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Molecular typing in screening of an outbreak of hospital infection

By

Ahmed Goma'a Ahmed

Demonstrator of Medical Microbiology and Immunology, Department of Medical Microbiology and Immunology, Faculty of Medicine, Mansoura University.

Supervised by
Prof. Dr. Samir Ahmed Khairallah Professor of Medical Microbiology And Immunology, Department of Medical Microbiology and Immunology, Faculty of Medicine, Mansoura University. Dr. Hamdia Yehia Askar Lecturer of Medical Microbiology And Immunology, Department of Medical Microbiology and Immunology, Faculty of Medicine, Mansoura University.

Infection control history

Even before the birth of the germ theory, the

courageous people who cared for the sick tried as hard as

they could to protect themselves from contagion. Modern concepts of disease transmission are more sophisticated, and

personal protective gear has evolved accordingly. In the

industrialized world, high-performance gloves, waterimpermeable gowns, highly efficient masks, and impervious

face shields are all part of the contemporary health care

workers standard gear.

Introduction to nosocomial infection and nosocomial Outbreaks

The

National

Nosocomial

Infections

Surveillance
condition

System

(NNIS)

system)

denes

nosocomial infection as a localized or systemic

1) that results from adverse reaction to the presence of an infectious agent(s) or its toxin(s) and 2) that was not present or incubating at the time of admission to the hospital.

For most bacterial nosocomial infections, this means that the infection usually becomes evident 48 hours (i.e., the typical incubation period) or more after admission. However, because the incubation period varies with the type of pathogen and to some extent with the patients underlying condition, each infection

must be assessed individually for evidence that links

it to the hospitalization

NI may be

endogenous, arising from an infectious agent present

within a patients body, or exogenous, transmitted from another source within the hospital.
In addition to patient-to-patient spread, others may

be involved, including staff, students, visitors, and

voluntary workers.

Infections that are in the incubation period at the time of admission to

are not classed as nosocomial, but communityhospital

acquired infection of patients or staff can be an important source of nosocomial infection.

Nosocomial outbreaks
An outbreak may be defined as
a relatively rapid increase in the incidence of infection or colonization by a certain bacterial strain,

caused by enhanced patientto-patient transmission.

Hospital-acquired outbreaks account for approximately 2-10% of all hospital-acquired infections.

Just

like

endemic

hospital-acquired

infections, infections in outbreaks contribute signicantly to morbidity and mortality.

Socio-economic impact
Socio-economic impacts of nosocomial infection

(NI), including prolongation of hospital stay, increment

of medical costs and deaths, are well recognized. However, most studies focusing on fatality among patients with NI have been discrete and specific in nature (e.g. elderly patients; stroke victims; patients

in intensive care units [ICUs];

specific body sites including blood, respiratory tract or surgical wound; and infections with multidrug resistant micro-

organisms

including

methicillin-resistant
extended-spectrum and

Staphylococcus
lactamase

aureus,

producing

Enterobacteriaceae,

Pseudomonas aeruginosa).

Severity

of

underlying

disease

classification was evaluated at the time of NI occurrence, using an established modified risk

stratification regimen, as

rapidly fatal

ultimately fatal

death anticipated within 5 years

disease unlikely to be fatal within 5 years or absence of underlying disease.

non-fatal

Deaths were considered to be associated with NI if

active the NI was still active and being treated with

antibiotics when death occurred and no other obvious causes of death were evident.

Hospital-Associated Methicillin-Resistant Staphylococcus aureus(MRSA)

Infection control measures have been

put into place in many hospitals with

varying success however; studies have

shown that even with strict hand-washing

and contact isolation guidelines, MRSA is

still capable of spreading.

For instance, even after proper hand washing and glove usage, MRSA could still be found on the hands of health-care workers.

Lack of education and poor compliance of the

health-care workers factors. might be contributing

Source Tracing (Typing)

Over development

the and

past

several of

years,

the

application

molecular

diagnostic techniques has initiated a revolution in the diagnosis and monitoring of infectious diseases.

bacteriophage

chromatographic profiles

Microbial phenotypic characteristics,

biotyping

susceptibility testing

Nucleic

acid

techniques,

such

as

plasmid

profiling, various methods for generating restriction

fragment length polymorphisms, and the polymerase chain reaction (PCR), are making increasing inroads into

clinical laboratories.
PCR-based systems to detect the etiologic agents of disease directly from clinical samples, without the need for culture, have been useful in rapid detection of unculturable or fastidious microorganisms.

