Intercenter reproducibility of binary typing for Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus typing methods: which should be the international standard?

Antimicrobial Resistance in the Intensive Care Unit: Mechanisms, Epidemiology, and Management of Specific Resistant Pathogens

Leishmaniasis, caused by Leishmania protozoan parasites transmitted by Phlebotomine sand flies, is a significant public health concern in the Mediterranean basin. Effective monitoring of Leishmania-infected sand flies requires standardized tools for comparing their distribution and infection prevalence. Consistent quantitative real-time PCR (qPCR) parameters and efficient DNA extraction protocols are crucial for reliable results over time and across regions. However, the absence of standardized technical recommendations for Leishmania DNA detection hinders effective surveillance. This study aimed to compare different DNA extraction protocols and conduct a qPCR-based External Quality Assessment (EQA) through a multicenter study involving nine reference laboratories, with a focus on optimizing Leishmania DNA detection in sand fly. EQA samples consisted of Leishmania infantum and L. major species, at concentrations ranging from 101 to 104 parasites/mL. All but one center detected all concentrations, demonstrating strong diagnostic proficiency. The ability to detect low concentrations highlighted the robustness of the qPCR assay used, though variations in Cq values indicated differences in sensitivity related to technical capabilities or DNA extraction kit performance. A comparative analysis of seven DNA extraction methods identified the EZ1 DSP Virus Kit and QIAamp DNA mini-kit as the most efficient, supporting their use in standardized protocols. The study also assessed the effects of lyophilization and shipment conditions, showing no significant compromise in Leishmania detection despite slight variations in Cq values. Experimentally infected sand flies were included to simulate field conditions, and all centers successfully detected positive samples with varying Cq values, probably reflecting differences in infection load. This study emphasizes the importance of standardized DNA extraction protocols and continuous quality assurance for accurate Leishmania DNA detection. The results highlight the superior performance of certain extraction kits and the need for ongoing technical training, essential for reliable leishmaniasis surveillance, particularly in field settings with low infection densities.

Methicillin-resistant Staphylococcus aureus (MRSA) is a universal threat. Once being well established in the healthcare setting, MRSA has undergone various epidemiological changes. This includes the emergence of more aggressive community-acquired MRSA (CA-MRSA) and the occurrence of MRSA which have their origin in animal breeding, called livestock-associated MRSA (LA-MRSA). Emergence of new clones as well as changes in the occurrence of some clonal lineages also describes the fluctuating dynamic within the MRSA family. There is paucity of data describing the possible impact of the COVID-19 pandemic on the MRSA dynamics. The aim of the study was the analysis of MRSA isolates in a three-year time period, including the pre-COVID-19 years 2018 and 2019 and the first year of the pandemic 2020. The analysis includes prevalence determination, antibiotic susceptibility testing, spa typing, and detection of genes encoding the PVL toxin. The MRSA rate remained constant throughout the study period. In terms of a dynamic within the MRSA family, only a few significant changes could be observed, but all except one occurred before the start of the COVID-19 pandemic. In summary, there was no significant impact of the COVID-19 pandemic on MRSA in Austria.

A number of high-resolution molecular typing systems have been developed in recent years. Their availability raises the new issues of selecting the method (s) best suited for a particular purpose and interpreting and communicating typing results. Most of the currently available methods are comparative only: they allow testing of a sample of isolates for delineation of those closely related from those markedly different in genomic backgrounds. This approach is adequate for outbreak investigation, allowing determination of clonal spread in a microenvironment and identification of the source of infection. Comparative methods with sufficient resolution for most pathogens include restriction fragment-length polymorphism (RFLP), pulsed-field gel electrophoresis (PFGE), and arbitrarily primed and randomly amplified polymorphic DNA-polymerase chain reaction (PCR) analysis. For surveillance systems, monitoring clonal spread and prevalence in populations over extended periods of time requires library typing systems. These must be standardized, must have a high throughput, and must use a uniform nomenclature. Promising or validated methods include serotyping, insertion sequence fingerprinting, ribotyping, PFGE, amplified fragment-length polymorphism (AFLP), infrequent-restriction-site amplification PCR, interrepetitive element PCR typing (rep-PCR) and PCR-RFLP of polymorphic loci. PCR methods generating arrays of size-specific amplicons (AFLP, rep-PCR) can be more reproducibly analyzed by using denaturing polyacrylamide gel or capillary electrophoresis with automated laser detection. Binary probe typing systems appear optimal and should be enhanced further through use of DNA chip technology. In these systems, amplification of polymorphic regions is followed by solid-phase hybridization with a reference panel of sequence-variant specific probes. The resulting binary type results allow determination of reproducible, numeric profiles. However, interpretation and nomenclature of typing results for large-scale surveillance purposes still require a better understanding of population structure and microevolution of most microbial pathogens.

