Overview of molecular typing methods for outbreak detection and epidemiological surveillance. Eurosurveillance, Volume. Issue. 2. 4 January 2. Review articles. Overview of molecular typing methods for outbreak detection and epidemiological surveillance. A J Sabat. 1, A Budimir. ![]() ![]() D Nashev. 3, R S. Overview of molecular typing methods for outbreak detection and epidemiological surveillance. Available online: http: //www. View. Article. aspx? CAPTURE STITCH PUBLISH Sign in. Article. Id=2. 03. Date of submission. June 2. 01. 2Typing 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. CCNR is a not-for-profit organization, federally incorporated in 1978. It is dedicated to education and research on all issues related to nuclear energy, whether.
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. Traditional typing systems based on phenotype, such as serotype, biotype, phage- type or antibiogram, have been used for many years. However, the methods that examine the relatedness of isolates at a molecular level have revolutionised our ability to differentiate among bacterial types (or subtypes). The choice of an appropriate molecular typing method (or methods) depends significantly on the problem to solve and the epidemiological context in which the method is going to be used, as well as the time and geographical scale of its use. Importantly, human pathogens of one species can comprise very diverse organisms. Therefore, typing techniques should have excellent typeability to be able to type all the isolates studied . In outbreak investigations, a typing method must have the discriminatory power needed to distinguish all epidemiologically unrelated isolates. Ideally, such a method can discriminate very closely related isolates to reveal person- to- person strain transmission, which is important to develop strategies to prevent further spread. At the same time it must be rapid, inexpensive, highly reproducible, and easy to perform and interpret . When typing is applied for continuous surveillance, the respective method must yield results with adequate stability over time to allow implementation of efficient infection control measures. Moreover, a typing method that is going to be used in international networks should produce data that are portable (i. Additionally, a typing method used for surveillance should rely on an internationally standardised nomenclature, and it should be applicable for a broad range of bacterial species. There should also be procedures in place to check and validate, by using quantifiable internal and external controls, that the typing data are of high quality. A clear advantage for a typing approach is the availability of software that: (i) enables automated quality control of raw typing data, (ii) allows pattern/type assignment, (iii) implements an algorithm for clustering of isolates based on the obtained data, (iv) provides assistance in the detection of outbreaks of infections, and (v) facilitates data management and storage. To date, many different molecular methods for epidemiological characterisation of bacterial isolates have been developed. However, none of them is optimal for all forms of investigation. Thus, a thorough understanding of the advantages and limitations of the available typing methods is of crucial importance for selecting the appropriate approaches to unambiguously define outbreak strains. Here, we present an overview of the typing methods that are currently used in bacterial disease outbreak investigations and active surveillance networks, and we specify their advantages and disadvantages. Importantly, we focus on those methods that have the strongest impact on public health, or for which there is a growing interest in relation to clinical use. Pub. Med database searches. To investigate the impact of typing methods in public health, we first queried the Pub. Med database using a combination of specific keywords to retrieve the relevant articles without any constraints on the time of publication. Furthermore, in order to reveal a growing interest in particular typing methods, we subsequently restrictively searched Pub. Med for articles published between January 2. December 2. 01. 2). We considered a method as a method of growing interest when the number of articles published between January 2. Specifically, an electronic search was conducted using the following combinations of keywords: PFGE . Also, to identify the impact of particular typing methods on outbreak investigations currently conducted, we searched the Pub. Med database with a restriction to articles published between January 2. PFGE . The results of these literature searches have been included in the following sections of this review that address the respective typing methods. Pulsed- field gel electrophoresis Pulsed- field gel electrophoresis (PFGE) has been considered as the . For most bacterial species, the technique was adopted as an epidemiological tool in the 1. Today, it is still the most frequently used approach to characterise bacterial isolates in outbreaks . In total, 1. 83 hits were obtained for the terms . For many years, PFGE has been a primary typing tool to analyse centre- to- centre transmission events, and it has been used successfully in large- scale epidemiological investigations . The success of PFGE results from its excellent discriminatory power and high epidemiological concordance. Moreover, it is a relatively inexpensive approach with excellent typeability and intra- laboratory reproducibility. In the past decade, protocols for PFGE have been standardised and inter- laboratory comparison has been undertaken through several initiatives, such as Pulse. Net . It has also been possible to establish international fingerprinting databases, which allowed fast detection of emerging clones and monitoring of the spread of pathogenic bacterial strains through different regions or countries. To perform PFGE, a highly purified genomic DNA sample is cleaved with a restriction endonuclease that recognises infrequently occurring restriction sites in the genome of the respective bacterial species. The resulting restriction fragments, which are mostly large, can be separated on an agarose gel by 'pulsed- field' electrophoresis in which the orientation of the electric field across the gel is changed periodically. The separated DNA fragments can be visualised on the gel as bands, which form a particular pattern on the gel, the PFGE pattern. For most bacteria PFGE can resolve DNA fragments with sizes ranging from about 3. Mb . Large restriction fragments are thus separated in a size- dependent manner and the method yields relatively few bands on the gel, which makes analysis of the results easier. A clear advantage of the PFGE method is that it addresses a large portion of an investigated genome (> 9. Accordingly, insertions or deletions of mobile genetic elements as well as large recombination events within genomic DNA will result in changes in the PFGE patterns. Usually, plasmid DNA does not interfere with the macrorestriction profiles of the chromosomal DNA, which is responsible for the particular PFGE pattern, as the fragments generated by restriction of plasmid DNA are too small to affect the profile. However, in some bacteria, differences in the carriage of large plasmids (over 5. PFGE profiles . Unfortunately, although widely used, PFGE suffers from several limitations. The method is technically demanding, labour- intensive and time- consuming, and it may lack the resolution power to distinguish bands of nearly identical size (i. Moreover, the analysis of PFGE results is prone to some subjectivity and the continuous quality control and portability of data are limited compared to sequence- based methods. Amplified fragment length polymorphism. In the amplified fragment length polymorphism (AFLP) method, genomic DNA is cut with two restriction enzymes, and double- stranded adaptors are specifically ligated to one of the sticky ends of the restriction fragments . Subsequently, the restriction fragments ending with the adaptor are selectively amplified by polymerase chain reaction (PCR) using primers complementary to the adaptor sequence, the restriction site sequence and a number of additional nucleotides (usually 1–3 nucleotides) from the end of the unknown DNA template. At the start of the amplification, highly stringent conditions are used to ensure efficient binding of primers to fully complementary nucleotide sequences of the template. AFLP allows the specific co- amplification of high numbers (typically between 5.
