Evaluation of the Use of Singleplex and Duplex CerTest VIASURE Real-Time PCR Assays to Detect Common Intestinal Protist Parasites.

Cryptosporidium spp., Giardia duodenalis and Entamoeba histolytica are species of protozoa- causing diarrhoea that are common worldwide, while Entamoeba dispar, Dientamoeba fragilis and Blastocystis sp. appear to be commensal parasites whose role in pathogenicity remains controversial. We conducted the clinical evaluation of five singleplex and one duplex CerTest VIASURE Real-Time PCR Assays against a large panel of positive DNA samples (n = 358), and specifically to Cryptosporidium spp. (n = 96), G. duodenalis (n = 115), E. histolytica (n = 25) E. dispar (n = 11), Blastocystis sp. (n = 42), D. fragilis (n = 37), and related parasitic phylum species such as Apicomplexa, Euglenozoa, Microsporidia and Nematoda. DNA samples were obtained from clinical stool specimens or cultured isolates in a national reference centre. Estimated diagnostic sensitivity and specificity values were 0.94-1 for Cryptosporidium spp., 0.96-0.99 for G. duodenalis, 0.96-1 for E. histolytica, 1-1 for E. dispar, and 1-0.99 for D. fragilis in the evaluated singleplex assays. In the duplex assay for the simultaneous detection of Blastocystis sp. and D. fragilis these values were 1-0.98 and 1-0.99, respectively. Measures of diagnostic precision for repeatability and reproducibility were found to be under acceptable ranges. The assays identified six Cryptosporidium species (C. hominis, C. parvum, C. canis, C. felis, C. scrofarum, and C. ryanae), four G. duodenalis assemblages (A, B, C, and F), and six Blastocystis subtypes (ST1-ST5, and ST8). The evaluated singleplex and duplex VIASURE Real-Time PCR assays provide sensitive, practical, and cost-effective choices to the molecular diagnosis of the main diarrhoea-causing intestinal protists in clinical microbiology and research laboratories.

Clinical Performance Evaluation of a Rapid Real-Time PCR Assay for Monkeypox Diagnosis: a Retrospective and Comparative Study.

In an increasingly globalized and interconnected world, the outbreak of an infectious disease in one country can become a worrying health emergency for the whole world. A current example is the 2022 monkeypox virus (mpox) outbreak affecting multiple areas across the world. In this context, strategies to interrupt transmission as soon as possible by identifying cases, clusters, and sources of infection should be developed around the world to prevent these crises. The aim of this retrospective and collaborative study was to perform external clinical validation of the VIASURE monkeypox virus real-time PCR detection kit (CerTest Biotec, Spain) with ready-to-use reagents designed for the rapid detection of mpox. A total of 165 samples with suspected infection were used for this analysis. The standard procedures of the clinical microbiology laboratory of the Miguel Servet University Hospital, using the RealStar Orthopoxvirus PCR kit v1.0 (Altona Diagnostics) and bidirectional Sanger sequencing (STAB VIDA, Caparica, Portugal), were considered reference techniques. Furthermore, a subset of 67 mpox-negative samples and 13 mpox-positive samples were routinely tested for clinical diagnosis of other rash/ulcerative pathologies. Accuracy testing resulted in appropriate clinical validation values, as follows: sensitivity, 1 (95% confidence interval [CI], 0.97 to 1); specificity, 1 (95% CI, 0.98 to 1); positive predictive value, 1 (95% CI, 0.93 to 1); negative predictive value, 1 (95% CI, 0.95 to 1). The strength of agreement between assays was almost perfect. The added value is the useful support for the specific diagnosis of mpox infections due to the diagnostic specificity data obtained. IMPORTANCE Given that a large number of mpox outbreaks have been reported worldwide since 2022 in countries in which the disease is not endemic, the main concern for clinicians and global health systems should be to develop effective, available, and easy-to-implement diagnostic strategies to interrupt mpox transmission as soon as possible. This retrospective study demonstrates the satisfactory clinical parameters of a commercially available molecular diagnostic kit for routine testing for mpox in clinical diagnostic laboratories.

Usefulness of Non-Skin Samples in the PCR Diagnosis of Mpox (Monkeypox).

