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Simple Summary: African swine fever virus is a pathogen capable of spreading among swine populations, which is usually fatal since no vaccines are currently available on the market. Outbreaks caused by this virus are the reason for massive economic losses on pig farms and are a matter of worry for the swine sector worldwide. Effective and reliable detection of the virus is relevant to prevent uncontrolled contagion, especially when no effective preventive measures can be applied. In order to confront the spread of African swine fever, several rapid real-time polymerase chain reaction detection assays were developed by different research groups. In the present study, we focused on the validation of the ASFV MONODOSE dtec-qPCR kit using reference genetic material provided by national and international reference laboratories, and results were compared with those obtained from using internationally accepted reference methods. The data obtained indicated that the kit can be used in veterinary samples. Technical innovations enable a user-friendly and rapid management of samples, reducing the probability of human errors happening and transport costs. African swine fever virus is considered an emerging virus that causes African swine fever, a disease characterised by high mortality and elevated transmission rates and that, as it is for most other viral diseases, cannot be treated with specific drugs. Effective and reliable detection of the virus is relevant to prevent uncontrolled contagion among boar populations and to reduce economic losses. Moreover, animal health laboratories are demanding standardisation, optimisation and quality assurance of the available diagnostic assays. In the present study, the ASFV MONODOSE dtec-qPCR kit was validated following the UNE-EN ISO/IEC 17025:2005 guidelines. Analytical validation terms include in silico and in vitro specificity, sensitivity, efficiency and reliability (repeatability/reproducibility). Diagnostic validation of the method was assessed through the analysis of a total of 181 porcine samples originating from six different matrix types doped with African swine fever virus DNA received from the European reference laboratory for African Swine Fever (INIA-CISA, Madrid, Spain): whole blood, blood serum, kidney, heart, liver and tonsil. Results agreed with those obtained from a reference detection method also based on real-time PCR, endorsed by WOAH, but the ASFV MONODOSE dtec-qPCR kit incorporates some technical innovations and improvements which may benefit end-users. This kit, available worldwide with full analytical and diagnostic validation, can recognise all known ASFV genotypes and brings additional benefits to the current qPCR technology. [ABSTRACT FROM AUTHOR]

Human mpox is caused by the Monkeypox virus, a microorganism closely related to the Variola virus, both belonging to the Orthopoxvirus genus. Mpox had been considered a rare disease until a global outbreak occurred in 2022. People infected with the virus present similar symptoms to patients suffering smallpox and other rash illnesses, hindering diagnosis. The WHO indicated that no commercial PCR or serology kits are currently widely available. In the present study, the MPXV MONODOSE dtec-qPCR kit was validated following guidelines of the UNE/EN ISO/IEC 17025:2005. The parameters evaluated for the acceptance of the assay were in silico and in vitro specificity, quantitative phase analysis, reliability, and sensitivity. The assay passed validation criteria and yielded an efficiency of 95.8%, high repeatability, reproducibility, and a Limit of Detection and Quantification of at least 10 copies. Results from the validation of the MPXV dtec-qPCR kit were satisfactory. The use of the MONODOSE format (dehydrated single PCR-tubes, ready to use) provided considerable advantages allowing the detection of the Monkeypox virus to be accurately achieved. This detection kit may be considered a reliable, fast, simple, and universally available option. [ABSTRACT FROM AUTHOR]