A number of high-resolution molecular typing systems have been developed in recent years. Their availability raises the new issues of selecting the method (s) best suited for a particular purpose and interpreting and communicating typing results. Most of the currently available methods are comparative only: they allow testing of a sample of isolates for delineation of those closely related from those markedly different in genomic backgrounds. This approach is adequate for outbreak investigation, allowing determination of clonal spread in a microenvironment and identification of the source of infection. Comparative methods with sufficient resolution for most pathogens include restriction fragment-length polymorphism (RFLP), pulsed-field gel electrophoresis (PFGE), and arbitrarily primed and randomly amplified polymorphic DNA-polymerase chain reaction (PCR) analysis. For surveillance systems, monitoring clonal spread and prevalence in populations over extended periods of time requires library typing systems. These must be standardized, must have a high throughput, and must use a uniform nomenclature. Promising or validated methods include serotyping, insertion sequence fingerprinting, ribotyping, PFGE, amplified fragment-length polymorphism (AFLP), infrequent-restriction-site amplification PCR, interrepetitive element PCR typing (rep-PCR) and PCR-RFLP of polymorphic loci. PCR methods generating arrays of size-specific amplicons (AFLP, rep-PCR) can be more reproducibly analyzed by using denaturing polyacrylamide gel or capillary electrophoresis with automated laser detection. Binary probe typing systems appear optimal and should be enhanced further through use of DNA chip technology. In these systems, amplification of polymorphic regions is followed by solid-phase hybridization with a reference panel of sequence-variant specific probes. The resulting binary type results allow determination of reproducible, numeric profiles. However, interpretation and nomenclature of typing results for large-scale surveillance purposes still require a better understanding of population structure and microevolution of most microbial pathogens.

Antibiotic resistance is a cause of concern worldwide. Community settings are important reservoir of drug resistant microorganisms like Staphylococcus aureus. The present study was to determine the prevalence, phenotypic and genotypic antibiotic resistance pattern of S. aureus isolated from different community settings of university campus. A total of 300 swab samples were collected for isolation of S. aureus from different community settings at university campus of Guru Angad Dev Veterinary and Animal Sciences University and Punjab Agriculture University, Ludhiana, India. Confirmed S. aureus isolates were further subjected to antibiotic sensitivity by Epsilometer test (E-test) and detection of antibiotic resistance genes. The prevalence of S. aureus in the community samples was 12% (36/300). Methicillin Resistant S. aureus (MRSA) contamination among community was 3.33% (10/300). Among S. aureus isolates from community samples 63.8% (23/36) and all the MRSA isolates were multidrug resistant (MDR). Five out of 10 MRSA carried SCCmec type IVa, and 4 were pvl positive gene, therefore, designated as community associated MRSA (CA-MRSA). Phenotypic resistance to antibiotics ciprofloxacin, chloramphenicol, ceftriaxone clindamycin and trimethoprim-sulfamethoxazole was 69.4% (MIC ≥32 μg/ml), 63.9% (MIC 32 μg/ ml), 16.7% (MIC 16–64 μg/ml), 16.7% (MIC 256 μg/ml) and 8.3% (MIC 12–64 μg/ml), respectively. Resistance genes blaZ, mecA, tetK, tetM, ermB and aacA-aphD were present. Presence of MRSA and MDR variant in community settings is a public health concern, as cell phone, offices telephone, computer keyboard and tap faucet are commonly shared or touched by people. Therefore, have potential to disseminate widely, not only in the community settings but also in hospitals environment, complicating treatment.

