Comparison of molecular typing methods for Candida albicans.

This is the published version. Copyright 1992 American Society for Microbiology.Four molecular approaches to determining the types of Candida albicans strains were compared. The strains used were those whose repeated DNA (ribosomal and mitochondrial) EcoRI restriction fragment length polymorphisms (RFLP) were determined by Stevens et al. (D. A. Stevens, F. C. Odds, and S. Scherer, Rev. Infect. Dis. 12:258-266, 1990). Scherer and Stevens (S. Scherer and D. A. Stevens, Proc. Natl. Acad. Sci. USA 85:1452-1456, 1988) used the same strains to examine the Southern blots of genomic EcoRI digests probed with the repeated sequence 27A. The results of these investigators were compared with determinations of RFLPs generated from repeated DNA by the enzyme HinfI and examination of the karyotypes of strains under two sets of conditions, one for the smaller chromosomes and one for the larger ones. Analysis of RFLPs of repeated DNA is most convenient but shows the lowest degree of resolution. Use of the repeated sequence and use of karyotype have very high resolution, but the former method is more convenient than the latter. HinfI digestion is more sensitive than EcoRI digestion but equally convenient. By using all four methods, separate types were identified for 18 of the 20 strains examined

An Evaluation of Hepatitis E Virus Molecular Typing Methods

Background: Hepatitis E virus (HEV) is a major cause of acute viral hepatitis. Better understanding of HEV subtypes involved in hepatitis E infections is essential. Investigation of sources and routes of transmission and the identification of potential clusters/outbreaks rely upon molecular typing of viral strains. A study was carried out to evaluate the ability of laboratories to undertake molecular typing with genotype and subtype determination. Methods: A blinded panel of 11 different Orthohepevirus A strains was distributed to 28 laboratories performing HEV sequence analysis. Laboratories used their routine HEV sequencing and genotyping methods. Results: Results were returned by 25 laboratories. Overall, 93% samples were assigned to the correct genotype and 81% were assigned to the correct subtype. Fragments amplified for typing ranged in size and the sequencing assays targeted both the structural and non-structural protein-coding regions. There was good agreement between the reported sequences where methods targeted overlapping fragments. In some cases, incorrect genotypes/subtypes were reported, including those not contained in the panel, and in one case, a genotype was reported for a blinded control sample containing Zika virus; collectively these data indicate contamination problems. Conclusions: In general, identification of genotypes was good; however, in a small number of cases, there was a failure to generate sequences from some of the samples. There was generally broad agreement between the use of online typing tools such as the one provided by HEVnet and curated lists of published HEV reference sequences; however, going forward harmonization between these resources is essential.This work was supported by a service contract ECD.9621 from European Centre for Disease Prevention and Control. N. Nasheri and J. Harlow, Health Canada internal funds: Internal funding was used to perform viral detection and genotyping. K. Schilling-Loeffler, R. Johne, and V.M. Corman, grant of the German Federal Ministry of Health with regard to a decision of the German Bundestag by the Federal Government (CHED project grant no. ZMVI1-2518FSB705) to institution; G. La Rosa and E. Suffredini, Italian Ministry of Health; I.L.A. Boxman, R.A.M. Dirks, Dutch Food and Consumer Safety Authority; B. Hogema, Sanquin; A. Avellon, Diasorin Iberica, Abbott; G. Sanchez and E.C. Ferrando, MICIU project AGL2017-82909. The study was funded by ECDC with PEI receiving these funds to cover sample preparation, pre-testing and transport costs.S

Update on commonly used molecular typing methods for Clostridioides Difficile

This review aims to provide a comprehensive overview of the significant Clostridioides difficile molecular typing techniques currently employed in research and medical communities. The main objectives of this review are to describe the key molecular typing methods utilized in C. difficile studies and to highlight the epidemiological characteristics of the most prevalent strains on a global scale. Geographically distinct regions exhibit distinct strain types of C. difficile, with notable concordance observed among various typing methodologies. The advantages that next-generation sequencing (NGS) offers has changed epidemiology research, enabling high-resolution genomic analyses of this pathogen. NGS platforms offer an unprecedented opportunity to explore the genetic intricacies and evolutionary trajectories of C. difficile strains. It is relevant to acknowledge that novel routes of transmission are continually being unveiled and warrant further investigation, particularly in the context of zoonotic implications and environmental contamination

