Ahmed Gomaa's Protocol

8/8/2019 Ahmed Gomaa's Protocol

http://slidepdf.com/reader/full/ahmed-gomaas-protocol 1/7

Introduction:

A nosocomial infection (derived from the Greek words nosos [disease] and

komein [to care for], and later the Latin word for hospital nosocomium) is defined as an

infection that is not present or incubating when the patient is admitted to hospital or other

health-care facility ( Garner et al., 1988). Hospital-acquired infections (HAI) increase

morbidity and mortality and constitute a high financial burden on health care systems

( Willemse-Erix et al., 2009). Vincent (2003) stated that the time frame for diagnosis of a

nosocomial infection will thus clearly be dependent on the incubation period of the

specific infection; 48–72 h after admission is generally deemed indicative of nosocomial,

rather than community acquired infection. Although generally associated with hospital

admission (hence the term hospital-acquired infection), nosocomial infections can arise

after admission to any health-care facility, and the term health-care associated infection is

increasingly being used. Such infections are common and associated with great morbidity

and mortality. Indeed, one provocative headline stated “Hospital acquired infections kill

5000 patients a year in England (Mayor , 2000).

Any organism can be implicated in nosocomial infection, and many infections are

polymicrobial ( Vincent et al., 1995). Recent years have seen a swing in the pattern of

infecting organisms towards gram-positive infections ( Friedman et al., 1998). The

surveillance and control of pathogens of epidemiologic importance project (SCOPE) data( Edmond et al., 1999) revealed that gram-positive cocci were isolated in 64% of 10617

episodes of nosocomial bacteraemia, whereas gram-negative bacilli were isolated in only

27% of cases. The European Prospective Investigation into Cancer and Nutrition(EPIC)

study ( Vincent et al, 1995) identified the following as the most commonly reported

nosocomial pathogens: Staphylococcus aureus (30%), Pseudomonas aeruginosa (29%),

coagulase-negative staphylococci (19%), yeasts (17%), Escherichia coli (13%),

enterococci (12%), Acinetobacter spp. (9%), and Klebsiella spp. (8%) ( Spencer et al.,

1996). Other studies have noted similar patterns of causative microorganisms ( Richards et

al., 2000).

An effective weapon against HAI is early detection of potential outbreaks and

sources of contamination ( Willemse-Erix et al., 2009).

1



Typing of nosocomial pathogens includes:

I. Phenotyping methods.

II. Molecular methods.

I. Phenotyping methods:These methods examine the physical attributes, biochemical products, and

chemical requirements of microorganisms (Leung et al., 2004; Environmental

Protection Agency EPA, 2005). Antimicrobial susceptibility testing is a common practice

in the clinical microbiology laboratory. The resultant antibiogram indicates the pattern of

in vitro resistance or susceptibility of an organism to a panel of antimicrobial agents

(Barenfanger et al., 1999). Serotyping uses a series of antibodies to detect antigens on

the surface of bacteria that have been shown to demonstrate antigenic variability (Ko et

al., 2000; Babl et al., 2001). Bacteriophage and bacteriocin typing as epidemiologic tools

are limited to bacteria. Bacteriophage (phage) typing classifies bacteria based on the

pattern of resistance or susceptibility to a certain set of phages (Hopkins et al., 2004).

II. Molecular methods:

Strommenger et al. (2008) issued that the use of efficient and accurate

epidemiological typing methods is a prerequisite for monitoring and for limiting the

occurrence and spread of epidemic clones within and between hospitals. Identification by

molecular methods allows for more rapid and accurate identification of etiologic agents

in a much shorter time than traditional methods. For example, a protocol using real-time

PCR to detect and differentiate Gram-positive from Gram-negative bacteria could yield

results in less than 3 hours, inclusive of preparation time (Klaschik et al., 2002). Such

rapid identification would allow for the earlier initiation of a focused antimicrobial

regimen, and decrease the likelihood of disease progression (Doern et al., 1994).

Therefore, the use of strain typing in infection control decisions is based onseveral assumptions: (i) isolates associated with the outbreak are recent progeny of a

single (common) precursor or clone, (ii) such isolates will have the same genotype, and

(iii) epidemiologically unrelated isolates will have different genotypes ( Singh et al.,

2006).

2



Many microbial typing methods are available: PCR multilocus enzyme

electrophoresis (MLEE), multilocus sequence typing (MLST), pulsed-field gel

electrophoresis (PFGE), restriction fragment length polymorphisms (RFLP), DNA

sequencing, ribotyping, restriction fragment length polymorphism studies, randomly

amplified polymorphism DNA (RAPD), amplified fragment length polymorphism

(AFLP) and repetitive sequence-based PCR (REP-PCR) ( Healy et al., 2005 ).

Typing Methods Using Polymerase chain reaction (PCR)

During the past decade, advances in PCR technology and other DNA signal and

target amplification techniques have resulted in these molecular diagnostics becoming

key procedures ( Wagar , 1996). Such techniques are conceptually simple, highly specific,

sensitive, and amenable to full automation ( Klapper et al., 1998).

Multiplex PCR

In multiplex PCR more than one target sequence can be amplified by including

more than one pair of primers in the reaction and it has the potential to produce

considerable savings of time and effort within the laboratory without compromising test

utility ( Elnifro et al., 2000). Since its introduction, multiplex PCR has been successfully

applied in many areas of nucleic acid diagnostics, including gene deletion analysis

( Chamberlain et al., 1989), mutation and polymorphism analysis ( Rithidech et al., 1997),

quantitative analysis ( Sherlock et al., 1998), and RNA detection (Zou et al., 1998).The multiplex PCR assay offers a rapid, simple, and accurate identification of

antibiotic resistance profiles and could be used in clinical diagnosis as well as for the

surveillance of the spread of antibiotic resistance determinants in epidemiological studies

(Strommenger et al., 2003).