ABSTRACT: DNA was extracted from single-cultivar of cold-pressed (virgin) unfiltered and cotton-filtered olive oils that were stored at 4 °C for up to a year using different DNA extraction kits and protocols. DNA was amplified using original and nested primers designed on 6 microsatellites loci of the UDO series. The most consistent results in terms of successful single sequence repeat amplifications were achieved using the Qiagen QIAamp DNA stool extraction kit, slightly modified and applied to oil sample amounts as small as 200 μL without any pretreatment. The kit allowed getting polymerase chain reaction (PCR) amplicons visible on gel and scorable peaks at the automatic sequencer for all 6 markers analyzed. Less consistent results were achieved with other kits, such as the Promega Wizard Magnetic DNA Purification System for Food, the LB Link-Biotech ExtMan 50–100 Evolution, the Qiagen Plant Mini kit, and the standard cetyltrimethyl-ammonium bromide-based DNA extraction protocol. The integration in the protocols of further tools, such as the hexane-based phase separation, the addition of water or NaCl solutions to the oil, the precipitation and the use of the pellet, and others, did not result in any substantial use. PCR amplifications that gave low DNA yields were improved by adopting the nested PCR technique, which uses the product of the 1st PCR as a template for a 2nd PCR carried out by means of internal primers. Conclusions are drawn as to the applicability of the method to trace the identity of single-cultivar virgin olive oils. Further work is required to check the sensitivity of the method in determining the varietal composition of blended oils, especially in detecting alleles from cultivars present in only small amounts.

Genetic counseling in direct-to-consumer exome sequencing: a case report.

Tung tree (Vernicia fordii) is a native and oil-producing woody plant in China. The oil is industrially important and promising biodiesel raw material. However, until recently the lack of effective protocols for the extraction of genomic DNA had made DNA-based molecular studies of tung tree difficult. Here, four conventional protocols and one novel protocol were compared for their capacity in isolating DNA from tung tree leaves of different age. Our results showed that all the four conventional protocols could isolate DNA from old leaves, two from matured leaves, but none from young leaves. However, the detectable DNA samples contained many contaminations, leading to overestimation of DNA concentration measured by ultraviolet spectrophotometer, also interfering with the downstream PCR reaction. The novel protocol could produce high-yield and good-quality DNA from tung tree leaves regardless of leaf age. Its key steps were that a single leaf tissue sample could be recycled for DNA extraction for up to four times, and correspondingly four DNA precipitations (termed as the 1st, 2nd, 3rd and 4th DNA sample, respectively) were conducted. All the four DNA samples of a single tissue were good template for PCR reaction. The novel protocol is an effective method for genomic DNA isolation of tung tree.

Novel amperometric genosensor based on peptide nucleic acid (PNA) probes immobilized on carbon nanotubes-screen printed electrodes for the determination of trace levels of non-amplified DNA in genetically modified (GM) soy

Antimicrobial susceptibility and molecular characteristics of methicillin-resistant Staphylococcus aureus in a Japanese secondary care facility.

Soy is a product of great economic importance, since it covers a variety of marketing fields that permeate the human and animal consumption for the processed food industry. Thus, to maximize production and improve product quality, the legume is a frequent target for genetic modification. The concern with the long-term effect of the transgenic product, beyond the consumer's right to know about the product you eat demanded a law regulating the presence of these products in the market. Existing legislation in Brazil indicates that rates of GM products, with more than 1% should be informed on food labels. To ensure compliance with the law are necessary protocols able to identify and quantify the waste. Therefore, this project aims to evaluate the protocols of DNA extraction by CTAB method proposed by Ferreira and Grattapaglia [1] and the method proposed by FERRARI et al [2], as well as detect PCR transgenic residues in processed foods derived from soybeans. After extraction, DNA was quantified by spectrophotometry and absorbance ratio 260/280 nm was analyzed to determine the quality of the samples. Furthermore, the technique was used to eletrofrese agarose gel to compare the quality of DNA samples of soybeans with fresh samples processed soybeans. The protocol for DNA extraction from FERREIRA & GRATTAPLAGLIA was more efficient compared to the yield of DNA extracted from different samples, but in relation to quality of DNA both protocols showed similar results. The detection of residues in transgenic samples showed that the PCR amplified only the lectin gene (endogenous), confirming that the samples contained soy, but did not amplify the exogenous gene, suggesting that the samples did not contain residues of transgenic.