The species Aeromonas lusitana was first described in 2016 with five strains recovered from untreated water and vegetables from Portugal. Since then, no further records exist of this species. During a surveillance study on the presence of Aeromonas in fish farms in Mexico, a new strain (ESV-351) of the mentioned species isolated from a rainbow trout was recovered. It was identified because it clustered phylogenetically with the type strain of A. lusitana based on the analysis of the rpoD gene sequences. In the present study, phenotypic characteristics, antimicrobial resistance profiles, and the presence of putative virulence genes of this novel strain (ESV-351) were determined in parallel to the five isolates from the original species description. Phenotypic differential characteristics exhibited by A. lusitana ESV-351 depicted an evident similarity to the characteristics exhibited by the other evaluated strains. However, the novel strain was positive for the production of indole using conventional methods, while the rest of the strains, including the type strain, were negative for its production. Furthermore, intermediate resistance to ampicillin, amoxicillin-clavulanic acid and cephalothin was detected in both the novel and the type strain. Five different virulence-related genes were detected in the novel strain and in the previously described strains, with the type strain exhibiting the highest number of virulence-related genes. In addition to this, the genome of the novel strain (ESV-351) was sequenced and compared with the genomes from the type strain (A. lusitana CECT 7828T) and other Aeromonas spp. The genomic analysis defined Aeromonas tecta as the closest species to A. lusitana with a highly similar number of predicted proteins. The genomic size, the number of protein-encoding genes and the number of different tRNAs, among other characteristics, make it possible to propose that the ESV-351 strain could potentially have the capacity to adapt to different environments. Genome comparison of the ESV-351 strain with the type strain revealed that both possess a similar sequence of the citrate synthase gene. In addition to this finding, the chromosomal region containing the citrate synthase locus of the novel strain exhibits some similarity to the chromosomal region in the genome of the A. hydrophila type strain and other known human pathogens, such as Vibrio cholerae. This could suggest a possible virulence role for the citrate synthase gene in A. lusitana (ESV-351). [ABSTRACT FROM AUTHOR]

Summary: Candida auris is an emerging multidrug resistant pathogenic fungus that causes candidaemia with high mortality rates and exhibits the ability to persist within the hospital environment. Candida auris is phylogenetically closely related to Candida haemulonii, C. lusitaniae, C. pseudohaemulonii and C. duobushaemulonii and is frequently misidentified by commercial identification methods. In the present study, the GPS™MONODOSE dtec‐qPCR kit (dried single‐dose PCR tubes) for the detection of C. auris was validated following the guidelines of the UNE/EN ISO/IEC 17025:2005, the French Standard NF T90‐471:2010 and using fast‐cycling protocols. Validation terms included in vitro specificity (inclusivity/exclusivity), quantitative phase analysis (10‐106 standard DNA copies), reliability (repeatability/reproducibility) and sensitivity (detection/quantification limits). GPS™ dtec‐qPCR kits passed validation with strict acceptance criteria (n ≥ 10 repetitions). In silico specificity was proven by the designer (GPS™). Experimental inclusiveness was achieved in two independent laboratories by testing strain JCM15448T and 117 clinical C. auris isolates from Asia, the Middle‐East Africa, Latin America and Europe. Exclusiveness was evaluated with 25 strains of closely related Candida spp. Use of the MONODOSE format provided considerable advantages allowing the detection of C. auris to be accurately achieved in less than an hour. The GPS™MONODOSE dtec‐qPCR kit is ready to undergo clinical evaluation. [ABSTRACT FROM AUTHOR]

Abstract: Previous studies indicate that Aeromonas aquariorum and Aeromonas hydrophila subsp. dhakensis are the same taxon and suggest that they should be synonymized. Using a polyphasic approach, the phenotypic and phylogenetic relationship of A. aquariorum with the 3 defined A. hydrophila subspecies (i.e. dhakensis, hydrophila, ranae) was investigated. Phylogenetic trees derived from the 16S rRNA, rpoD or gyrB genes and a multilocus phylogenetic analysis (with the concatenated sequences of gyrB, rpoD, recA, dnaJ and gyrA) confirmed that both A. aquariorum and A. hydrophila subsp. dhakensis are a unique taxon, different from the other A. hydrophila subspecies, corroborating the phenotypic and DNA–DNA hybridization (DDH) results. A formal synonymization of A. aquariorum and A. hydrophila subsp. dhakensis and a reclassification of both as Aeromonas dhakensis sp. nov. comb nov. is therefore proposed. [Copyright &y& Elsevier]

