it discuss common hospital acquired infections ,laboratory diagnosis and infection control guideline of these infection esp MRSA , CRE ,VRE , MDRTB

1. "
َ‫ل‬ َ‫م‬ْ‫ل‬ِ‫ع‬ َ‫ال‬ َ‫َك‬‫ن‬‫ا‬َ‫ح‬ْ‫ب‬ُ‫س‬ ْ‫ا‬‫و‬ُ‫ل‬‫ا‬َ‫ق‬
َ‫ن‬َ‫ت‬ْ‫م‬َّ‫ل‬َ‫ع‬ ‫ا‬َ‫م‬ َّ‫ال‬ِ‫إ‬ ‫َا‬‫ن‬
َ‫ك‬َّ‫ن‬ِ‫إ‬ ‫ا‬
ُ‫م‬‫ي‬ِ‫ك‬َ‫ح‬ْ‫ال‬ ُ‫م‬‫ي‬ِ‫ل‬َ‫ع‬ْ‫ال‬ َ‫نت‬َ‫أ‬
"
‫البقرة‬ ‫سورة‬
(
32
)

2. Hospital acquired infection :
Molecular study and infection
control guidlines
Abd Alla Ibrahim Ahmed Shady
Resident in Clinical Pathology Department
Faculty of Medicine-Mansoura University

3. Supervisors
Prof. Dr. Lotfy Abdel-Naby Mahmoud
Prof. of Clinical Pathology
Faculty of Medicine-Mansoura University
Dr. Wafaa Mohamed Elemshaty
Associate Professor of Clinical Pathology
Faculty of Medicine -Mansoura University

4. Introduction
Hospital-acquired infections (HAIs) or
nosocomial infections (NI) are a major
challenge to patient safety and contribute
significantly to morbidity and mortality, as
well as to excess costs for hospital stay.They
affect both developed and resource-poor
countries and constitute a significant burden
both for the patient and for the health care
system.

5. Aim of work
The aim of this essay is to review morbidity,
mortality , infections routes and medical cost
associated with hospital acquired infections ,
with stress on molecular studies of these
infections along with infection control
guidelines.

6. Definitions
Nosocomial infection (NI) or hospital
acquired infection (HAI) can be defined as an
infection acquired in hospital by a patient who
was admitted for a reason other than that
infection . This includes infections acquired in
the hospital but appearing after discharge, and
also occupational infections among staff of the
facility .

7. Frequency of HAIs
Among the more industrialized and developed
nations, the World Health Organization found 8.7 %
of all hospitalized patients to have nosocomial
infections. While HAI are an important health care
concern worldwide , they are especially troublesome
in developing nations. Nosocomial infection rates
range from 1% in Northern Europe, especially the
Netherlands, which introduced extremely aggressive
infection control measures, to 40% in some parts of
Asia, South America, and sub-Saharan Africa .

8. Impact of HAIs
Nosocomial infections (NI) contribute significantly to
morbidity and mortality, as well as to excess costs for
hospitalized patients. According to the available
evidence, the impact of Health care associated
infection (HCAI) implies prolonged hospital stay,
long-term disability, increased resistance of
microorganisms to antimicrobials, massive additional
financial burden for health systems, high costs for
patients and their family, and unnecessary deaths

9. Routes of transmission
Direct transmission from another host (healthy or ill)
or from an environmental reservoir or surface by
direct contact or direct large-droplet spread of
infectious secretions is the simplest route of agent
spread. Examples of direct-contact transmission
routes include kissing (infectious mononucleosis),
shaking hands [common cold (rhinovirus)], or other
skin contact (e.g., contamination of a wound with
Staphylococci or Enterococcus spp. during trauma,
surgical procedures or dressing changes) .

10. Potentially pathogenic micro-organisms can colonize
environmental surfaces in the hospital environment
and so act as a source for outbreaks of nosocomial
infection. Studies have presented evidence that the
majority of Gram-positive bacteria, including
Staphylococcus aureus and Enterococcus spp., are
able to survive for months on dry surfaces. Gram-
negative bacteria, such as Klebsiella spp., Escherichia
coli, and Acinetobacter spp. can also survive for a
relatively long time on inanimate surfaces, while
common fungi such as Candida spp. have similar
properties.

11. Predisposing factor for HAIs
The highest prevalence of HAI occurred in
ICUs and acute care surgical and orthopedic
settings. Old age, multiple morbidities or
disease severity, and decreased immunity
increase patient susceptibility. Poor infection
control measures are an overall risk factor as
are certain invasive procedures including
central venous or urinary catheter placements.
Antimicrobial misuse is associated with drug-
resistant HAI .

12. Common HAIs
 Urinary tract, respiratory tract, surgical site, skin and
bloodstream infections are currently recognized as the
major nosocomial infections.
 However, it is becoming increasingly clear that
gastroenteritis outbreaks are also a major burden on
the health services of industrialized nations .