A comparative genomic framework for the in silico design and assessment of molecular typing methods using whole-genome sequence data with application to Listeria monocytogenes

xiii, 100 leaves : ill. ; 29 cmAlthough increased genome sequencing e orts have increased our understanding of genomic variability within many bacterial species, there has been limited application of this knowledge towards assessing current molecular typing methods and developing novel molecular typing methods. This thesis reports a novel in silico comparative genomic framework where the performance of typing methods is assessed on the basis of the discriminatory power of the method as well as the concordance of the method with a whole-genome phylogeny. Using this framework, we designed a comparative genomic ngerprinting (CGF) assay for Listeria monocytogenes through optimized molecular marker selection. In silico validation and assessment of the CGF assay against two other molecular typing methods for L. monocytogenes (multilocus sequence typing (MLST) and multiple virulence locus sequence typing (MVLST)) revealed that the CGF assay had better performance than these typing methods. Hence, optimized molecular marker selection can be used to produce highly discriminatory assays with high concordance to whole-genome phylogenies. The framework described in this thesis can be used to assess current molecular typing methods against whole-genome phylogenies and design the next generation of high-performance molecular typing methods from whole-genome sequence data

Overview of molecular typing methods for outbreak detection and epidemiological surveillance

Typing methods for discriminating different bacterial isolates of the same species are essential epidemiological tools in infection prevention and control. Traditional typing systems based on phenotypes, such as serotype, biotype, phage-type, or antibiogram, have been used for many years. However, more recent methods that examine the relatedness of isolates at a molecular level have revolutionised our ability to differentiate among bacterial types and subtypes. Importantly, the development of molecular methods has provided new tools for enhanced surveillance and outbreak detection. This has resulted in better implementation of rational infection control programmes and efficient allocation of resources across Europe. The emergence of benchtop sequencers using next generation sequencing technology makes bacterial whole genome sequencing (WGS) feasible even in small research and clinical laboratories. WGS has already been used for the characterisation of bacterial isolates in several large outbreaks in Europe and, in the near future, is likely to replace currently used typing methodologies due to its ultimate resolution. However, WGS is still too laborious and time-consuming to obtain useful data in routine surveillance. Also, a largely unresolved question is how genome sequences must be examined for epidemiological characterisation. In the coming years, the lessons learnt from currently used molecular methods will allow us to condense the WGS data into epidemiologically useful information. On this basis, we have reviewed current and new molecular typing methods for outbreak detection and epidemiological surveillance of bacterial pathogens in clinical practice, aiming to give an overview of their specific advantages and disadvantages

Genotyping and antifungal susceptibility of Dipodascus capitatus isolated in a neonatal intensive care unit of a sicilian hospital

In August 2015, Dipodascus capitatus was isolated from two patients admitted to the neonatal intensive care unit. Nosocomial acquisition of the fungus was suspected and epidemiological studies were undertaken. The patients were simultaneously hospitalized, and the comparison of the two isolates by two independent molecular typing methods have confirmed clonal dissemination of a single strain of D. capitatus. Antimicrobial susceptibility testing was useful for identifying the appropriated antifungal therapy in micafungin. To our knowledge these are the first described cases of neonatal D. capitatus infection and also the first report of successful treatment by micafungin.In August 2015, Dipodascus capitatus was isolated from two patients admitted to the neonatal intensive care unit. Nosocomial acquisition of the fungus was suspected and epidemiological studies were undertaken. The patients were simultaneously hospitalized, and the comparison of the two isolates by two independent molecular typing methods have confirmed clonal dissemination of a single strain of D. capitatus. Antimicrobial susceptibility testing was useful for identifying the appropriated antifungal therapy in micafungin. To our knowledge these are the first described cases of neonatal D. capitatus infection and also the first report of successful treatment by micafungin

Application of Molecular Typing Methods to the Study of Medically Relevant Gram-Positive Cocci

The development of molecular genotyping methods has been a landmark in the possibility of classifying microorganisms below the species level. The ability to differentiate efficiently related bacterial isolates is essential for the control of infectious diseases and has become a necessary technology for clinical microbiology laboratories. The aim of this chapter is to provide an overview of the methods available for analyzing bacterial isolates, focusing on those methods employed for typing Streptococcus pneumoniae and Staphylococcus aureus. Different molecular approaches have been used to better understand the epidemiology of these medically relevant gram-positive cocci.Fil: Bonofiglio, Laura. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gardella, Noella Mariel. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; ArgentinaFil: Mollerach, Marta Eugenia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