Cases of mpox have been reported in several European countries, including Spain. Our objective was to evaluate the usefulness of serum and nasopharyngeal samples for diagnosis of mpox. The presence of MPXV DNA was studied using real-time PCR (CerTest Biotec, Zaragoza, Spain) in 106 samples from 50 patients: 32 skin, 31 anogenital, 25 sera, and 18 nasopharyngeal/pharyngeal, in the Hospital Clínico Universitario of Zaragoza (Spain). Sixty-three samples from twenty-seven patients were MPXV PCR-positive. The real-time PCR Ct values in the anogenital and skin samples were lower than serum and nasopharyngeal samples. More than 90% of anogenital (95.7%), serum (94.4%), and skin (92.9%) samples were real-time PCR-positive. Eighteen (66.7%) of the twenty-seven patients who were MPXV PCR-positive had antecedents or presented with one to three sexually transmitted infection (STI) agents. Our results indicate that the use of serum samples can help facilitate the diagnosis of MPXV infections.

Retrospective and Comparative Study of Three Molecular Assays for the Macrolide Resistance Detection in Mycoplasma genitalium Positive Urogenital Specimens.

The capacity of Mycoplasma genitalium to develop resistance to macrolides makes detection of macrolide resistance genes by rapid real-time PCR assays increasingly necessary in clinical diagnostic laboratories so as to initiate appropriate treatment as rapidly as possible. The aim of this retrospective and comparative study was to conduct the clinical evaluation of three commercially available kits for macrolide resistance detection. A total of 111 M. genitalium positive samples analyzed in the Clinical Microbiology Laboratory of the Miguel Servet University Hospital, Zaragoza (Spain) were used. After M. genitalium molecular confirmation, the three assays under study were evaluated and discrepant results were resolved via sequencing. The clinical sensitivity for resistance detection was 83% (95% confidence interval, 69% to 93%) for the ResistancePlus® MG panel kit (SpeeDx Pty Ltd., Sydney, Australia), 95% (84% to 99%) for AllplexTM MG & AziR Assay (Seegene®, Seoul, Korea), and 97% (88% to 99%) for the VIASURE macrolide resistance-associated mutations (23SrRNA) Real time PCR detection kit (Certest Biotec, Zaragoza, Spain). The clinical specificity was 100% (94% to 100%) for Allplex and VIASURE assays and 95% (86% to 99%) for SpeeDx assay. The results arising from this study are cause for strong consideration for the implementation of rapid real-time PCR assays in clinical diagnosis laboratories to eliminate treatment failure and transmission as soon as possible.

Evaluation of a Novel Commercial Real-Time PCR Assay for the Simultaneous Detection of Cryptosporidium spp., Giardia duodenalis, and Entamoeba histolytica.

Cryptosporidium spp., Giardia duodenalis, and Entamoeba histolytica are the most common diarrhea-causing protozoan species globally. Misdiagnosis is a concern for asymptomatic and chronic infections. Multiplexing, i.e., the detection of more than one parasite in a single test by real-time PCR, allows high diagnostic performance with favorable cost-effectiveness. We conducted a clinical evaluation of the VIASURE Cryptosporidium, Giardia, & E. histolytica real-time PCR assay (CerTest Biotec, San Mateo de Gállego, Spain) against a large panel (n = 358) of well-characterized DNA samples positive for Cryptosporidium spp. (n = 96), G. duodenalis (n = 115), E. histolytica (n = 25), and other parasitic species of the phyla Amoebozoa (n = 11), Apicomplexa (n = 14), Euglenozoa (n = 8), Heterokonta (n = 42), Metamonada (n = 37), Microsporidia (n = 4), and Nematoda (n = 6). DNA samples were obtained from clinical stool specimens or cultured isolates in a national reference center. Estimated sensitivity and specificity were 0.96 and 0.99 for Cryptosporidium spp., 0.94 and 1 for G. duodenalis, and 0.96 and 1 for E. histolytica, respectively. Positive and negative predictive values were calculated as 1 and 0.98 for Cryptosporidium spp., 0.99 and 0.98 for G. duodenalis, and 1 and 0.99 for E. histolytica, respectively. The assay identified six Cryptosporidium species (Cryptosporidium hominis, Cryptosporidium parvum, Cryptosporidium canis, Cryptosporidium felis, Cryptosporidium scrofarum, and Cryptosporidium ryanae) and four G. duodenalis assemblages (A, B, C, and F). The VIASURE assay provides rapid and accurate simultaneous detection and identification of the most commonly occurring species and genetic variants of diarrhea-causing parasitic protozoa in humans. IMPORTANCE Thorough independent assessment of the diagnostic performance of novel diagnostic assays is essential to ascertain their true usefulness and applicability in routine clinical practice. This is particularly true for commercially available kits based on multiplex real-time PCR aimed to detect and differentiate multiple pathogens in a single biological sample. In this study, we conducted a clinical evaluation of the VIASURE Cryptosporidium, Giardia, & E. histolytica real-time PCR assay (CerTest Biotec) for the detection and identification of the diarrhea-causing enteric protozoan parasites Cryptosporidium spp., G. duodenalis, and E. histolytica. A large panel of well-characterized DNA samples from clinical stool specimens or cultured isolates from a reference center was used for this purpose. The VIASURE assay demonstrated good performance for the routine testing of these pathogens in clinical microbiological laboratories.

