Genotyping methods of nosocomial infections pathogen
- 1. Genotyping methods of nosocomial infections pathogen 1. This two aricles are recommended for future reading. These are used mainly in preparation of this presentation 2. Nosocomial infections are an important source of morbidity and mortality in hospital settings, afflicting an estimated 2 million patients in United States each year. This number represents approximately 5% of hospitalized patients and results in an estimated 88,000 deaths and 4.5 billion dollars in excess health care costs. 3. Although viruses, fungi, and parasites are recognized as sources of nosocomial infections, bacterial agents remain the most commonly recognized cause of hospital- acquired infections. Major antimicrobial resistance problems are typically associated with gram-positive nosocomial pathogens, which include glycopeptide (vancomycin) methicillin-resistant Staphylococcus aureus (MRSA), and, more recently,glycopeptide-intermediate and- resistant S. aureus. 4. Among the gram-negative bacilli, extended-spectrum-beta-lactamase-producing strains of Escherichia coli and Klebsiella pneumoniae and multidrag-resistant strains of Pseudomonas aeruginosa and Acinetobacter baumannii. CRE have raise special attention and have become the primary concern throught the world. 5. Understanding pathogen distribution and relatedness is essential for determining the epidemiology of nosocomial infections and aiding in the design of rational pathogen control methods. The role of pathogen typing is to determine if epidemiologically related isolates are also genetically related. Historically, this analysis of nosocomial pathogens has relied on a comparison of phenotypic characteristics such as biotypes, serotypes, bacteriophage or bacteriocin types, and antimicrobial susceptibility profiles. 6. Genotyping is the process of determining differences in the genetic make-up (genotype) of an individual by examining the individual's DNA sequence using biological assays and comparing it to another individual's sequence or a reference sequence. It reveals the alleles an individual has inherited from their parents. Traditionally genotyping is the use of DNA sequences to define biological populations by use of molecular tools. It does not usually involve defining the genes of an individual. 7. DNA based molecular methodologies include pulsed-field gel electrophoresis (PFGE) and other restriction-based methods, as well as plasmid analysis and PCR based typing methods. Establishing clonality of pathogens can aid in the identification of the source (environmental or personnel) of organisms, distinguish infectious from noninfectious strains and distinguish relapse from reinfection. In recent years, molecular or genotypic techniques have received increased attention as means of analyzing epidemiologic interrelationships. In the examination of the genotypic methods for their application to the study and control of hospital acquired infections, the goal of genotyping studies is that epidemiologically related isolates collected during an outbreak of nosocomial disease are able to be linked to one another.
- 2. In other words, isolates involved in a nosocomial outbreak are genetically related and thus originate from the same strain. 8. PFGE, Southern Blot Analysis-Ribotyping, Plasmid Analysis, Typing Methods Using PCR, Multiplex PCR, Nested PCR, Arbitrarily primed PCR (AP-PCR), Amplified fragment length polymorphism (AFLP), Variable number tandem repeat (VNTR) typing, Spoligotyping for Mycobacterium tuberculosis are some of most frequently used genotypic techniques. 9. 10. There have been some recent advances in molecular epidemiology, such as NUCLEOTIDE SEQUENCE-BASED ANALYSIS, single-locus sequence typing (SLST),multi-loculus sequence typing (MLST) 11. The chromosome is the most fundamental component of identity of the cell and therefore represents a preferred measure for assessing strain interrelatedness. One approach has been to digest chromosomal DNA with restriction enzymes, resulting in a series of fragments of different sizes that form different patterns when analyzed by agarose gel electrophoresis. Differences in these patterns are referred to as restriction fragment length polymorphisms (RFLPs). Enzymes used to cleave DNA often recognize numerous sites within the bacterial chromosome, resulting in too many band fragments to efficiently and accurately compare following conventional agarose gel electrophoresis 12. Restriction enzyme is a type of enzyme that always cleansnucleotide sequence in DNA at the same site. In this example,splitting occurs between thymine and guanine in the sequence TGATCA. The same sequence of nucleotides may appear in DNA of many organisms or multiple times on the same strain of DNA. 13. In conventional agarose gel electrophoresis, DNA molecules that are more than 40 to 50 kb in size fail to migrate efficiently. By periodically changing the direction of the electrical field in which the DNA is separated, PFGE allows the separation of DNA molecules of over 1,000 kbp in length (often referred to as megabase-sized DNA). PFGE methods differ in the way the pulsed electric field is delivered to the agarose gel. Two of the most commonly utilized approaches are contour-clamped homogenous electric field (CHEF) and field inversion gel electrophoresis. Field inversion gel electrophoresis utilizes a conventional electrophoresis chamber in which the orientation of the electric field is periodically inverted by 180 degrees. 