Each DBS-DNA extraction method was capable of accuracy in detecting all PWS and AS individuals. However, the efficiency to detect healthy individuals varied according to methodology. In our experience, DNA extracted from DBS analyzed by the MS-HRM methodology provides an accurate approach for genetic screening of imprinting related disorders in newborns, offering several benefits compared to traditional whole blood methods.

A screening platform to monitor RNA processing and protein-RNA interactions in ribonuclease P uncovers a small molecule inhibitor.

Ribonucleoprotein (RNP) complexes and RNA-processing enzymes are attractive targets for antibiotic development owing to their central roles in microbial physiology. For many of these complexes, comprehensive strategies to identify inhibitors are either lacking or suffer from substantial technical limitations. Here, we describe an activity-binding-structure platform for bacterial ribonuclease P (RNase P), an essential RNP ribozyme involved in 5' tRNA processing. A novel, real-time fluorescence-based assay was used to monitor RNase P activity and rapidly identify inhibitors using a mini-helix and a pre-tRNA-like bipartite substrate. Using the mini-helix substrate, we screened a library comprising 2560 compounds. Initial hits were then validated using pre-tRNA and the pre-tRNA-like substrate, which ultimately verified four compounds as inhibitors. Biolayer interferometry-based binding assays and molecular dynamics simulations were then used to characterize the interactions between each validated inhibitor and the P protein, P RNA and pre-tRNA. X-ray crystallographic studies subsequently elucidated the structure of the P protein bound to the most promising hit, purpurin, and revealed how this inhibitor adversely affects tRNA 5' leader binding. This integrated platform affords improved structure-function studies of RNA processing enzymes and facilitates the discovery of novel regulators or inhibitors.

External guide sequence technology: a path to development of novel antimicrobial therapeutics.

RNase P is a ribozyme originally identified for its role in maturation of tRNAs by cleavage of precursor tRNAs (pre-tRNAs) at the 5'-end termini. RNase P is a ribonucleoprotein consisting of a catalytic RNA molecule and, depending on the organism, one or more cofactor proteins. The site of cleavage of a pre-tRNA is identified by its tertiary structure; and any RNA molecule can be cleaved by RNase P as long as the RNA forms a duplex that resembles the regional structure in the pre-tRNA. When the antisense sequence that forms the duplex with the strand that is subsequently cleaved by RNase P is in a separate molecule, it is called an external guide sequence (EGS). These fundamental observations are the basis for EGS technology, which consists of inhibiting gene expression by utilizing an EGS that elicits RNase P-mediated cleavage of a target mRNA molecule. EGS technology has been used to inhibit expression of a wide variety of genes, and may help development of novel treatments of diseases, including multidrug-resistant bacterial and viral infections.

Survey of infectious agents in the endangered Darwin's fox (Lycalopex fulvipes): high prevalence and diversity of hemotrophic mycoplasmas.

Very little is known about the diseases affecting the Darwin's fox (Lycalopex fulvipes), which is considered to be one of the most endangered carnivores worldwide. Blood samples of 30 foxes captured on Chiloé Island (Chile) were tested with a battery of PCR assays targeting the following pathogens: Ehrlichia/Anaplasma sp., Rickettsia sp., Bartonella sp., Coxiella burnetti, Borrelia sp., Mycoplasma sp., Babesia sp., Hepatozoon canis, Hepatozoon felis, Leishmania donovani complex, and Filariae. Analysis of the 16S rRNA gene revealed the presence of Mycoplasma spp. in 17 samples (56.7%, 95% Confidence Intervals= 38.2-73.7). Of these, 15 infections were caused by a Mycoplasma belonging to the M. haemofelis/haemocanis (Mhf/Mhc) group, whereas two were caused by a Mycoplasma showing between 89% and 94% identity with different Candidatus Mycoplasma turicensis from felids and rodents hemoplasmas. The analysis of the sequence of the RNA subunit of the RNase P gene of 10 of the foxes positive for Mhf/Mhc showed that eight were infected with M. haemocanis (Mhc), one with a Mycoplasma showing 94% identity with Mhc, and one by M. haemofelis (Mhf). One of the foxes positive for Mhc was infected with a Ricketssia closely related to R. felis. All foxes were negative for the other studied pathogens. Our results are of interest because of the unexpectedly high prevalence of Mycoplasma spp. detected, the variability of species identified, the presence of a potentially new species of hemoplasma, and the first time a hemoplasma considered to be a feline pathogen (Mhf) has been identified in a canid. Though external symptoms were not observed in any of the infected foxes, further clinical and epidemiological studies are necessary to determine the importance of hemoplasma infection in this unique species.

Inhibition of cell division induced by external guide sequences (EGS Technology) targeting ftsZ.