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The city, in Los Angeles County, was riven with internecine gang warfare at the time, and verbal conflict could quickly escalate to violence. Yet this was not one of Compton's then- common violent scenes. For a start, the schoolkids had been bused in specifically to the local tennis courts. This was Richard Williams doing his worst to toughen up his beloved daughters Venus and Serena, who in just a few years would begin their domination of women's tennis. I'm going to do what I want to do.'. Growing up in Shreveport, Louisiana, in the 1. Another died after being run over by a white woman who claimed it was the victim's fault. I was close to being killed so many times. A hell of a lot of times. As Serena walked on court for the final of the Indian Wells event in the well- heeled Californian desert community, the watching crowd let rip. Boos rained down on the then 1. Venus and her father received a similar welcome as they took their seats in the stand. The crowd was unhappy that Venus had pulled out of the semifinal clash against her sister a day earlier due to injury. Some suspected that matches between the duo were fixed by their father to maintain family harmony. Accusations of racism would follow, with Serena writing in her autobiography that . Despite the poisonous nature of the occasion, Williams knew his youngest daughter was ready for what unfolded that day. Serena would go on to defeat fellow teen Kim Clijsters 4- 6 6- 4 6- 2 despite being treated like what he describes as a . It's easy to say, but very difficult to be done. She was showing me ways of how it could be done. They're going to be tennis players. But it was decided that Compton was the best place for their early education. Serena's unquestionable power, aggression and on- court mental fortitude certainly attest to that. Yet those deep wells of strength have been required in more troublesome circumstances too. The Indian Wells debacle was followed by the 2. Venus and Serena's half sister Yetunde Price in a drive- by shooting in Compton. In 2. 01. 1, meanwhile, Serena received the shock news that she had a number of blood clots in her lungs, while still recovering from a serious foot injury caused by treading on broken glass at a restaurant the previous year. She required immediate surgery, and those closest to her feared for her life. Yet Williams believes one of his unorthodox suggestions played its part in the recuperation. And Serena felt better, I believe, from that day on. Serena remains the dominant force in women's tennis and Venus has recaptured some of her best form in recent years as she copes with Sjogren's syndrome, their father and one- time coach now struggles to watch his girls play. He's seldom in the players' box at tournaments these days and feels his presence is no longer necessary on the biggest occasions. That's enough to make you cry. I tried to get them to believe that they have pushed themselves, not me. Wars. 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Welcome noobs! Also, all groups that contain elements of advertising will be deleted.). Michael Cudlitz Spells Trouble for Rich. Richard Speight Jr.’s Kings of Con character Richard Slate as Comic- Con HQ’s breakout freshman comedy returns with its first new episode of the new year! As Season 1 resumes, Rich and Rob (Rob Benedict) make their way to Piano, Texas where they’re welcomed into a luxury hotel while in town for their latest convention appearance. On Cloud 9 from the welcome change in scenery (and cleanliness of the hotel), Rob and Rich soon bump into a “frisky” fan who seems to be head- over- heels for Rich who eagerly invites them to join her hotel room party.
Needless to say, Rich jumps at the chance to spend a little time with the overly enthusiastic fan in hopes that their time at the party will lead to a “happy ending.” What Rich never expected was to come face to torso with the fan’s rough and tough husband – played by The Walking Dead. As seen in our sneak peek from this week’s new episode, Cudlitz’s character is far from pleased to discover Rich in his hotel room with his pants around his ankles! Unfortunately, Rob’s time at the hotel is turning out to be just as unenjoyable as Rich’s thanks to the surprise arrival of the convention company’s owner (Sex and the City? Thoughts on Michael Cudlitz’s special guest appearance?
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