Abstract: It is well known that water constitutes an important contamination route for microorganisms. This is especially true for Aeromonas which are widespread in untreated and treated waters. In this study, Portuguese untreated waters not regularly monitored were screened for the presence and diversity of aeromonads. A total of 206 isolates were discriminated by RAPD-PCR and 80 distinct strains were identified by gyrB based phylogenetic analysis. The most frequently detected species were Aeromonas hydrophila, Aeromonas bestiarum and Aeromonas media. The antibiotic susceptibility profile of these strains was determined and showed a typical profile of the genus. Nonetheless, the percentage of resistant strains to tetracycline, chloramphenicol and/or trimethoprim/sulfamethoxazole was lower than that reported for clinical isolates and isolates recovered from aquacultures and other environments historically subjected to antibiotic contamination. This suggests that the existence of such pressures in those environments selects for resistant Aeromonas. A similar trend for integron presence was found. Genes coding for CphA and TEM, and tet(A), (E), (C) or (D) genes were found in 28%, 1%, and 10% of the strains, respectively. 10% of the strains contained an integron. Variable regions of seven class 1 integrons and one class 2 integron were characterised. Furthermore, strains displayed virulence related phenotypes such as extracellular lipolytic and proteolytic activities as well as aerolysin related genes (43% of strains). The ascV and aexT genes were found in 16% and 3% of strains respectively and, in some cases, concomitantly in the same specimen. This study shows that diverse Aeromonas spp. presenting distinct antibiotic resistance features and putative virulence traits are frequently present in waters for human and animal consumption in Portugal. Genes associated to antibiotic resistance and microbial virulence previously identified in organisms with human health significance were detected in these aeromonads, suggesting that these waters may act as a pivotal route for infections. [Copyright &y& Elsevier]

Abstract: Five Aeromonas strains, isolated from both clinical and environmental sources and characterized by a polyphasic approach, including phylogenetic analysis derived from gyrB, rpoD, and 16S rRNA gene sequencing, as well as DNA–DNA hybridization, extensive biochemical and antibiotic susceptibility tests, were recognized as members of an unknown, or undescribed, Aeromonas species. These “Aeromonas eucrenophila-like” strains were closely related to the species A. eucrenophila and Aeromonas encheleia, but they were negative for indole and acid from glycerol tests. Therefore, based on the results of the phylogenetic analyses and DNA–DNA pairing data of these strains, a novel species of the genus Aeromonas is described, for which the name Aeromonas tecta is proposed with isolate F518T (CECT7082T, DSM17300T, MDC91T) as the type strain. [Copyright &y& Elsevier]

A new species of the genus Aeromonas, Aeromonas sharmana sp. nov., was recently described on the basis of a single isolate, strain GPTSA-6⊃ T, obtained from a warm spring in India. The description of this new species included biochemical characterization, antibiotic susceptibility, cellular fatty-acid profiles, and 16S rRNA gene sequencing, but not DNADNA hybridization data. In the present article, phylogenetic analysis (branch distances in the tree and nucleotide signatures) of the 16S rRNA of isolate GPTSA-⊃ T, together with certain phenotypic characteristics of the isolate reported in the earlier description, clearly indicate that this microorganism does not belong to the genus Aeromonas as known to date, although it falls within the radiation of the family Aeromonadaceae. Emendation from the List of Prokaryotic Names with Standing in Nomenclature is consequently propose [ABSTRACT FROM AUTHOR]

The taxonomy of the "Aeromonas hydrophila" complex (comprising the species A. hydrophila, A. bestiarum, A. salmonicida, and A. popoffii) has been controversial, particularly the relationship between the two relevant fish pathogens A. salmonicida and A. bestiarum. In fact, none of the biochemical tests evaluated in the present study were able to separate these two species. One hundred and sixteen strains belonging to the four species of this complex were identified by 16S rDNA restriction fragment length polymorphism (RFLP). Sequencing of the 16S rDNA and cluster analysis of the 16S--23S intergenic spacer region (ISR)-RFLP in selected strains of A. salmonicida and A. bestiarum indicated that the two species may share extremely conserved ribosomal operons and demonstrated that, due to an extremely high degree of sequence conservation, 16S rDNA cannot be used to differentiate these two closely related species. Moreover, DNA--DNA hybridization similarity between the type strains of A. salmonicida subsp. salmonicida and A. bestiarum was 75.6 %, suggesting that they may represent a single taxon. However, a clear phylogenetic divergence between A. salmonicida and A. bestiarum was ascertained from an analysis based on gyrB and rpoD gene sequences, which provided evidence of a lack of congruence of the results obtained from 16S rDNA, 16S--23S ISR-RFLP, DNA--DNA pairing, and biochemical profiles. [ABSTRACT FROM AUTHOR]