Nested PCR

Nested PCR involves the sequential use of two PCR primer sets. The first primer

set is used 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 ( Singh et al., 2006). Ginevra et al.

(2009) found that in Legionnaires' disease nested PCR-based SBT (NPSBT) applied

directly to clinical specimens improved significantly epidemiological typing as compared

to the initial Sequence-based typing (SBT) in particular when no isolates are available.3



Arbitrarily primed PCR

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 ( Eribe et al., 2000). Grattard et al. (1994) found that AP- PCR was a very

discriminatory tools for the investigation of nosocomial outbreaks caused by

Enterobacter cloacae . The DNA polymorphism assay most recently introduced for the

typing of Group A streptococcus (GAS; Streptococcus pyogenes ) is random amplified

polymorphic DNA (RAPD) analysis ( Welsh et al., 1990), which has also been called

arbitrarily primed PCR.

Multilocus enzyme electrophoresis ( MLEE )

The application of multilocus enzyme electrophoresis (MLEE) to meningococcal

isolate collections ( Selander et al., 1986) has established the existence of particular

meningococcal lineages associated with invasive disease and played a seminal role in

elucidating the epidemiology and population biology of the meningococcus ( Caugant et

al., 1986); however, for largely practical reasons this technique has not been widely

adopted by reference laboratories and has rarely been employed during outbreak

investigations (Tzanakaki et al., 2001).

Repetitive sequence-based PCR (REP-PCR)

An alternative approach to PCR-based fingerprinting, repetitive-element PCR

(rep-PCR), uses as primers oligonucleotides homologous to defined sequences which are

present in multiple copies in the bacterial genome (Versalovic et al., 1994). Indeed, rep-

PCR's same-day reproducibility and discriminating power have sufficed for small-scale

epidemiological and phylogenetic studies involving wild-type E. coli strains (Johnson et

al., 1998).

Pulsed-field gel electrophoresis (PFGE)

Pulsed-field gel electrophoresis has been used effectively as a molecular

subtyping tool in outbreak investigations (CDC, 2001) and surveillance ( Swaminathan et

4





amplification of the fragments with corresponding primers; and 3) electrophoretic

separation of the products on a high resolution gel (Janssen et al., 1996; Liscum and

Oeller, 2006).

Selective amplification of Apa I/Taq I templates with primer combination A02-T02

(both having an additional C at their 3' end) generated auto radiographic AFLP

fingerprints that were grouped by numerical analysis in two main AFLP clusters allowing

clear separation of M. ulcerans (cluster I) from the M. tuberculosis complex members

M. bovis and M. tuberculosis (cluster II) ( Huys et al., 2000).

Plasmid Analysis:

Plasmid typing was the first molecular method to be used as a bacterial typing

tool (Archer et al.,1984). Typing is performed through the isolation of plasmid DNA and

comparison of the numbers and sizes of the plasmids by agarose gel electrophoresis

( Singh et al., 2006). Plasmid analysis has been applied in clinical situations to determine

the evolution and spread of antibiotic resistance among isolates with different PFGE

profiles or among different species of organisms within hospitals (Donabedian et al.,

2003; Feil et al., 2003). Singh et al. (1992) had found a high rate of fecal colonization

with trimethoprim resistance (Tmpr) Escherichia coli and, using total plasmid content

analysis, had shown that this was due to a diversity of strains.

RECENT ADVANCES IN MOLECULAR TYPING: NUCLEOTIDE

SEQUENCE-BASED ANALYSIS

Sequence-based molecular epidemiology is attractive in offering the promise of

reproducible typing profiles that are highly amenable to standardization, uniform

interpretation, and database cataloging, since they are based on simple quaternary data

(A, T, G, and C) ( Kemp et al., 2005).

Single-locus sequence typing (SLST)

Sequence data for specific loci (genes for virulence, pathogenicity, drug

resistance, etc.) from different strains of the same species have revealed variability in a

6

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Singh%20KV%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus





specific gene ( Singh et al., 2006). At present, the single-locus sequence typing (SLST)

approach with most promise involves analysis of a particular region of the staphylococcal

protein A gene ( spa ) which is polymorphic due to 24-bp repeat sequences that may vary

in both the number of repeats and the overall sequence in the polymorphic X or short

sequence repeat region ( Koreen et al., 2004).

Multi-locus sequence typing (MLST)

MLST was designed based on the principles of multilocus enzyme electrophoresis

(MLEE) in which the electrophoretic mobilities of housekeeping enzymes of isolates of

interest are compared (Enright et al., 1999). Based on the resulting migration pattern of

each enzyme, an allele number is assigned for each housekeeping locus. Once arranged

into a string of integers, the allele numbers at each locus define the electrophoretic type

(ET) of a strain (Selander et al., 1980). Being sequence-based, MLST provides a

definitive characterization of bacterial isolates that is consistent from one laboratory to the

next. The nuclei acid sequences are typically stored in a public database that can be

readily accessed via the Internet (González-Escalona et al., 2008).

Recently, a method based on the unique lengths of the intergenic regions

containing repetitive DNA loci (Tenover et al., 2007; van Belkum, 2007), known as

multiple-locus variable-number tandem-repeat analysis (MLVA), was introduced anddescribed.

7

Documents

Ahmed Gomaa's Protocol