A Genetic Lab-on-Phone Test for Point-of-Care Diagnostic of Lactose Intolerance near Patient and in less than 90 Minutes.

BACKGROUND: The -13910 C/T single nucleotide polymorphism located within the MCM6 gene, an enhancer region located upstream of the lactase-phlorizin hydrolase gene, is associated with lactase persistence/non-persistence traits among the Caucasian population. The performance of a new point-of-care CE-IVD (In Vitro Diagnostic) marked isothermal lab-on-phone lactose intolerance assay, using crude samples, was assessed in comparison with Sanger sequencing using purified DNA, as reference method. METHODS: The study was conducted following a non-probability sampling using direct buccal swab (n = 63) and capillary blood (n = 43) clinical samples from a total of 63 volunteers. A 3 × 3 confusion matrix/contingency table was used to evaluate the performance of the isothermal lab-on-phone lactose intolerance assay. RESULTS: The isothermal lab-on-phone lactose intolerance assay successfully detected the -13910 C/T variant with a limit of detection of 5 cells/assay and demonstrated an overall accuracy of 98.41% (95% CI, 91.47%-99.96%) for buccal swab samples and 100% (95% CI, 91.19%-100%) for capillary blood, taking just 90 min from sample to result, with only 2 min hands-on. CONCLUSIONS: The lab-on-phone pocket-sized assay displayed good performance when using direct buccal swab and capillary blood samples, enabling a low-cost, real-time, and accurate genotyping of the -13910 C/T region for the rapid diagnosis of primary lactose intolerance at point-of-care, which enables a prompt implementation of appropriate diet habits and/or intolerance therapies. To our knowledge, this is the first point-of-care genetic test for lactose intolerance to be made available on the market.

MedSecurance Project: Advanced Security-for-Safety Assurance for Medical Device IoT (IoMT).

The MedSecurance project focus on identifying new challenges in cyber security with focus on hardware and software medical devices in the context of emerging healthcare architectures. In addition, the project will review best practice and identify gaps in the guidance, particularly the guidance stipulated by the medical device regulation and directives. Finally, the project will develop comprehensive methodology and tooling for the engineering of trustworthy networks of inter-operating medical devices, that shall have security-for-safety by design, with a strategy for device certification and certifiable dynamic network composition, ensuring that patient safety is safeguarded from malicious cyber actors and technology "accidents".

An isothermal lab-on-phone test for easy molecular diagnosis of SARS-CoV-2 near patients and in less than 1 hour.

OBJECTIVES: The performance of a new point-of-care CE-IVD-marked isothermal lab-on-phone COVID-19 assay was assessed in comparison to a gold standard real-time reverse transcriptase-PCR method. METHODS: The study was conducted following a nonprobability sampling of ≥16-year-old volunteers from three different laboratories, using direct mouthwash (N = 24) or nasopharyngeal (N = 191) clinical samples. RESULTS: The assay demonstrated 95.19% sensitivity and 100% specificity for detection of SARS-CoV-2 in direct nasopharyngeal crude samples and 78.95% sensitivity and 100% specificity in direct mouthwash crude samples. It also successfully detected currently predominant SARS-CoV-2 variants of concern (Beta B.1.351, Delta B.1.617.2, and Omicron B.1.1.529) and demonstrated to be inert against potential cross-reactions of other common respiratory pathogens that cause infections that present similar symptoms to COVID-19. CONCLUSION: This lab-on-phone pocket-sized assay relies on an isothermal amplification of SARS-CoV-2's N and E genes, taking just 50 minutes from sample to result, with only 2 minutes of hands-on time. It presents good performance when using direct nasopharyngeal crude samples, enabling a low-cost, real-time, rapid, and accurate identification of SARS-CoV-2 infections at the point of care, which is important for both clinical management and population screening, as a tool to break the chain of transmission of COVID-19 pandemic, especially in low-resources environments.

LungCARD - Report on worldwide research and clinical practices related to lung cancer.