‘Next-Generation’ surveillance: an epidemiologists’ perspective on the use of molecular information in food safety and animal health decision-making

Advances in the availability and affordability of molecular and genomic data are transforming human health care. Surveillance aimed at supporting and improving food safety and animal health is likely to undergo a similar transformation. We propose a definition of ‘molecular surveillance’ in this context and argue that molecular data are an adjunct to rather than a substitute for sound epidemiological study and surveillance design. Specific considerations with regard to sample collection are raised, as is the importance of the relation between the molecular clock speed of genetic markers and the spatiotemporal scale of the surveillance activity, which can be control- or strategy-focused. Development of standards for study design and assessment of molecular surveillance system attributes is needed, together with development of an interdisciplinary skills base covering both molecular and epidemiological principles

Application of four molecular typing methods for analysis of Mycobacterium fortuitum group strains causing post-mammaplasty infections

A cluster of cases of post-augmentation mammaplasty surgical site infections occurred between 2002 and 2004 in Campinas, in the southern region of Brazil. Rapidly growing mycobacteria were isolated from samples from 12 patients. Eleven isolates were identified as Mycobacterium fortuitum and one as Mycobacterium porcinum by PCR-restriction digestion of the hsp65 gene. These 12 isolates, plus six additional M. fortuitum isolates from non-related patients, were typed by pulsed-field gel electrophoresis (PFGE) and three PCR-based techniques: 16S-23S rRNA internal transcribed spacer (ITS) genotyping; randomly amplified polymorphic DNA (RAPD) PCR; and enterobacterial repetitive intergenic consensus (ERIC) PCR. Four novel M. fortuitum allelic variants were identified by restriction analysis of the ITS fragment. One major cluster, comprising six M. fortuitum isolates, and a second cluster of two isolates, were identified by the four methods. RAPD-PCR and ITS genotyping were less discriminative than ERIC-PCR. ERIC-PCR was comparable to PFGE as a valuable complementary tool for investigation of this type of outbreak.Universidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, SP, BrazilFleury Ctr Med Diagnost, São Paulo, BrazilInst Adolfo Lutz Registro, São Paulo, BrazilInst Vozza Med & Diagnose LTDA, Campinas, BrazilCtr Vigilancia Epidemiol Prof Alexandre Vranjac, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, SP, BrazilWeb of Scienc

Molecular characterisation of Vibrio parahaemolyticus carrying tdh and trh genes using ERIC-, RAPD- and BOX-PCR on local Malaysia bloody clam and Lala

Molecular typing methods have been widely applied for many purposes. In this study, such methods were adopted as DNA fingerprinting tools to determine the origin and divergence of virulent Vibrio parahaemolyticus strains found in local seafood. Although not all strain carry virulent tdh and trh gene, increasing prevalence demands an effective fingerprinting scheme which can constantly monitor and trace the sources of such emerging food pathogens. By using ERIC-, RAPD-, and BOX-PCR methods, 33 Vibrio parahaemolyticus isolates from local Malaysia bloody clam (Anadara granosa) and Lala (Orbicularia orbiculata) with confirmed presence of tdh and trh gene were characterised, followed by determination of clonal relatedness among virulent strains using cluster analysis and discriminatory index. This study also involved application of Immunomagnetic Separation (IMS) Method which significantly improved the specificity of strain isolation. Cluster analysis using Unweighted Pair Group Mathematical Averaging (UPGMA) and Dice Coefficient shown clustering according to isolation food source, IMS level and haemolysin gene possessed. Nevertheless, different DNA fingerprinting methods generated different clustering at different similarity cut-off percentage, regardless as individual or as composite dendrograms. ERIC- and RAPD-PCR composite fingerprinting relatively shown the highest discriminatory index at following similarity cut- off percentage: 0.68 at 50%; 0.83 at 65%; and 0.93 at 75%. Discriminatory power increased with similarity cut-off percentage. However, result also suggested that BOX-PCR might be an effective fingerprinting tool, as it generated three clusters with no single-colony isolate at 70% similarity cut-off. This study not only achieved its objective to determine clonal relatedness among virulent strains from local seafood via characterisation, but also speculated the best possible combination of molecular typing methods to effectively do s