Sample pooling for SARS-CoV-2 RT-PCR screening de Salazar, Adolfo; Aguilera, Antonio; Trastoy, Rocio ...
Clinical microbiology and infection,
12/2020, Letnik:
26, Številka:
12
Journal Article
Recenzirano
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To evaluate the efficacy of sample pooling compared to the individual analysis for the diagnosis of coronavirus disease 2019 (COVID-19) by using different commercial platforms for nucleic acid ...extraction and amplification.
A total of 3519 nasopharyngeal samples received at nine Spanish clinical microbiology laboratories were processed individually and in pools (342 pools of ten samples and 11 pools of nine samples) according to the existing methodology in place at each centre.
We found that 253 pools (2519 samples) were negative and 99 pools (990 samples) were positive; with 241 positive samples (6.85%), our pooling strategy would have saved 2167 PCR tests. For 29 pools (made out of 290 samples), we found discordant results when compared to their correspondent individual samples, as follows: in 22 of 29 pools (28 samples), minor discordances were found; for seven pools (7 samples), we found major discordances. Sensitivity, specificity and positive and negative predictive values for pooling were 97.10% (95% confidence interval (CI), 94.11–98.82), 100%, 100% and 99.79% (95% CI, 99.56–99.90) respectively; accuracy was 99.80% (95% CI, 99.59–99.92), and the kappa concordant coefficient was 0.984. The dilution of samples in our pooling strategy resulted in a median loss of 2.87 (95% CI, 2.46–3.28) cycle threshold (Ct) for E gene, 3.36 (95% CI, 2.89–3.85) Ct for the RdRP gene and 2.99 (95% CI, 2.56–3.43) Ct for the N gene.
We found a high efficiency of pooling strategies for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA testing across different RNA extraction and amplification platforms, with excellent performance in terms of sensitivity, specificity and positive and negative predictive values.
Recombination is a pervasive process generating diversity in most viruses. It joins variants that arise independently within the same molecule, creating new opportunities for viruses to overcome ...selective pressures and to adapt to new environments and hosts. Consequently, the analysis of viral recombination attracts the interest of clinicians, epidemiologists, molecular biologists and evolutionary biologists. In this review we present an overview of three major areas related to viral recombination: (i) the molecular mechanisms that underlie recombination in model viruses, including DNA-viruses (Herpesvirus) and RNA-viruses (Human Influenza Virus and Human Immunodeficiency Virus), (ii) the analytical procedures to detect recombination in viral sequences and to determine the recombination breakpoints, along with the conceptual and methodological tools currently used and a brief overview of the impact of new sequencing technologies on the detection of recombination, and (iii) the major areas in the evolutionary analysis of viral populations on which recombination has an impact. These include the evaluation of selective pressures acting on viral populations, the application of evolutionary reconstructions in the characterization of centralized genes for vaccine design, and the evaluation of linkage disequilibrium and population structure.
CTX-M Enzymes: Origin and Diffusion Cantón, Rafael; González-Alba, José María; Galán, Juan Carlos
Frontiers in microbiology,
01/2012, Letnik:
3
Journal Article
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CTX-M β-lactamases are considered a paradigm in the evolution of a resistance mechanism. Incorporation of different chromosomal bla(CTX-M) related genes from different species of Kluyvera has derived ...in different CTX-M clusters. In silico analyses have shown that this event has occurred at least nine times; in CTX-M-1 cluster (3), CTX-M-2 and CTX-M-9 clusters (2 each), and CTX-M-8 and CTX-M-25 clusters (1 each). This has been mainly produced by the participation of genetic mobilization units such as insertion sequences (ISEcp1 or ISCR1) and the later incorporation in hierarchical structures associated with multifaceted genetic structures including complex class 1 integrons and transposons. The capture of these bla(CTX-M) genes from the environment by highly mobilizable structures could have been a random event. Moreover, after incorporation within these structures, β-lactam selective force such as that exerted by cefotaxime and ceftazidime has fueled mutational events underscoring diversification of different clusters. Nevertheless, more variants of CTX-M enzymes, including those not inhibited by β-lactamase inhibitors such as clavulanic acid (IR-CTX-M variants), only obtained under in in vitro experiments, are still waiting to emerge in the clinical setting. Penetration and the later global spread of CTX-M producing organisms have been produced with the participation of the so-called "epidemic resistance plasmids" often carried in multi-drug resistant and virulent high-risk clones. All these facts but also the incorporation and co-selection of emerging resistance determinants within CTX-M producing bacteria, such as those encoding carbapenemases, depict the currently complex pandemic scenario of multi-drug resistant isolates.