PURPOSE: The management of advanced lung cancer has evolved tremendously over the past two decades. Increasing understanding of the molecular changes that drive tumor progression has transformed the treatment of this disease. Nevertheless, various countries differ in the degree of implementation of genetic tests and the availability of innovative drugs. The LungCARD consortium created a questionnaire to collect information about the local research and clinical practices related to lung cancer diagnosis and therapy. METHODS: A survey composed of 37 questions related to specific lung cancer pharmacogenomics and therapy, was distributed among 18 countries. RESULTS: All together 36 responses were gathered, answered mainly by clinicians. The majority attends 50-200 cancer cases per month, 20-50% of all cancer cases are lung cancer patients, and more than 80% are with non-small-cell lung cancer (NSCLC). Targeted therapy is applied to 50% on average of all NSCLC patients. Forty five percent of participating medical oncologists are treating their patients with immunotherapy. More than 90% of the respondents are guided by results of genetic tests in introducing targeted treatment. As expected, the majority orders EGFR gene testing (85%), followed by ALK (58%) and KRAS testing (32%). Almost all (96%) agreed that more biomarkers should be included in routine genetic testing (ROS1, anti-PDL1, KRAS, MET, HER2, BRAF...), and that blood test is useful in pharmacogenomic testing. CONCLUSION: There is a great variation between countries with respect to all discussed topics. However, the majority recognized a necessity of introducing next generation sequencing (NGS)-based diagnostics and potential of testing from blood. The biggest problem in the treatment of NSCLC is still an access to innovative drugs.

Characterization and in vitro evaluation of new bacteriophages for the biocontrol of Escherichia coli.

In the present study two new phages (phT4A and ECA2) were characterized and their efficacy was evaluated separately and in cocktail (phT4A/ECA2) to control Escherichia coli. The isolated phages, phT4A and ECA2, belonged to the Myoviridae and Podoviridae family, respectively and both are safe (no integrase and toxin codifying genes) to be used in bacterial control. In general, the increase of multiplicity of infection (MOI) from 1 to 100 promoted a significant increase in the efficiency of phage phT4A and phage cocktail phT4A/ECA2. Both phages were effective against E. coli, but phage phT4A (reduction of 5.8 log CFU/mL after 8h treatment) was more effective than phage ECA2 phage (reduction of 4.7 log CFU/mL after 8h treatment). The use of a cocktail phT4A/ECA2 was significantly more effective (reductions of 6.2 log CFU/mL after 6h treatment) than the use single phage suspensions of phT4A and ECA2 (reductions 5.3 log CFU/mL and 4.9 log CFU/mL, respectively, after 6h treatment). The rate of emergence of phage-resistant mutants was lower for phage phT4A when compared with phage ECA2 and phage cocktail phT4A/ECA2.The results indicate that in addition to the efficacy, the potential development of phage-resistant mutants must also be considered in the design of phage cocktails.

Bacteriophages with potential to inactivate Salmonella Typhimurium: Use of single phage suspensions and phage cocktails.

The aim of this study was to compare the dynamics of three previously isolated bacteriophages (or phages) individually (phSE-1, phSE-2 and phSE-5) or combined in cocktails of two or three phages (phSE-1/phSE-2, phSE-1/phSE-5, phSE-2/phSE-5 and phSE-1/phSE-2/phSE-5) to control Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) in order to evaluate their potential application during depuration. Phages were assigned to the family Siphoviridae and revealed identical restriction digest profiles, although they showed a different phage adsorption, host range, burst size, explosion time and survival in seawater. The three phages were effective against S. Typhimurium (reduction of ∼2.0 log CFU/mL after 4h treatment). The use of cocktails was not significantly more effective than the use of single phages. A big fraction of the remained bacteria are phage-resistant mutants (frequency of phage-resistant mutants 9.19×10(-5)-5.11×10(-4)) but phage- resistant bacterial mutants was lower for the cocktail phages than for the single phage suspensions and the phage phSE-1 presented the highest rate of resistance and phage phSE-5 the lowest one. The spectral changes of S. Typhimurium resistant and phage-sensitive cells were compared and revealed relevant differences for peaks associated to amide I (1620cm(-1)) and amide II (1515cm(-1)) from proteins and from carbohydrates and phosphates region (1080-1000cm(-1)). Despite the similar efficiency of individual phages, the development of lower resistance indicates that phage cocktails might be the most promising choice to be used during the bivalve depuration to control the transmission of salmonellosis.

