The challenge of COVID-19 for a Clinical Microbiology Department.

OBJECTIVES: To quantify the workload and cost overload that the COVID-19 pandemic has meant for a Clinical Microbiology laboratory in a real-life scenario. METHODS: We compared the number of samples received, their distribution, the human resources, and the budget of a Microbiology laboratory in the COVID pandemic (March-December 2020) with the same months of the previous year. RESULTS: the total number of samples processed in the Clinical Microbiology laboratory in March to December 2020 increased 96.70% with respect to 2019 (from 246,060 to 483,993 samples), reflecting an increment of 127.50% when expressed as samples/1000 admissions (from 6057 to 13,780). The increase in workload was mainly at the expense of the virology (+2058%) and serology (+86%) areas. Despite additional personnel hiring, the samples processed per technician increased 12.5%. The extra cost attributed to Microbiology amounts to 6,616,511 euros (114.8%). CONCLUSIONS: This is the first study to provide quantitative figures about workload and cost increase caused by the COVID-19 in a Microbiology laboratory.

Evaluation of the Alfred AST® system for rapid antimicrobial susceptibility testing directly from positive blood cultures.

To assess the concordance of antimicrobial susceptibility testing results obtained by the Alfred AST® system performed directly from positive blood cultures in comparison with the standard susceptibility test results performed from isolated colonies by an automated broth microdilution method and to determine the applicability of Alfred AST® system in the routine of our blood culture laboratory. This system is based on the detection of growth by turbidimetry through a technology based on light scattering. Antimicrobial susceptibility testing was performed directly from positive bottles by the Alfred AST® system (Alifax, Padova, Italy). The broth microdilution method (MicroScan, Beckman Coulter, CA, USA) performed to the isolates was considered the standard for comparison. We evaluated 115 significant episodes of bacteremia produced by 51 Gram-negative Enterobacterales, 8 Pseudomonas spp., 2 non-fermenting Gram-negative rods, 7 Staphylococcus aureus, 23 coagulase-negative Staphylococcus, 12 Enterococcus spp., and 12 Streptococcus spp. We performed 828 susceptibility determinations with a categorical agreement with the standard method of 97.1%. Only 24 errors (2.9%) were detected. It should be pointed out that for staphylococci and glycopeptides the correlation was only 87% and for non-fermenting Gram-negative rods and piperacillin/tazobactam was only 88.9%. Time to get antibiogram results by Alfred AST® system was 5 versus 48 h for the standard microdilution method from the isolated colonies. The Alfred AST® system is a useful and rapid method to obtain antimicrobial susceptibility results within the same work shift after blood culture positivity.

Evaluation of combined use of the MALDI-TOF and GenomEra MRSA/SA assay for the direct detection of methicillin resistance in Staphylococcus aureus from positive blood culture bottles / Evaluación del uso combinado de ensayo de MALDI-TOF y Genomera MRSA/SA para la detección directa de la resistencia a la meticilina en Staphylococcus aureus de botellas de hemocultivo positivo

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Design of a homogeneous multifunctional supported lipid membrane on layer-by-layer coated microcarriers.

Key challenges in the development of drug delivery systems are the prevention of serum compartment interaction and the targeted delivery of the cargo. Layer-by-Layer microcarriers offer many advantages due to various options in drug assembly and multifunctional design. Surface modification with a supported lipid membrane enhances biocompatibility, drug protection ability, and specific functionality. However, the integration of functionalized lipids strongly influences the membrane formation and is often accompanied by submicrometer irregularities: The accessibility of underlying polymers to serum components may change the carrier's properties and enhances the susceptibility to opsonization. Therefore, the formation of a tightly assembled multifunctional lipid membrane has been emphasized. A phosphatidylserine/phosphatidylcholine (POPS/POPC) bilayer equipped with phosphatidylethanolamine-polyethylene glycol-biotin (PE-PEG-Biotin) was used to facilitate a biotin/streptavidin binding site for a variable attachment of an additional function, such as antibodies for specific targeting. Thus, a prefunctionalized carrier where only the outer functionality needs to be replaced without disturbing the underlying structure could be created.

Study of KRAS new predictive marker in a clinical laboratory.

BACKGROUND: The presence of somatic mutations in the KRAS gene has been identified as a reliable strong negative predictor for the response to targeting the epidermal growth factor receptor (EGFR), in patients with metastatic colorectal cancer and the use of anti-EGFR monoclonal antibodies such as Cetuximab and Panitumumab is now restricted to patients with no detectable KRAS mutations. Between 30 and 40 % of colorectal cancers contain a mutated KRAS oncogene. The aim of this study was to evaluate concordance between three methods to analyze KRAS mutational status in regard to clinical testing. METHODS: We analyzed KRAS mutations in codons 12 and 13 of exon 2 in one hundred formalin-fixed paraffin-embedded (FFPE) colorectal cancer samples by three different methods: Direct Sequencing and two commercial kits on allele-specific oligonucleotide hybridization (KRAS StripAssay, Vienna Lab.) and Amplification Refractory Mutation System/Scorpions (ARMS/S; TheraScreen KRAS Mutation kit DxS) based on q-PCR. RESULTS: We have found similar frequencies of KRAS mutations by TheraScreen and Strip-Assay (44 and 48 %), with a κ value of 0.90, indicating almost perfect agreement between methods. The frequency by direct sequencing was much lower (26 %) and the κ values were 0.67 (compared to TheraScreen) and 0.57 (compared to Strip-Assay) indicating low sensitivity. CONCLUSIONS: On analyzing KRAS mutation in FFPE tumor samples, direct sequencing sensitivity is too low to be used in a clinical setting. Choosing between ARMS/S; TheraScreen KRAS Mutation kit DxS and KRAS StripAssay, Vienna Lab, will depend on laboratory facilities and expertise.