We appreciate the comments by Berg et al. regarding our article. First, the aim of the quality assessment program reported in this study was not to standardize the methodology across the network of laboratories involved but to assess the outcome of laboratories testing clinical material using their laboratory’s in-house standard protocol for determination of HIV-1 tropism using proviral DNA. As MARCH is an international study, the different HIV subtype distribution necessitated having nonstandardized methodologies and, hence, use of different primers required to amplify different clades. We agree that samples had a high degree of variability, with some samples having a very wide range in their false-positive rates (FPRs), and these were solid findings. Our external quality assessment (EQA) samples were derived from real patient samples and deliberately selected to be similar to those recruited to MARCH, i.e., long-term aviremic patients. The use of only clones or other homogeneous samples would therefore not be a true representation of the genetic diversity of real patient samples and would have led to a misrepresentation of the performance of laboratories testing such samples for diagnostics. One could have separately evaluated the two different issues, diversity of samples and diversity of laboratory performance. However, we presented the data in one single examination to assess the overall impact of such material as an international quality assessment program. Furthermore, it was interesting that Berg et al. compared our study to that of Svicher et al. This study used samples from viremic patients (10,000 copies/ml) and tested HIV RNA, which is more likely to deliver uniform results because the circulating virus is likely to reflect the fittest clone/quasispecies circulating in that patient at that time. The DNA in long-term aviremic patients is likely to be far more heterogeneous, reflecting multiple quasispecies of archived virus of varying fitness. We felt very strongly that a clonal panel would not be helpful for the purposes of our program. The observed variability validates the importance of performing this EQA, and these data will be critical for interpreting the results of the MARCH study itself. We acknowledge that triplicate testing is costly and not feasible in all laboratory settings. Nevertheless, we felt triplicate testing was essential, considering both the inherent variability of proviral DNA and the lack of information from rigorously conducted randomized clinical trials, to the clinical relevance of this test. Therefore, we consider it far more important to detect X4 viruses and be conservative in terms of patient safety. Overall, and for the reasons described both in the article and in this response, we do not believe that singlicate testing of a clonal standard would reliably ensure that a patient’s virus would likely respond to the protocol drug, maraviroc. In summary, we feel that Berg et al. have misinterpreted the intent of the EQA program established specifically for MARCH. Proviral DNA, obtained from real patient samples, was associated with a high degree of variability, and the most suitable approach for quality assessment for determination of viral tropism was developed for this study.

Environmental context Arsenic can be found in all environmental compartments in a large number of chemical forms of varying toxicity. We performed an inter-laboratory comparison study focusing on quantitation of some of the most toxic arsenic forms in seven different biological materials and found very good agreement among the submitted results. Certification of the studied materials will provide suitable quality control samples for environmentally relevant concentrations of arsenic in food products and biota. Rationale Arsenic (As) speciation analysis in biological matrices has been performed for several decades; however, there are very few matrix certified reference materials available for the validation of analytical methods. The literature data on the mass fractions of As species in the existing certified reference materials are inconsistent and suggest method extraction dependency. Methodology In the present study, an international round-robin study was organised to identify the possible sources of discrepancies in quantitation of several As species in different matrices of biological reference materials: one plant tissue, three marine and three terrestrial biological tissues. Each participating laboratory was provided with a set of identical calibration standard solutions, and analysed the samples by following a common group extraction method as well as using an in-house protocol. Results The results showed that significant biases can be introduced by insufficient verification of the analyte’s mass fractions in the calibration standard solutions. The choice of extraction method seems to have very little impact on the quantitation of As species in the studied plant and terrestrial biological tissues. However, following a prescribed extraction method led to significant reduction of uncertainties in more complex samples such as marine animal tissues. Discussion Some differences in the mass fractions of As species extracted from marine animal tissues in water (with and without H2O2) were observed and should be further investigated. Despite the variety of extraction methods used, very good agreement between reported mass fractions was achieved and the combined consensus values will be published as certified reference values of As species in the studied materials.