Comparative RNA-seq-Based Transcriptome Analysis of the Virulence Characteristics of Methicillin-Resistant and -Susceptible Staphylococcus pseudintermedius Strains Isolated from Small Animals.

Staphylococcus pseudintermedius is often associated with pyoderma, which can turn into a life-threatening disease. The dissemination of highly resistant isolates has occurred in the last 10 years and has challenged antimicrobial treatment of these infections considerably. We have compared the carriage of virulence genes and biofilm formation between methicillin-resistant and methicillin-susceptible S. pseudintermedius (MRSP and MSSP, respectively) isolates and their in vitro gene expression profiles by transcriptome sequencing (RNA-seq). Isolates were relatively unevenly distributed among the four agr groups, and agr type III predominated in MRSP. Five virulence genes were detected in all isolates. Only the spsO gene was significantly associated with MSSP isolates (P = 0.04). All isolates produced biofilm in brain heart infusion broth (BHIB)-4% NaCl. MSSP isolates produced more biofilm on BHIB and BHIB-1% glucose media than MRSP isolates (P = 0.03 and P = 0.02, respectively). Virulence genes encoding surface proteins and toxins (spsA, spsB, spsD, spsK, spsL, spsN, nucC, coa, and luk-I) and also prophage genes (encoding phage capsid protein, phage infection protein, two phage portal proteins, and a phage-like protein) were highly expressed in the MRSP isolate (compared with the MSSP isolate), suggesting they may play a role in the rapid and widespread dissemination of MRSP. This study indicates that MRSP may upregulate surface proteins, which may increase the adherence of MRSP isolates (especially sequence type 71 [ST71]) to corneocytes. MSSP isolates may have an increased ability to form biofilm under acidic circumstances, through upregulation of the entire arc operon. Complete understanding of S. pseudintermedius pathogenesis and host-pathogen signal interaction during infections is critical for the treatment and prevention of S. pseudintermedius infections.

Next-generation sequencing and comparative analysis of Pyrrhocorax pyrrhocorax and Pyrrhocorax graculus (Passeriformes: Corvidae) mitochondrial genomes.

The complete mitochondrial genomes of Red-billed Chough (Pyrrhocorax pyrrhocorax) and Yellow-billed Chough (Pyrrhocorax graculus) were sequenced using the Ion Torrent PGM platform. These mitogenomes contain 16,889 bp (Red-billed Chough) and 16,905 bp (Yellow-billed Chough), including 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and a control region (D-loop). The gene content, orientation, and structure are similar to a wide range of other vertebrate species and the nucleotide composition is very similar to other Passeriformes. All PCGs start with ATG, except for COX1 that starts with GTG, and four stop codons and one incomplete stop codon are used (TAA, TAG, AGG, AGA, and T-). The size of PCGs is the same in both mitogenomes, except for ND6 that has one codon less in the Yellow-billed Chough. All the tRNAs can fold into a typical cloverleaf secondary structure. These mitogenomic data can be of great value in complementing forthcoming approaches on molecular ecology, comparative and functional genomics.

Deaths of individuals aged 16-24 years in the UK after using mephedrone.

OBJECTIVE: Mephedrone is a stimulant drug chemically related to amphetamine, with effects similar to those of amphetamine and cocaine. This study aims to analyse fatalities following ingestion of mephedrone in the UK amongst 16- to 24-year-olds in 2009-2013, providing an update on data presented at the 2nd International Conference on Novel Psychoactive Substances. METHODS: A literature search was undertaken to identify published information on pharmacology, toxicity and fatalities associated with mephedrone. Fatalities involving mephedrone were extracted from the National Programme on Substance Abuse Deaths database, which receives information on drug-related deaths from coroners in the UK and Islands and other data suppliers. Selection criteria are as follows: deceased aged 16-24 years at time of death and mephedrone directly implicated in the cause of death and/or mentioned in the coroner's verdict. RESULTS: Thirty cases met the study criteria, and when known, all were of White ethnicity, most (85%) had a history of drug use and 73% were male. Two-thirds (63%) were accidental poisonings. Mephedrone was used with other substances in most cases (87%); other substances were implicated in 60% of deaths. CONCLUSIONS: Mephedrone use can have potentially fatal consequences, especially in combination with other substances. Deaths from its use in the 16-24 years' age group continue to occur in the UK, despite it being a controlled drug. Health professionals and potential consumers should be alert to this risk.