14. Figure represents the Dendrogram of pulsed-field gel electrophoresis analysis of Acinetobacter baumannii isolates isolated form various clinical sites analized by GelCompare software. Most analysis programs also contain algorithms that allow for phylogenic analysis of strains, which allows for the detection of strain evolution and ancestral relationships among isolates. 15. Plasmid typing was the first molecular method to be used as a bacterial typing tool. Typing is performed through the isolation of plasmid DNA and comparison of the numbers and sizes of the plasmids by agarose gel electrophoresis. Evaluation of plasmid content is not generally useful in delineation of strain relatedness. Plasmid analysis has been applied in clinical situations to determine the evolution and spread of antibiotic
- 3. resistance among isolates with different PFGE profiles or among different species of organisms within hospitals. 16. In addition to the use of rare-cutting restriction enzymes and PFGE to utilize RFLP for isolate typing, Southern blotting can be used to make the number of bands resolved manageable for interpretation. As described above, typical restriction enzyme digestion of chromosomal DNA results in the generation of too many fragments to efficiently analyze and make genetic comparisons among strains. One of the most common targets for Southern blotting is the gene for the rRNA, and the targeting of the rRNA gene is referred to as ribotyping. Typically, the discriminatory power of ribotyping has been shown to be less that of PFGE or some PCR-based method.Southern blotting has also been used to detect specific antimicrobial resistance genes or virulence factors. 17. Polymerase chain reaction is the most used molecular technique in clinical microbiology today. It utilize to sets of primers and DNA polymerase which provides amplification od target sequence prom one copy to billions of copies. In order to increase the efficiency of PCR typing and reduce reagent costs, multiple sets of primers can be included in a single reaction tube in a process termed multiplex PCR. The amplification products that primers produce need to be of noticeably different sizes to facilitate interpretation. If the amplification products were too close in size, it would be difficult to determine the identity of the amplification product. An additional concern with multiplex PCR is that the mixing of different primers can potentially cause interference in the amplification process, thus making optimization of the reaction difficult. 18. When there is an extreme need for sensitivity and specificity in PCR, the process of nested PCR can be carried out. Nested PCR involves the sequential use of two PCR primer sets.The first primer set isused to amplify a target sequence (which increases the sensitivity for the second primer set); the amplicon generated then serves as the template for a second amplification using primers internal to those of the first amplicon. This secondary amplification proceeds only if the intended target was initially amplified. 19. Arbitrarily primed PCR (AP-PCR) and the very similar randomly amplified polymorphic DNA assay are variations of the PCR technique in which a random primer, which is not targeted to amplify any specific bacterial DNA sequence, is used for amplification. The key to the random priming is that low annealing temperatures are used (at least initially) during amplification, allowing imperfect hybridization at multiple random chromosomal locations to occur and initiate DNA synthesis. 20. Amplified fragment length polymorphism (AFLP) is a typing method that utilizes a combination of restriction enzyme digestion and PCR. In the AFLP procedure, the DNA is digested with two different restriction endonucleases, usually chosen so that one cuts more frequently than the other. In order to make the interpretation of the results more feasible, only a specific subset is used for isolate comparison. 21. Cost-effectiveness of this methods in a control and prevention of helthcare assciated infectionas are proven in a example of Northwestern Memorial Hospital in Chicago, Illinois, where the typing was integrated into its infection control program. The effectiveness of their integrated infection control program was assessed by examining data on nosocomial infections during a 24-month period before and a 60-month period following implementation of the program.
- 4. 22. Following implementation, infections per 1,000 patient days fell 13 percent, and the number of hospitalized patients with nosocomial infections decreased 23 percent. The rate of infection fell to 43 percent below the national average, and approximately 50 deaths were avoided during the 5-year period. The cost of implementing the typing program was $400,000 per year; however, this expenditure led to a savings of $5.00 for each dollar spent on the program because of the large reduction of nosocomial infections. 23. Conclusions are that molecular technique will be and continue to be an essential tool in nosocomial infection control program, has proven to be medically justified and cost- effective and is powerful tool for combating the spread of resistant pathogens in the hospital environment