The ribosomal RNA multigene family in Escherichia coli comprises seven rrn operons of similar, but not identical, sequence. Four operons ( rrnC, B, G, and E) contain genes in the 16S–23S intergenic spacer region (ISR) for tRNAGlu-2 and three ( rrnA, D, and H) contain genes for tRNAIle-1 and tRNAAla-1B. To increase our understanding of their molecular evolution, we have determined the ISR sequence of the seven operons in a set of 12 strains from the ECOR collection. Each operon was specifically amplified using polymerase chain reaction primers designed from genes or open reading frames located upstream of the 16S rRNA genes in E. coli K12. With a single exception (ECOR 40), ISRs containing one or two tRNA genes were found at the same respective loci as those of strain K12. Intercistronic heterogeneity already found in K12 was representative of most variation among the strains studied and the location of polymorphic sites was the same. Dispersed nucleotide substitutions were very few but 21 variable sites were found grouped in a stem-loop, although the secondary structure was conserved. Some regions were found in which a stretch of nucleotides was substituted in block by one alternative, apparently unrelated, sequence (as illustrated by the known putative insertion of rsl in K12). Except for substitutions of different sizes and insertions/deletions found in the ISR, the pattern of nucleotide variation is very similar to that found for the 16S rRNA gene in E. coli. Strains K12 and ECOR 40 showed the highest intercistronic heterogeneity. Most strains showed a strong tendency to homogenization. Concerted evolution could explain the notorious conservation of this region that is supposed to have low functional restrictions. [ABSTRACT FROM AUTHOR]

Hepatitis E virus (HEV) is one of the causative agents of water-borne human viral hepatitis and considered in Europe an emerging zoonotic pathogen. Analysis of bottled water through a standard method validated for HEV can contribute towards the risk management of this hazard. Putting some recent reports by the European Food Safety Authority in place, this study aimed to assess the performance of the concentration and extraction procedures described in ISO 15216-1:2017 for norovirus and hepatitis A virus on HEV detection. Following the ISO recommendation, the bottled water samples were spiked using serially diluted HEV fecal suspensions together with mengovirus as process control and concentrated by filtration via positively charged nylon membranes. In order to extract viral RNA from the resulting concentrates, two different methods were compared in this study: The one recommended in the ISO norm, NucliSens® MiniMag® system (NS), and an alternative commercially available kit NucleoSpin®RNA virus kit (MN). Finally, three reverse transcription quantitative PCR (RT-qPCR) assays were used to quantify HEV titers. The evaluated procedures resulted in average HEV recoveries of 14.08 ± 4.90% and 3.58 ± 0.30% for the MN and NS methods, respectively. The limit of detection (LoD95%) was 1.25 × 104 IU/L for both extraction methods combined with the three RT-qPCR assays tested, with the exception of NS extraction coupled with RT-qPCR1 that showed a LoD95% of 4.26 × 103 IU/L. The method characteristics generated in this study support the limited suitability of the ISO 15216-1:2017 concentration procedure coupled with the evaluated RT-qPCR assays for detecting HEV in bottled water. [ABSTRACT FROM AUTHOR]

Characterization of 999 Aeromonas strains using a published 16S rDNA RFLP identification method showed that 8.1% of the strains produced unexpected (hereafter called "atypical") restriction patterns, making their identification uncertain. Atypical patterns were due to the presence of nucleotide polymorphisms among the rrn operons of the 16S rRNA gene (so-called microheterogeneities). Double sequencing signals at certain positions revealed the nucleotide composition was responsible for the microheterogeneities. Although the number of microheterogeneities was relatively low (0.06-0.66%), trees inferred from the 16S rRNA gene led either to a misidentification or to an inconclusive result for the majority of these strains. Strains with atypical patterns were, however, correctly identified using the rpoD gene sequences, as belonging to Aeromonas caviae, A. veronii, and A. media. All of them, but particularly the two former species, are associated with human disease. Microheterogeneities in 16S rRNA gene sequence were significantly (P < 0.01) more prevalent in clinical than in environmental strains. This work also analyzed the effects of these microheterogeneities on the taxonomic position of the investigated strains. The results suggest the need for recording microheterogeneities in the 16S rRNA gene. [ABSTRACT FROM AUTHOR]