Analysis of the oxidative damage repair genes NUDT1, OGG1, and MUTYH in patients from mismatch repair proficient HNPCC families (MSS-HNPCC).

PURPOSE: Several studies have described molecular differences between microsatellite stable hereditary nonpolyposis colorectal cancer (MSS-HNPCC) and microsatellite unstable Lynch syndrome tumors (MSI-HNPCC). These differences highlight the possibility that other instability forms could explain cancer susceptibility in this group of families. The base excision repair (BER) pathway is the major DNA repair pathway for oxidative DNA damage. A defect in this pathway can result in DNA transversion mutations and a subsequent increased cancer risk. Mutations in MUTYH have been associated with increased colorectal cancer (CRC) risk while no association has been described for OGG1 or NUDT1. EXPERIMENTAL DESIGN: We performed mutational screening of the three genes involved in defense against oxidative DNA damage in a set of 42 MSS-HNPCC families. RESULTS: Eight rare variants and 5 frequent variants were found in MSS-HNPCC patients. All variants were previously described by other authors except variant c.285C>T in OGG1. Segregation studies were done and in silico programs were used to estimate the level of amino acid conservation, protein damage prediction, and possible splicing alterations. Variants OGG1 c.137G>A; MUTYH c.1187G>A were detected in Amsterdam I families and cosegregate with cancer. Analysis of OGG1 c.137G>A transcripts showed an inactivation of the splicing donor of exon 1. CONCLUSIONS: Two rare variants (OGG1 c.137G>A; MUTYH c.1187G>A) and one common polymorphism (NUDT1 c.426C>T) were associated with CRC risk. We show that the BER pathway can play a significant role in a number of MSS-HNPCC colorectal cancers. More studies could be of interest in order to gain further understanding of yet unexplained CRC susceptibility cases.

Microtubes self-assembled from a cholesterol-modified nucleoside.

We describe the formation of lipid microtubes from a novel cholesterol-modified nucleoside in binary mixture with phospholipids. Stable cylindrical structures with an outer diameter of 2-3 microm and a length of 20-40 microm were formed. By varying the preparation conditions, thinner tubules with nanometre-scale diameters could also be obtained.

Lipid membranes carrying lipophilic cholesterol-based oligonucleotides--characterization and application on layer-by-layer coated particles.

Cholesterol-based lipophilic oligonucleotides incorporated into lipid membranes were studied using solid-state NMR, differential scanning calorimetry, and fluorescence methods. Lipophilic oligonucleotides can be used to build nanotechnological structures on membrane surfaces, taking advantage of the specific Watson-Crick base pairing. We used a cholesteryl-TEG anchor first described by Pfeiffer and Hook (J. Am. Chem. Soc. 2004, 126, 10224-10225). The cholesterol-based anchor molecules were found to incorporate well into lipid membranes without disturbing the bilayer structure and dynamics. In contrast to cholesterol, which is known to induce significant condensation of the membrane lipids, the cholesteryl-TEG anchor does not display this property. When the cholesteryl-TEG moiety was covalently bound to an oligonucleotide, the resulting lipophilic DNA molecules inserted spontaneously into lipid membranes without altering their structure. The duplex formed by two complementary cholesteryl-TEG oligonucleotides had increased thermodynamic stability compared to the same oligonucleotides without the anchor, both in solution and incorporated into lipid membranes. Since the cholesteryl-TEG anchor lacks the characteristic properties of cholesterol, oligonucleotides modified with this anchor are equally distributed between liquid-disordered and liquid-ordered domains in "raft" forming membranes. As an example of an application of these lipophilic oligonucleotides, cholesteryl-TEG-DNA was incorporated into supported lipid bilayers formed on polyelectrolyte-coated silica microparticles. The modified oligonucleotides were stably inserted into the lipid membrane and retained their recognition properties, therefore enabling further functionalization of the particles.

Efficiency of a bienzyme sequential reaction system immobilized on polyelectrolyte multilayer-coated colloids.

We assembled multilayer films of glucose oxidase (GOx) and horseradish peroxidase (HRP) coimmobilized together with polyelectrolyte layers on the surface of silica microparticles. The influence of different polyelectrolyte combinations on the immobilization and functionality of the enzymes was examined for several multilayer configurations. Precomplexation of the enzymes with a polyvinylpyridine-based polyamine allowed the stable adsorption of enzyme layers without affecting their catalytic activity. The efficiency of the sequential reaction between GOx and HRP on the surface of the colloids was quantitatively analyzed and rationalized in terms of the kinetic parameters of both enzymes and the reaction-diffusion kinetics of the system. In the optimized configuration, with GOx and HRP coimmobilized in the same layer, the overall rate of hydrogen peroxide conversion was around 2.5 times higher than for GOx and HRP in separate layers or for equivalent amounts of both enzymes free in solution.