Rv0180c contributes to Mycobacterium tuberculosis cell shape and to infectivity in mice and macrophages.

Mycobacterium tuberculosis, the main causative agent of human tuberculosis, is transmitted from person to person via small droplets containing very few bacteria. Optimizing the chance to seed in the lungs is therefore a major adaptation to favor survival and dissemination in the human population. Here we used TnSeq to identify genes important for the early events leading to bacterial seeding in the lungs. Beside several genes encoding known virulence factors, we found three new candidates not previously described: rv0180c, rv1779c and rv1592c. We focused on the gene, rv0180c, of unknown function. First, we found that deletion of rv0180c in M. tuberculosis substantially reduced the initiation of infection in the lungs of mice. Next, we established that Rv0180c enhances entry into macrophages through the use of complement-receptor 3 (CR3), a major phagocytic receptor for M. tuberculosis. Silencing CR3 or blocking the CR3 lectin site abolished the difference in entry between the wild-type parental strain and the Δrv0180c::km mutant. However, we detected no difference in the production of both CR3-known carbohydrate ligands (glucan, arabinomannan, mannan), CR3-modulating lipids (phthiocerol dimycocerosate), or proteins in the capsule of the Δrv0180c::km mutant in comparison to the wild-type or complemented strains. By contrast, we established that Rv0180c contributes to the functionality of the bacterial cell envelope regarding resistance to toxic molecule attack and cell shape. This alteration of bacterial shape could impair the engagement of membrane receptors that M. tuberculosis uses to invade host cells, and open a new perspective on the modulation of bacterial infectivity.

Identifying causal models between genetically regulated methylation patterns and gene expression in healthy colon tissue.

BACKGROUND: DNA methylation is involved in the regulation of gene expression and phenotypic variation, but the inter-relationship between genetic variation, DNA methylation and gene expression remains poorly understood. Here we combine the analysis of genetic variants related to methylation markers (methylation quantitative trait loci: mQTLs) and gene expression (expression quantitative trait loci: eQTLs) with methylation markers related to gene expression (expression quantitative trait methylation: eQTMs), to provide novel insights into the genetic/epigenetic architecture of colocalizing molecular markers. RESULTS: Normal mucosa from 100 patients with colon cancer and 50 healthy donors included in the Colonomics project have been analyzed. Linear models have been used to find mQTLs and eQTMs within 1 Mb of the target gene. From 32,446 eQTLs previously detected, we found a total of 6850 SNPs, 114 CpGs and 52 genes interrelated, generating 13,987 significant combinations of co-occurring associations (meQTLs) after Bonferromi correction. Non-redundant meQTLs were 54, enriched in genes involved in metabolism of glucose and xenobiotics and immune system. SNPs in meQTLs were enriched in regulatory elements (enhancers and promoters) compared to random SNPs within 1 Mb of genes. Three colorectal cancer GWAS SNPs were related to methylation changes, and four SNPs were related to chemerin levels. Bayesian networks have been used to identify putative causal relationships among associated SNPs, CpG and gene expression triads. We identified that most of these combinations showed the canonical pathway of methylation markers causes gene expression variation (60.1%) or non-causal relationship between methylation and gene expression (33.9%); however, in up to 6% of these combinations, gene expression was causing variation in methylation markers. CONCLUSIONS: In this study we provided a characterization of the regulation between genetic variants and inter-dependent methylation markers and gene expression in a set of 150 healthy colon tissue samples. This is an important finding for the understanding of molecular susceptibility on colon-related complex diseases.