Niches are spaces for the biological units of selection, from cells to complex communities. In a broad sense, "species" are biological units of individuation. Niches do not exist without individual ...organisms, and every organism has a niche. We use "niche" in the Hutchinsonian sense as an abstraction of a multidimensional environmental space characterized by a variety of conditions, both biotic and abiotic, whose quantitative ranges determine the positive or negative growth rates of the microbial individual, typically a species, but also parts of the communities of species contained in this space. Microbial organisms ("species") constantly diversify, and such diversification (radiation) depends on the possibility of opening up unexploited or insufficiently exploited niches. Niche exploitation frequently implies "niche construction," as the colonized niche evolves with time, giving rise to new potential subniches, thereby influencing the selection of a series of new variants in the progeny. The evolution of niches and organisms is the result of reciprocal interacting processes that form a single unified process. Centrifugal microbial diversification expands the limits of the species' niches while a centripetal or cohesive process occurs simultaneously, mediated by horizontal gene transfers and recombinatorial events, condensing all of the information recovered during the diversifying specialization into "novel organisms" (possible future species), thereby creating a more complex niche, where the selfishness of the new organism(s) establishes a "homeostatic power" limiting the niche's variation. Once the niche's full carrying capacity has been reached, reproductive isolation occurs, as no foreign organisms can outcompete the established population/community, thereby facilitating speciation. In the case of individualization-speciation of the microbiota, its contribution to the animal' gut structure is a type of "niche construction," the result of crosstalk between the niche (host) and microorganism(s). Lastly, there is a parallelism between the hierarchy of niches and that of microbial individuals. The increasing anthropogenic effects on the biosphere (such as globalization) might reduce the diversity of niches and bacterial individuals, with the potential emergence of highly transmissible multispecialists (which are eventually deleterious) resulting from the homogenization of the microbiosphere, a possibility that should be explored and prevented.
Recent research on Parkinson disease (PD) detection has shown that vocal disorders are linked to symptoms in 90% of the PD patients at early stages. Thus, there is an interest in applying vocal ...features to the computer-assisted diagnosis and remote monitoring of patients with PD at early stages. The contribution of this research is an increase of accuracy and a reduction of the number of selected vocal features in PD detection while using the newest and largest public dataset available. Whereas the number of features in this public dataset is 754, the number of selected features for classification ranges from 8 to 20 after using Wrappers feature subset selection. Four classifiers (k nearest neighbor, multi-layer perceptron, support vector machine and random forest) are applied to vocal-based PD detection. The proposed approach shows an accuracy of 94.7%, sensitivity of 98.4%, specificity of 92.68% and precision of 97.22%. The best resulting accuracy is obtained by using a support vector machine and it is higher than the one, which was reported on the first work to use the same dataset. In addition, the corresponding computational complexity is further reduced by selecting no more than 20 features.
Triple negative breast cancer (TNBC) is an incurable disease where novel therapeutic strategies are needed. Proteolysis targeting chimeric (PROTAC) are novel compounds that promote protein ...degradation by binding to an ubiquitin ligase. In this work, we explored the antitumoral activity of two novel BET-PROTACs, MZ1 and ARV-825, in TNBC, ovarian cancer and in a BET inhibitor resistant model.
OVCAR3, SKOV3, BT549, MDA-MB-231 cell lines and the JQ1 resistant cell line MDA-MB-231R were evaluated. MTTs, colony-forming assay, three-dimensional cultures in matrigel, flow cytometry, and western blots were performed to explore the anti-proliferative effect and biochemical mechanism of action of MZ1 and ARV-825. In vivo studies included BALB/c nu/nu mice engrafted with MDA-MB-231R cells.