Genome sequence of the model sulfate reducer Desulfovibrio gigas: a comparative analysis within the Desulfovibrio genus.

Desulfovibrio gigas is a model organism of sulfate-reducing bacteria of which energy metabolism and stress response have been extensively studied. The complete genomic context of this organism was however, not yet available. The sequencing of the D. gigas genome provides insights into the integrated network of energy conserving complexes and structures present in this bacterium. Comparison with genomes of other Desulfovibrio spp. reveals the presence of two different CRISPR/Cas systems in D. gigas. Phylogenetic analysis using conserved protein sequences (encoded by rpoB and gyrB) indicates two main groups of Desulfovibrio spp, being D. gigas more closely related to D. vulgaris and D. desulfuricans strains. Gene duplications were found such as those encoding fumarate reductase, formate dehydrogenase, and superoxide dismutase. Complexes not yet described within Desulfovibrio genus were identified: Mnh complex, a v-type ATP-synthase as well as genes encoding the MinCDE system that could be responsible for the larger size of D. gigas when compared to other members of the genus. A low number of hydrogenases and the absence of the codh/acs and pfl genes, both present in D. vulgaris strains, indicate that intermediate cycling mechanisms may contribute substantially less to the energy gain in D. gigas compared to other Desulfovibrio spp. This might be compensated by the presence of other unique genomic arrangements of complexes such as the Rnf and the Hdr/Flox, or by the presence of NAD(P)H related complexes, like the Nuo, NfnAB or Mnh.

Anti-Aß-MAb and dually decorated nanoliposomes: effect of Aß1-42 peptides on interaction with hCMEC/D3 cells.

Anti-Aß-MAb (Aß-MAb)-decorated immunoliposomes (LIP) and dually decorated ones (dd-LIP) with OX-26 and Aß-MAb were constructed. In both cases, the biotin-streptavidin ligation method was applied. All LIP types were characterized for size distribution, zeta potential, and integrity during incubation with serum proteins. Uptake and transcytosis of both LIP types and control vesicles by human brain endothelial hCMEC/D3 cells were measured. All LIP types had mean diameters below 150-200 nm and low polydispersity. Aß-MAb-LIP uptake was higher than control PEGylated liposomes, while uptake of dd-LIP was similar to that of OX-26-LIP. Aß-MAb-LIP and dd-LIP uptake increased significantly when cells were pre-incubated with Aß1-42 peptides; OX-26-LIP uptake was not modulated. Transcytosis of Aß-MAb-LIP through monolayers was 2.5 times higher when monolayers were pre-incubated with Aß1-42. Transport of both probes, FITC-dextran and rhodamine-lipid, was equivalent, indicating that Aß-MAb-LIP are transferred intact through the BBB model. The Aß peptide-induced increase in binding (and transport) is regulated by the membrane receptors for Aß1-42 peptides (RAGE), as proven after blocking RAGE by a specific MAb. Aß1-42 peptides did not modulate the barrier tightness and integrity, as determined by transendothelial resistance and Lucifer Yellow permeability. Additionally, hCMEC/D3 cell viability was not affected by Aß peptides or by Aß-MAb-LIP.

The binding affinity of anti-Aß1-42 MAb-decorated nanoliposomes to Aß1-42 peptides in vitro and to amyloid deposits in post-mortem tissue.