Additionally, amplified

sequence DNA

analysis allows

of for

microbial

identification and better characterization of the

pathogen. Subspecies variation, identified by

various techniques, has been shown to be important in the prognosis of certain diseases.

Other important advances include the determination of viral load and the direct detection of of genes and or gene mutations software widely

responsible for drug resistance. Increased use

automation these user-friendly more makes available. technologies

In all, the detection of infectious agents at the nucleic acid level represents

The true synthesis of clinical chemistry and clinical microbiology techniques.

Criteria to Evaluate Typing Systems

At this time, no typing system completely fullls all the criteria described below, and the advantages and disadvantages of a technique should be understood before use Typeability
:

Reproducibility

Discriminatory power Ease of interpretation and performance

1.

Typeability: an unambiguous result for each isolate analyzed. Nontypeable isolates have a null or

uninterpretable result.

2. Reproducibility: the same result when the same strain is is tested by repeatedly. technical Reproducibility inuenced

factors (day-to-day variation) and biological

factors (variation in the stability of the typing characteristic).

3. Discriminatory power: differentiates between

unrelated strains

but varies as species vary in genetic stability under the inuence of selective pressure.

4. Ease of interpretation: multiple users of the typing system obtain the same results and reach the same conclusions.

R C

5. Ease of performance:
the rapidity

convenience,
cost of equipment, and personnel needed to perform a technique .

Capsule Polysaccharides Fimbirae (M-protein) Teichoid acid Lipoteichoic acid

GENOTYPE (Chromosomal & plasmid DNA)

PHENOTYPE

Different Enzymes Peptidoglycan

Advantages of molecular typing over other typing methods

The difficulty in assigning an organism to a biologically meaningful category should be well considered before the use of any molecular identification tool. Researchers should be aware of the evolutionary history and taxonomic

position of the specimen under study.

Ideally, they should understand the order of branching and ages of divergence (phylogeny) of the organisms. Terms such as

strain, variant, subspecies or breed could

be highly subjective in some circumstances and be used as synonyms by different investigators to describe the same biological entity.

However, with the enormous inuence of molecular biology in the 1970s it became increasingly clear that phenotypic

characterization was only an expression of the

organisms underlying genotype, several levels removed from the true genetic identity of the organism.

Thus, molecular methods began to nd application in clinically relevant areas including epidemiological analysis, giving rise to the term

molecular epidemiology.

This transition can be viewed as a multi-step

process
the rst iteration of which was analysis of isolate plasmid content by

agarose-gel electrophoresis

However, the bacterial genome is the most fundamental molecule of identity in the cell and thus represents

the

ultimate

metric

for

assessing

isolate

interrelatedness.

This principal is the foundation for

second-generation (restriction enzymes and probes),

third-generation (pulsed eld gel electrophoresis [PFGE] and PCR), and

fourth-generation (DNA sequence)

All these are molecular approaches to epidemiological analysis

It should be realized that a continuous genetic variability does exist among individuals under study has to be properly assessed before

undertaking any taxonomic identification in

order to guarantee that there is no overlap between intraspecies variation and interspecies divergence.

If a new method or genetic marker will be employed

for the first time, the genetic composition of all species of

that taxon should be determined. If possible, a representative sample of individuals should be genotyped, preferentially from different geographic locations (or from different hosts, in cases of internal parasites). The preservation of voucher specimens to serve as a future reference is also highly recommended.

It is also important to keep in mind that there is no perfect DNA-typing method

So the choice of a particular technique is often a compromise that depends on a number of factors, including:
the resources of the laboratory. financial constraints. available expertise.

time limitations.
And more importantly, the research question pursued.