Crambescin C1 Acts as A Possible Substrate of iNOS and eNOS Increasing Nitric Oxide Production and Inducing In Vivo Hypotensive Effect.

Crambescins are guanidine alkaloids from the sponge Crambe crambe. Crambescin C1 (CC) induces metallothionein genes and nitric oxide (NO) is one of the triggers. We studied and compared the in vitro, in vivo, and in silico effects of some crambescine A and C analogs. HepG2 gene expression was analyzed using microarrays. Vasodilation was studied in rat aortic rings. In vivo hypotensive effect was directly measured in anesthetized rats. The targets of crambescines were studied in silico. CC and homo-crambescine C1 (HCC), but not crambescine A1 (CA), induced metallothioneins transcripts. CC increased NO production in HepG2 cells. In isolated rat aortic rings, CC and HCC induced an endothelium-dependent relaxation related to eNOS activation and an endothelium-independent relaxation related to iNOS activation, hence both compounds increase NO and reduce vascular tone. In silico analysis also points to eNOS and iNOS as targets of Crambescin C1 and source of NO increment. CC effect is mediated through crambescin binding to the active site of eNOS and iNOS. CC docking studies in iNOS and eNOS active site revealed hydrogen bonding of the hydroxylated chain with residues Glu377 and Glu361, involved in the substrate recognition, and explains its higher binding affinity than CA. The later interaction and the extra polar contacts with its pyrimidine moiety, absent in the endogenous substrate, explain its role as exogenous substrate of NOSs and NO production. Our results suggest that CC serve as a basis to develop new useful drugs when bioavailability of NO is perturbed.

Push-pull driving of the Central America Forearc in the context of the Cocos-Caribbean-North America triple junction.

Different kinematic models have been proposed for the triple junction between the North American, Cocos and Caribbean plates. The two most commonly accepted hypotheses on its driving mechanism are (a) the North American drag of the forearc and (b) the Cocos Ridge subduction push. We present an updated GPS velocity field which is analyzed together with earthquake focal mechanisms and regional relief. The two hypotheses have been used to make kinematic predictions that are tested against the available data. An obliquity analysis is also presented to discuss the potential role of slip partitioning as driving mechanism. The North American drag model presents a better fit to the observations, although the Cocos Ridge push model explains the data in Costa Rica and Southern Nicaragua. Both mechanisms must be active, being the driving of the Central American forearc towards the NW analogous to a push-pull train. The forearc sliver moves towards the west-northwest at a rate of 12-14 mm/yr, being pinned to the North American plate in Chiapas and western Guatemala, where the strike-slip motion on the volcanic arc must be very small.

Main crustal seismic sources in El Salvador.

We present a map and a data set containing information about intra-plate seismic sources in El Salvador. These are the results of the field campaigns and data analysis carried out by the research group of Planetary Geodinamics, Active Tectonics and Related Risks from Complutense University of Madrid during the last 12 years. We include two maps, the first map contains 1405 fault traces with evidences of Quaternary activity derived form morphometric, paleoseismological and geomorphological analysis together with field data mapping carried in El Salvador. The second map is a synthesis of the 29 intra-plate seismic sources selected from the quaternary faults map. The geometry of these sources was simplified and we also include a table where some available data of the proposed sources are included, such as their name, orientation, length and slip-rate. For further interpretation and discussion of these sources see (Alonso-Henar et al., 2018) [1, doi.org/10.1016/j.enggeo.2018.06.015].

Influence of implant neck surface and placement depth on crestal bone changes and soft tissue dimensions around platform-switched implants: A histologic study in dogs.