Amyloid ß (Aß) aggregates are considered as possible targets for therapy and/or diagnosis of Alzheimer disease (AD), and nanoparticles functionalized with Aß-specific ligands are considered promising vehicles for imaging probes and therapeutic agents. Herein, we characterized the binding properties of nanoliposomes decorated with an anti-Aß monoclonal antibody (Aß-MAb). The Aß-MAb was obtained in mice by immunization with Aß antigen followed by hybridoma fusion. Surface Plasmon Resonance (SPR) studies confirmed the very high affinity of purified Aß-MAb for both Aß monomers and fibrils (K(D) = 0.08 and 0.13 nm, respectively). The affinity of the biotinylated Aß-MAb, used thereafter for liposome decoration, was lower although still in the low nanomolar range (K(D) = 2.1 and 1.6 nm, respectively). Biotin-streptavidin ligation method was used to decorate nanoliposomes with Aß-MAb, at different densities. IgG-decorated liposomes were generated by the same methodology, as control. Vesicles were monodisperse with mean diameters 124-134 nm and demonstrated good colloidal stability and integrity when incubated with serum proteins. When studied by SPR, Aß-MAb-liposomes, but not IgG-liposomes, markedly bound to Aß monomers and fibrils, immobilized on the chip. K(D) values (calculated on Aß-MAb content) were about 0.5 and 2 nm with liposomes at high and low Aß-MAb density, respectively. Aß-MAb-liposome binding to Aß fibrils was additionally confirmed by ultracentrifugation technique, in which interactions occur in solution under physiological conditions. Moreover, Aß-MAb-liposomes bound amyloid deposits in post-mortem AD brain samples, confirming the potential of these nanoparticles for the diagnosis and therapy of AD.

Retrospective analysis of clinical yeast isolates in a hospital in the centre of Portugal: spectrum and revision of the identification procedures.

Abstract We conducted a four-year (2003-2006) retrospective study of yeasts recovered in a hospital laboratory in the centre of Portugal to evaluate the epidemiology of yeast infections. Clinical isolates and data were gathered from 751 patients corresponding to 906 episodes of yeast infection. The isolates were first identified using classical and commercial methods, routinely employed at the hospital laboratory. We then re-identified the same isolates using RFLP of the ITS 5.8S rRNA gene and sequence of the D1/D2 domain of the 26S rRNA gene. Candida parapsilosis isolates were re-identified using the Ban I digestion of the SADH gene. C. albicans was the most frequently isolated of the yeasts found in the analysed specimens, with an overall incidence of 69.6% and then in decreasing order, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei. C. parapsilosis was most frequently recovered from younger patients, decreasing with age, while C. glabrata occurrence increased with age. We found an increased number of cases of fungemia per 100,000 people per year, reaching a maximum of 4.4 during 2006.

A novel heterozygous missense mutation in the UMOD gene responsible for Familial Juvenile Hyperuricemic Nephropathy.

BACKGROUND: Familial Juvenile Hyperuricemic Nephropathy is an autosomal dominant nephropathy, characterized by decreased urate excretion and progressive interstitial nephritis. Mutations in the uromodulin coding UMOD gene have been found responsible for the disease in some families. CASE PRESENTATION: We here describe a novel heterozygous p.K307T mutation in an affected female with hyperuricemia, renal cysts and renal failure. The proband's only son is also affected and the mutation was found to segregate with the disease. CONCLUSIONS: This mutation is the fourth reported in exon 5. Initial studies identified a mutation clustering in exon 4 and it has been recommended that sequencing this exon alone should be the first diagnostic test in patients with chronic interstitial nephritis with gout or hyperuricemia. However, regarding the increasing number of mutations being reported in exon 5, we now suggest that sequencing exon 5 should also be performed.

Novel compound heterozygous mutations in SLC5A2 are responsible for autosomal recessive renal glucosuria.

Familial renal glucosuria is an inherited renal tubular disorder. A homozygous nonsense mutation in the SLC5A2 gene, encoding the sodium/glucose co-transporter SGLT2, has recently been identified in an affected child of consanguineous parents. We now report novel compound heterozygous mutations in the son of non-consanguineous parents. One allele has a p.Q167fsX186 mutation, which is expected to produce a truncated protein, and the other a p.N654S mutation involving a highly conserved residue. These findings confirm that mutations in the SLC5A2 gene are responsible for recessive renal glucosuria.