All points should be scrutinized carefully to avoid an inappropriate choice of a species

Performance Comparison of Representative Molecular Epidemiology Methods

Method Typing Capacity Plasmid analysis PFGE Chromosomal High High High Good High Good Moderate Goodmoderate High Moderatepoor Good Discriminatory Power Good Good Reproducibility Ease of Use High Ease of Interpretation Good

RFLP
Ribotyping PCR-RFLP RAPD High Good High High Moderate High High Good Poor Good High High High High Good-High

AFLP
Repetitive elements Sequencing

High
Good

High
Good

Good
High

Moderate
High

High
High

High

High

High

Moderate

Good-High

Schematic representation of most important procedural steps involved in the various DNA-based methods

Specimen Collection
Although viability is not as critical for molecular testing, the quality of nucleic acids may be compromised if the specimen is

improperly handled. DNA and especially RNA

can be damaged in lysed or nonviable cells.

Due to the sensitivity of molecular testing, it is also important to avoid contamination that could yield false-positive results

Sampling must include material from the original

infection. The time and site of collection must be optimal for

the likely presence of the infectious agent. For classical methods that include culturing of the

agent, a sufficient number of microorganisms (103/ml

specimen) must be obtained for agar or liquid culture growth. For molecular testing, however, minimum numbers (as few as

50 organisms) can be detected successfully.

The quantity of target organisms as well as the clinical implications should be taken into account when interpreting the signicance of

positive results, as molecular detection can

reveal infective agents at levels below clinical signicance.

First, depending on the microorganism, more rigorous lysis procedures may be required.

Mycobacteria and fungi in particular have thick cell walls that are more difficult to lyse than those of other bacteria and parasites.

Gram-positive bacteria having a thicker cell wall than gram-negative bacteria may require more rigorous cell lysis conditions.

Mycoplasma, on the other hand, lacks a cell wall, and so care must be taken with the sample to avoid spontaneous lysis of the cells and loss of nucleic acids.

Second, the concentration of organisms within the clinical sample must be considered. Samples should be centrifuged to concentrate

the uid.

Third, inhibitors of enzymes used in molecular analysis may be present in clinical specimens; removal or inactivation of inhibitors

must be a part of specimen preparation.

Finally,

if

RNA

is

to

be

analyzed,

inactivation or removal of RNases in the sample and in all reagents and materials that come into

contact with the sample must occur

Thus,

ensuring

that

the

integrity

of

specimens is maintained, i.e., that specimens are not contaminated by other specimens or

with the products of previous amplication

procedures, is critical to avoid false positives.

On the other hand, it is equally important to ensure that the lack of a product in an amplication procedure is due to the absence

of the target organism and not the presence of

inhibitors preventing the amplication of target sequences (false negative).

First, DNA is an extremely stable and long-lived

biological molecule that can be recovered from biological

material that has been under stress conditions (processed food products, mummified plant tissues, blood stains, etc.).

Second, DNA is found in all biological tissues or fluids

with nucleated cells (or non-nucleated cells with plastids and/or mitochondria), enabling its analysis from almost all

kinds of biological substrates (saliva, faeces, plant seeds,

milk, etc.).

Finally, DNA can provide more information than proteins due to the degeneracy of the genetic code and the presence of large non-

coding stretches

Future Perspective
High-speed methodological innovation in the

field of microbial typing will continue to shape the

field over the coming decade. However, the coming years will also define the position of sequencebased typing; the main question will be whether nucleic acid sequencing approaches will fill all the

niches in epidemiological typing.

It seems obvious that sequencing will remain the preferred technology for assessing phylogenetic relatedness and will define the

population genetics and dynamics of bacterial

species. However, novel technologies for direct characterization of primary cultures of microorganisms will become available.

This may generate a novel series of realtime typing procedures of specific relevance to the clinical microbiology laboratory. Such

methods will not be nucleic acid mediated, but

rather, will focus on the identification of proteins, glycans, lipids, or combinations thereof with entire cells as the ultimate target.

Finally, the quality of a typing system is not

defined

by

the technology

used but

by

the

timeliness of its data. From that perspective, the expectation is that we will continue to see many changes in the way in which epidemiological data are collected. It remains to be seen; however, which method will answer clinical questions with the highest degree of reliability.