OBJECTIVES: To analyse bone remodelling and peri-implant soft tissues around platform-switching implants with and without a machined collar placed at different levels in relation to bone crest. MATERIAL AND METHODS: All mandibular premolars and the first molars were extracted in five dogs. At 6 months, six implants with and without a machined neck (MACH and GBAE implants, respectively) were randomly inserted in each hemimandible positioning the implant-abutment interface in either a supracrestal (+1.5 mm), equicrestal, or subcrestal (-1.5 mm) position. After 6 months, animals were killed for histomorphometric analysis. RESULTS: When net bone loss (primary outcome variable) was compared between MACH and GBAE groups, the multivariable regression analysis revealed no significant differences between implants inserted at the same vertical position. The dimensions of the peri-implant mucosa were greater in MACH implants compared with GBAE implants; however, these differences failed to reach statistical significance. Regarding the number of inflammatory cells and collagen fibre orientation, no statistically significant differences were found between MACH and GBAE groups. CONCLUSIONS: The surface treatment of the implant neck does not seem to have an influence on net bone loss, and there were no statistically significant differences in the peri-implant soft tissues between platform-switching implants with and without a machined neck.

Protein O-mannosylation deficiency increases LprG-associated lipoarabinomannan release by Mycobacterium tuberculosis and enhances the TLR2-associated inflammatory response.

Protein O-mannosylation is crucial for the biology of Mycobacterium tuberculosis but the key mannosylated protein(s) involved and its(their) underlying function(s) remain unknown. Here, we demonstrated that the M. tuberculosis mutant (Δpmt) deficient for protein O-mannosylation exhibits enhanced release of lipoarabinomannan (LAM) in a complex with LprG, a lipoprotein required for LAM translocation to the cell surface. We determined that LprG is O-mannosylated at a unique threonine position by mass spectrometry analyses of the purified protein. However, although replacement of this amino acid by an alanine residue completely abolished LprG O-mannosylation, the increased release of the LAM/LprG complex was preserved. We found that the increased secretion of this complex is due to enhanced LAM production in the Δpmt M. tuberculosis and M. smegmatis mutants relative to their wild-type counterparts. This abnormal release of LAM/LprG has functional consequences on the induction of inflammatory responses and provides a possible explanation for the reduced virulence of the M. tuberculosis Δpmt mutant.

Crambescidin-816 acts as a fungicidal with more potency than crambescidin-800 and -830, inducing cell cycle arrest, increased cell size and apoptosis in Saccharomyces cerevisiae.

In this paper, we show the effect of crambescidin-816, -800, and -830 on Saccharomyces cerevisiae viability. We determined that, of the three molecules tested, crambescidin-816 was the most potent. Based on this result, we continued by determining the effect of crambescidin-816 on the cell cycle of this yeast. The compound induced cell cycle arrest in G2/M followed by an increase in cell DNA content and size. When the type of cell death was analyzed, we observed that crambescidin-816 induced apoptosis. The antifungal effect indicates that crambescidins, and mostly crambescidin-816, could serve as a lead compound to fight fungal infections.

Oral toxicity of okadaic acid in mice: study of lethality, organ damage, distribution and effects on detoxifying gene expression.

In vivo, after administration by gavage to mice and rats, okadaic acid has been reported to produce lesions in liver, small intestine and forestomach. Because several reports differ in the damage detected in different organs, and on okadaic acid distribution after consumption, we determined the toxicity of this compound after oral administration to mice. After 24 hours, histopathological examination showed necrotic foci and lipid vacuoles in the livers of intoxicated animals. By immunohistochemical analysis, we detected this toxin in the liver and kidneys of intoxicated animals. Okadaic acid induces oxidative stress and can be activated in vitro into reactive compounds by the post-mitochondrial S9 fraction, so we studied the okadaic effect on the gene expression of antioxidant and phase II detoxifying enzymes in liver. We observed a downregulation in the expression of these enzymes and a reduction of protein expression of catalase and superoxide dismutase 1 in intoxicated animals.

Mapping IS6110 in high-copy number Mycobacterium tuberculosis strains shows specific insertion points in the Beijing genotype.