EGS (external guide sequence) technology is a promising approach to designing new antibiotics. EGSs are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA by forming a precursor tRNA-like complex. The ftsZ mRNA secondary structure was modeled and EGSs complementary to two regions with high probability of being suitable targets were designed. In vitro reactions showed that EGSs targeting these regions bound ftsZ mRNA and elicited RNase P-mediated cleavage of ftsZ mRNA. A recombinant plasmid, pEGSb1, coding for an EGS that targets region "b" under the control of the T7 promoter was generated. Upon introduction of this plasmid into Escherichia coli BL21(DE3)(pLysS) the transformant strain formed filaments when expression of the EGS was induced. Concomitantly, E. coli harboring pEGSb1 showed a modest but significant inhibition of growth when synthesis of the EGSb1 was induced. Our results indicate that EGS technology could be a viable strategy to generate new antimicrobials targeting ftsZ.

Ribonucleases P/MRP and the expanding ribonucleoprotein world.

One of the hallmarks of life is the widespread use of certain essential ribozymes. The ubiquitous ribonuclease P (RNase P) and eukaryotic RNase MRP are essential complexes where a structured, noncoding RNA acts in catalysis. Recent discoveries have elucidated the three-dimensional structure of the ancestral ribonucleoprotein complex, suggested the possibility of a protein-only composition in organelles, and even noted the absence of RNase P in a non-free-living organism. With respect to these last two findings, import mechanisms for RNases P/MRP into mitochondria have been demonstrated, and RNase P is present in organisms with some of the smallest known genomes. Together, these results have led to an ongoing debate regarding the precise definition of how "essential" these ribozymes truly are.

Identification of an hepatitis delta virus-like ribozyme at the mRNA 5'-end of the L1Tc retrotransposon from Trypanosoma cruzi.

L1Tc is a non-LTR LINE element from Trypanosoma cruzi that encodes its transposition machinery and bears an internal promoter. Herewith, we report the identification of an in vitro active hepatitis delta virus-like ribozyme located in the first 77 nt at the 5'-end of the L1Tc mRNA (L1TcRz). The data presented show that L1TcRz has a co-transcriptional function. Using gel-purified uncleaved RNA transcripts, the data presented indicate that the kinetics of the self-cleaving, in a magnesium-dependent reaction, fits to a two-phase decay curve. The cleavage point identified by primer extension takes place at +1 position of the element. The hydroxyl nature of the 5'-end of the 3'-fragment generated by the cleavage activity of L1TcRz was confirmed. Since we have previously described that the 77-nt long fragment located at the 5'-end of L1Tc has promoter activity, the existence of a ribozyme in L1Tc makes this element to be the first described non-LTR retroelement that has an internal promoter-ribozyme dual function. The L1Tc nucleotides located downstream of the ribozyme catalytic motif appear to inhibit its activity. This inhibition may be influenced by the existence of a specific L1Tc RNA conformation that is recognized by RNase P.

Inhibition of aac(6')-Ib-mediated amikacin resistance by nuclease-resistant external guide sequences in bacteria.

Inhibition of bacterial gene expression by RNase P-directed cleavage is a promising strategy for the development of antibiotics and pharmacological agents that prevent expression of antibiotic resistance. The rise in multiresistant bacteria harboring AAC(6')-Ib has seriously limited the effectiveness of amikacin and other aminoglycosides. We have recently shown that recombinant plasmids coding for external guide sequences (EGS), short antisense oligoribonucleotides (ORN) that elicit RNase P-mediated cleavage of a target mRNA, induce inhibition of expression of aac(6')-Ib and concomitantly induce a significant decrease in the levels of resistance to amikacin. However, since ORN are rapidly degraded by nucleases, development of a viable RNase P-based antisense technology requires the design of nuclease-resistant RNA analog EGSs. We have assayed a variety of ORN analogs of which selected LNA/DNA co-oligomers elicited RNase P-mediated cleavage of mRNA in vitro. Although we found an ideal configuration of LNA/DNA residues, there seems not to be a correlation between number of LNA substitutions and level of activity. Exogenous administration of as low as 50 nM of an LNA/DNA co-oligomer to the hyperpermeable E. coli AS19 harboring the aac(6')-Ib inhibited growth in the presence of amikacin. Our experiments strongly suggest an RNase P-mediated mechanism in the observed antisense effect.

Identification of severe combined immunodeficiency by T-cell receptor excision circles quantification using neonatal guthrie cards.

OBJECTIVE: To assess the feasibility of T-cell receptor excision circles (TRECs) quantification for neonatal mass screening of severe combined immunodeficiency (SCID). STUDY DESIGN: Real-time PCR based quantification of TRECs for 471 healthy control patients and 18 patients with SCID with various genetic abnormalities (IL2RG, JAK3, ADA, LIG4, RAG1) were performed, including patients with maternal T-cell engraftment (n = 4) and leaky T cells (n = 3). RESULTS: TRECs were detectable in all normal neonatal Guthrie cards (n = 326) at the levels of 10(4) to 10(5) copies/microg DNA. In contrast, TRECs were extremely low in all neonatal Guthrie cards (n = 15) and peripheral blood (n = 14) from patients with SCID, including those with maternal T-cell engraftment or leaky T cells with hypomorphic RAG1 mutations or LIG4 deficiency. There were no false-positive or negative results in this study. CONCLUSION: TRECs quantification can be used as a neonatal mass screening for patients with SCID.