A considerable number of new SARS-CoV-2 lineages have emerged since the first COVID-19 cases were reported in Wuhan. As a few variants showed higher COVID-19 disease transmissibility and the ability to escape from immune responses, surveillance became relevant at that time. Single-nucleotide mutation PCR-based protocols were not always specific, and consequently, determination of a high number of informative sites was needed for accurate lineage identification. A detailed in silico analysis of SARS-CoV-2 sequences retrieved from GISAID database revealed the S gene 921 bp-fragment, positions 22784-23705 of SARS-CoV-2 reference genome, as the most informative fragment (30 variable sites) to determine relevant SARS-CoV-2 variants. Consequently, a method consisting of the PCR-amplification of this fragment, followed by Sanger's sequencing and a "single-click" informatic program based on a reference database, was developed and validated. PCR-fragments obtained from clinical SARS-CoV-2 samples were compared with homologous variant-sequences and the resulting phylogenetic tree allowed the identification of Alpha, Delta, Omicron, Beta, Gamma, and other variants. The data analysis procedure was automatized and simplified to the point that it did not require specific technical skills. The method is faster and cheaper than current whole-genome sequencing methods; it is available worldwide, and it may help to enhance efficient surveillance in the fight against the COVID-19 pandemic., Competing Interests: The authors declare no conflict of interest.

Candida auris is a multidrug-resistant pathogenic ascomycete yeast of increasing health concern. C. auris colonizes patient's skin and can persist for weeks on surfaces, so it can be transmitted within and between hospitals. The most common diagnostic platforms in microbiology use reference databases that have not yet incorporated C. auris, misidentifying it. This chapter describes how to detect C. auris by qPCR with the GPS™ CanAur MONODOSE dtec-qPCR Test (Alicante, Spain) in less than 45 min, using ready-to-use tubes with all the components dehydrated. This commercial kit was subjected to validation following the guidelines of the UNE-EN ISO/IEC 17025:2005 and French Standard NF T90-471:2010., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Candida auris is an emerging multidrug resistant pathogenic fungus that causes candidaemia with high mortality rates and exhibits the ability to persist within the hospital environment. Candida auris is phylogenetically closely related to Candida haemulonii, C. lusitaniae, C. pseudohaemulonii and C. duobushaemulonii and is frequently misidentified by commercial identification methods. In the present study, the GPS ™ MONODOSE dtec-qPCR kit (dried single-dose PCR tubes) for the detection of C. auris was validated following the guidelines of the UNE/EN ISO/IEC 17025:2005, the French Standard NF T90-471:2010 and using fast-cycling protocols. Validation terms included in vitro specificity (inclusivity/exclusivity), quantitative phase analysis (10-10 6  standard DNA copies), reliability (repeatability/reproducibility) and sensitivity (detection/quantification limits). GPS ™ dtec-qPCR kits passed validation with strict acceptance criteria (n ≥ 10 repetitions). In silico specificity was proven by the designer (GPS ™ ). Experimental inclusiveness was achieved in two independent laboratories by testing strain JCM15448 T and 117 clinical C. auris isolates from Asia, the Middle-East Africa, Latin America and Europe. Exclusiveness was evaluated with 25 strains of closely related Candida spp. Use of the MONODOSE format provided considerable advantages allowing the detection of C. auris to be accurately achieved in less than an hour. The GPS ™ MONODOSE dtec-qPCR kit is ready to undergo clinical evaluation., (© 2018 Blackwell Verlag GmbH.)

A Gram-negative, facultatively anaerobic bacillus, designated strain 266(T), was isolated from an irrigation water system in the south-west of Western Australia. Analysis of the 16S rRNA gene sequence confirmed that strain 266(T) belonged to the genus Aeromonas, with the nearest species being Aeromonas fluvialis (99.6% similarity to the type strain, with 6 nucleotide differences) followed by Aeromonas veronii and Aeromonas allosaccharophila (both 99.5%). Analysis of gyrB and rpoD sequences suggested that strain 266(T) formed a phylogenetic line independent of other species in the genus. This was confirmed using the concatenated sequences of six housekeeping genes (gyrB, rpoD, recA, dnaJ, gyrA and dnaX) that also indicated that A. veronii and A. allosaccharophila were the nearest relatives. DNA-DNA reassociation experiments and phenotypic analysis further supported the conclusion that strain 266(T) represents a novel species, for which the name Aeromonas australiensis sp. nov. is proposed, with type strain 266(T) (=CECT 8023(T) =LMG 26707(T)). [corrected].