The BET-PROTACs MZ1 and ARV-825 efficiently downregulated the protein expression levels of the BET protein BRD4, in MDA-MB-231 and MDA-MB-231R. MZ1 and ARV-825 also showed an antiproliferative effect on sensitive and resistant cells. This effect was corroborated in other triple negative (BT549) and ovarian cancer (SKOV3, OVCAR3) cell lines. MZ1 provoked G2/M arrest in MDA-MB-231. In addition, a profound effect on caspase-dependent apoptosis was observed in both sensitive and resistant cells. No synergistic activity was observed when it was combined with docetaxel, cisplatin or olaparib. Finally, in vivo administration of MZ1 rescued tumor growth in a JQ1-resistant xenograft model, reducing the expression levels of BRD4.
Using both in vitro and in vivo approaches, we describe the profound activity of BET-PROTACs in parental and BETi-resistant TNBC models. This data provides options for further clinical development of these agents in TNBC.
We aimed to evaluate the correct assignment of HCV genotypes by three commercial methods-Trugene HCV genotyping kit (Siemens), VERSANT HCV Genotype 2.0 assay (Siemens), and Real-Time HCV genotype II ...(Abbott)-compared to NS5B sequencing. We studied 327 clinical samples that carried representative HCV genotypes of the most frequent geno/subtypes in Spain. After commercial genotyping, the sequencing of a 367 bp fragment in the NS5B gene was used to assign genotypes. Major discrepancies were defined, e.g. differences in the assigned genotype by one of the three methods and NS5B sequencing, including misclassification of subtypes 1a and 1b. Minor discrepancies were considered when differences at subtype levels, other than 1a and 1b, were observed. The overall discordance with the reference method was 34% for Trugene and 15% for VERSANT HCV2.0. The Abbott assay correctly identified all 1a and 1b subtypes, but did not subtype all the 2, 3, 4 and 5 (34%) genotypes. Major discordances were found in 16% of cases for Trugene HCV, and the majority were 1b- to 1a-related discordances; major discordances were found for VERSANT HCV 2.0 in 6% of cases, which were all but one 1b to 1a cases. These results indicated that the Trugene assay especially, and to a lesser extent, Versant HCV 2.0, can fail to differentiate HCV subtypes 1a and 1b, and lead to critical errors in clinical practice for correctly using directly acting antiviral agents.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Phylogenetic analyses of the bacterial genomes based on the simple classification in core- genes and accessory genes pools could offer an incomplete view of the evolutionary processes, of which some ...are still unresolved. A combined strategy based on stratified phylogeny and ancient molecular polymorphisms is proposed to infer detailed evolutionary reconstructions by using a large number of whole genomes. This strategy, based on the highest number of genomes available in public databases, was evaluated for improving knowledge of the ancient diversification of E. coli. This staggered evolutionary scenario was also used to investigate whether the diversification of the ancient E. coli lineages could be associated with particular lifestyles and adaptive strategies.
Phylogenetic reconstructions, exploiting 6220 available genomes in Genbank, established the E. coli core genome in 1023 genes, representing about 20% of the complete genome. The combined strategy using stratified phylogeny plus molecular polymorphisms inferred three ancient lineages (D, EB1A and FGB2). Lineage D was the closest to E. coli root. A staggered diversification could also be proposed in EB1A and FGB2 lineages and the phylogroups into these lineages. Several molecular markers suggest that each lineage had different adaptive trajectories. The analysis of gained and lost genes in the main lineages showed that functions of carbohydrates utilization (uptake of and metabolism) were gained principally in EB1A lineage, whereas loss of environmental-adaptive functions in FGB2 lineage were observed, but this lineage showed higher accumulated mutations and ancient recombination events. The population structure of E. coli was re-evaluated including up to 7561 new sequenced genomes, showing a more complex population structure of E. coli, as a new phylogroup, phylogroup I, was proposed.
A staggered reconstruction of E. coli phylogeny is proposed, indicating evolution from three ancestral lineages to reach all main known phylogroups. New phylogroups were confirmed, suggesting an increasingly complex population structure of E. coli. However these new phylogroups represent < 1% of the global E. coli population. A few key evolutionary forces have driven the diversification of the two main E. coli lineages, metabolic flexibility in one of them and colonization-virulence in the other.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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