BACKGROUND: Mycobacterium tuberculosis Beijing strains are characterized by a large number of IS6110 copies, suggesting the potential implication of this element in the virulence and capacity for rapid dissemination characteristic of this family. This work studies the insetion points of IS6110 in high-copy clinical isolates specifically focusing on the Beijing genotype. RESULTS: In the present work we mapped the insertion points of IS6110 in all the Beijing strains available in the literature and in the DNA sequence databases. We generated a representative primer collection of the IS6110 locations, which was used to analyse 61 high-copy clinical isolates. A total of 440 points of insertion were identified and analysis of their flanking regions determined the exact location, the direct repeats (DRs), the orientation and the distance to neighboring genes of each copy of IS6110. We identified specific points of insertion in Beijing strains that enabled us to obtain a dendrogram that groups the Beijing genotype. CONCLUSIONS: This work presents a detailed analysis of locations of IS6110 in high-copy clinical isolates, showing points of insertion present with high frequency in the Beijing family and absent in other strains.

Bioengineered protein phosphatase 2A: update on need.

Harmful algal blooms caused by phytoplankton can occur in all aquatic environments. Some of the algae present in these blooms are capable of producing extremely potent toxins. Due to climate change and eutrophication, harmful algal blooms are increasing on a global scale. One kind of toxin producing algae are those that produce okadaic acid, its derivatives (dinophysistoxin-1 and 2), and microcystins. These toxins are potent inhibitors of protein phosphatase 2A, so this protein is used to detect the mentioned toxins in natural samples. Originally protein phosphatase 2A purified from animal tissues was used, but enzyme activity and stability fluctuations prevented the use of the enzyme in detection kits. Expression of the enzyme as a recombinant protein provided a solution to this problem. For this purpose, several strategies have been followed. We evaluated the activity, specificity and stability of the human protein phosphatase 2A catalytic subunit α expressed in insect larvae and showed that this expression system can be a reliable source of high quantities of stable enzyme.

Protein synthesis inhibition and oxidative stress induced by cylindrospermopsin elicit apoptosis in primary rat hepatocytes.

The increasing presence of cyanotoxin producers in several regions of the world is hazardous for humans and animals. Cylindrospermopsin (CYN) is nowadays recognized as a widely distributed freshwater cyanobacterial toxin. This toxin has been shown to induce protein synthesis inhibition as well as inhibition of glutathione synthesis. Given that the liver seems to be the main target of cylindrospermopsin, in this work we used cultures of primary rat hepatocytes to study the type of cell death induced by CYN nanomolar concentrations. The involvement of reactive oxygen species in toxin induced cell death, the relationship between protein synthesis inhibition and toxicity, and the cell endogenous antioxidant response regulation were studied. We show that cylindrospermopsin induces apoptosis in primary rat hepatocytes. At the concentrations used in this work, protein synthesis inhibition and oxidative stress were involved in the cytotoxic effect elicited by the toxin. Finally, activation of the cell antioxidant response was observed at the transcriptional and translational levels.

Rapid test for identification of a highly transmissible Mycobacterium tuberculosis Beijing strain of sub-Saharan origin.

The development of a rapid test to identify Mycobacterium tuberculosis Beijing isolates and specifically strain GC1237, coming from a sub-Saharan country, is needed due to its alarming wide spread on Gran Canaria Island (Spain). A rapid test that detects IS6110 present between dnaA and dnaN in the Beijing strains and in a specific site for GC1237 (Rv2180c) has been developed. This test would be a useful tool in the surveillance of subsequent cases.

Deciphering the role of IS6110 in a highly transmissible Mycobacterium tuberculosis Beijing strain, GC1237.

The capacity of infection and the ability of Mycobacterium tuberculosis strains belonging to the Beijing family to spread rapidly probably result from genetic advantages and unidentified mechanisms of virulence not yet thoroughly investigated. Among the mechanisms proposed to be responsible for the varying virulence phenotypes of M. tuberculosis strains we find IS6110 insertions, genetic reorganizations and deletions, which have strong influences on fitness. Beijing family is one of the lineages with the highest number of copies of IS6110. By studying genetic markers characteristic for this lineage, here we have characterized the clinical isolate M. tuberculosis GC1237 strain responsible for important epidemic outbreaks in the Gran Canary Island. We have identified and analyzed each point of insertion of IS6110 using a bacterial artificial chromosome (BAC) library of this strain, in addition to the use of other approximations. Nineteen copies of IS6110 have been localized in GC1237 genome of which, four copies of IS6110 can act as a promoter and we have focused in the characterization of one copy located 31 bp upstream of the essential gene Rv2179c and compared to the reference strain H37Rv.