Two clinical Aeromonas strains (A2-50(T) and A2-67(T)) recovered from the wounds of two patients in Taiwan could not be assigned to any known species of this genus based on their 16S rRNA gene sequences, which showed similarities of 99.6-99.8 % to those of the type strains of Aeromonas caviae, A. trota and A. aquariorum. The rpoD phylogenetic tree allocated these strains to two novel and independent phylogenetic lines, the neighbouring species being A. caviae, the type strain of which showed 93.2 % similarity (56 bp differences) to strain A2-50(T) and 92.2 % (63 bp differences) to strain A2-67(T). A multilocus phylogenetic analysis of five housekeeping genes (gyrB, rpoD, recA, dnaJ and gyrA; 3684 bp) confirmed that the two strains formed independent phylogenetic lineages within the genus. These data, together with phenotypic characterization and DNA-DNA reassociation results, revealed that these strains represent novel Aeromonas species, for which the names Aeromonas taiwanensis sp. nov. (type strain A2-50(T) =CECT 7403(T) =LMG 24683(T)) and Aeromonas sanarellii sp. nov. (type strain A2-67(T) =CECT 7402(T) =LMG 24682(T)) are proposed.

A Gram-stain-negative, facultatively anaerobic bacterial strain, designated 717(T), was isolated from a water sample collected from the Muga river, Girona, north-east Spain. Preliminary analysis of the 16S rRNA gene sequence showed that this strain belonged to the genus Aeromonas, the nearest species being Aeromonas veronii (99.5 % similarity, with seven different nucleotides). A polyphasic study based on a multilocus phylogenetic analysis of five housekeeping genes (gyrB, rpoD, recA, dnaJ and gyrA; 3684 bp) showed isolate 717(T) to be an independent phylogenetic line, with Aeromonas sobria, Aeromonas veronii and Aeromonas allosaccharophila as the closest neighbour species. DNA-DNA reassociation experiments and phenotypic analysis identified that strain 717(T) represents a novel species, for which the name Aeromonas fluvialis sp. nov. is proposed, with type strain 717(T) (=CECT 7401(T) =LMG 24681(T)).

Twenty-two human extraintestinal isolates (11 from blood) and three isolates recovered from patients with diarrhea were genetically characterized as Aeromonas aquariorum, a novel species known only from ornamental fish. The isolates proved to bear a considerable number of virulence genes, and all were resistant to amoxicillin (amoxicilline), cephalothin (cefalotin), and cefoxitin. Biochemical differentiation from the most relevant clinical species is provided.

A new species of the genus Aeromonas, Aeromonas sharmana sp. nov., was recently described on the basis of a single isolate, strain GPTSA-6T, obtained from a warm spring in India. The description of this new species included biochemical characterization, antibiotic susceptibility, cellular fatty-acid profiles, and 16S rRNA gene sequencing, but not DNADNA hybridization data. In the present article, phylogenetic analysis (branch distances in the tree and nucleotide signatures) of the 16S rRNA of isolate GPTSA-6T, together with certain phenotypic characteristics of the isolate reported in the earlier description, clearly indicate that this microorganism does not belong to the genus Aeromonas as known to date, although it falls within the radiation of the family Aeromonadaceae. Emendation from the List of Prokaryotic Names with Standing in Nomenclature is consequently proposed.

Bacterial infections caused by members of the genus Aeromonas, with a relatively high antibiotic resistance, are among the most common and troublesome diseases of fish raised in ponds with recirculation systems. In this study, carried out at an experimental aquaculture station in northern Portugal, 51 strains identified as belonging to the genus Aeromonas were isolated from 20 rainbow trout (Oncorhynchus mykiss) skin and kidney samples, as well as from raceway water samples. Macro- and microscopic examination of the fish tissues revealed lesions or cellular alterations in skin and kidney that seemed to correlate with the presence of those isolates. The sensitivity of all isolated strains to different groups of beta-lactam antibiotics (penicillins, cephalosporins, monobactams and carbapenems) was evaluated using the disc diffusion method. The highest rates of resistance were to amoxicillin, carbenicillin and ticarcillin. Unexpected resistance to imipenem, an antibiotic of clinical usage, was also detected, which suggests that resistance may have been transferred to the Aeromonas population from the environment.