13. Diagnosis of HAIs can be characterized in terms of
typeability, reproducibility, discriminatory power,
 Typeability refers to the ability of a technique to
assign an unambiguous result (type) to each isolate.
 The reproducibility of a method refers to the ability to
yield the same result upon repeat testing of a bacterial
strain.
 The discriminatory power of a technique refers to its
ability to differentiate among epidemiologically
unrelated isolates, ideally assigning each to a
different type

14. Diagnosis
A - Phenotypic methods
1-Biotyping
2-Antimicrobial susceptibility
testing
3-Serotyping
4-Bacteriophage and
bacteriocin typing
B-Genotypic methods
1- Plasmid Analysis
2- Pulsed-field gel electrophoresis
3-Southern Blot Analysis-Ribotyping
4-Northern blotting
5-Heteroduplex Migration Analysis
6-Single Strand Conformation Polymorphism
Analysis
7-Typing Methods Using PCR
8 -DNA arrays
9 -Pyrosequencing
10 –Spectroscopy
11-Proteomics and metabolomics
12- Nucleotide sequence- based analysis

15. A - Phenotypic methods
1-Biotyping
Biotyping is often used to determine the species of
microorganisms based upon their abilities to utilize
components in different growth media and carry out
certain chemical reactions, but it can also be used to
separate members of a particular species due to
biochemical differences among the organisms .
Biotyping cannot differentiate among strains where
biochemical diversity is uncommon, such as the
enterococci, and therefore the utility of biotyping in
epidemiologic studies is quite limited

16. 2-Antimicrobial susceptibility testing
Strains defined by this method should always be
confirmed by genomic typing, because unrelated
clones can undergo convergence to the same
resistance phenotype under antibiotic selective
pressure, through mutations and genetic exchanges.
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 . Antimicrobial susceptibility
testing is typically performed using either automated
broth microdilution or disk diffusion methods

17. Disk diffusion testing of a hyper b-lactamase
producing Staphylococcus aureus

18. 3-Serotyping
Serotyping uses a series of antibodies to detect
antigens on the surface of bacteria that have been
shown to demonstrate antigenic variability .
Serotyping methods have been used for decades for
the taxonomic grouping of a number of bacterial
pathogen species and remain important for typing
Salmonella, Legionella, Shigella, and Streptococcus
pneumoniae isolates.Serotyping also has been shown
to have epidemiologic value in differentiating strains
within species of nosocomial pathogens such as
Klebsiella and Pseudomonas

19. 4-Bacteriophage and bacteriocin typing
Bacteriophage (phage) typing classifies bacteria
based on the pattern of resistance or susceptibility to a
certain set of phages. Bacteriophages are viruses that
are able to attach to the cell walls of certain bacteria,
enter, multiply, and lyse the cells. The differential
ability of phages to infect certain cells is based upon
the availability of corresponding receptors on the cell
surface for the phage to bind. Often different strains
of pathogens have a different cohort of receptors,
leading to variable lysis profiles

20. B-Genotypic methods
1- Plasmid Analysis

21. 2- Pulsed-field gel electrophoresis

22. 3-Southern Blot Analysis-Ribotyping

24. 4-Northern blotting

25. 5-Heteroduplex Migration Analysis
Originally described as a PCR artifact , heteroduplex
migration has become a popular mutation –scanning
technique ,primarily because of its technical
simplicity .With this technique , the dsDNA
generated by PCR is denatured and then allowed to
reanneal , followed by electrophoresis under slightly
denaturing conditions (e.g. 15% urea ,40 c ) on
polyacrylamide gels .Detection is performed by silver
staining of the gel or by fluorescence detection if one
of the PCR primers is labeled .

26. 6-Single Strand Conformation Polymorphism Analysis
Similar to heteroduplex analysis , it first requires PCR
amplification . The amplification is then diluted ,
denatured with heat and formamide , and the resulting
ssDNA is separated by non denaturing
polyacrylamide electrophoresis (usually run at 40 c ) .
During electrophoresis the single –stranded molecules
fold into three-dimensional structures according to
their primary sequence . Electorphoretic mobility then
becomes a function of size and shape of the folded
single –stranded molecules .

27. 7-Typing Methods Using PCR
a-Multiplex PCR
A key strategy in the development of a multiplex PCR
assay is the design of the primers. Primers must be
designed such that all of the primers have very close
annealing temperature optimum, and the
amplification products that they produce need to be of
notice 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

28. b-Nested PCR

29. c- Arbitrarily primed PCR
Arbitrarily primed PCR (AP-PCR) typing and random
amplified polymorphic DNA analysis are based on
low-stringency PCR amplification of genomic DNA
with arbitrary sequence primer. Segments of DNA
lying between closely spaced annealing sites are
amplified to produce a strain-specific array of DNA
fragments

30. d- Amplified fragment length polymorphism

31. e- Restriction fragment length polymorphisms

32. f-Repetitive element PCR
This rep-PCR typing is based on the presence of
multiple copies of short repetitive sequences found in
microbial genomes. These sequences are interspersed
throughout the genome and are usually located in
non-coding regions of DNA . The number of
repetitive elements and their respective genomic
locations are used to genotype isolates and to
differentiate highly related strains.

33. g- Multi Locus Variable copy Numbers of Tandem Repeats
Analysis
MLVA is based on the detection of short sequence
repeats that vary in copy number in the microbial
genome at various loci. MLVA detects
polymorphisms at five different sites in the genome.
Four regions of detection are on the bacterial
chromosome and one is located on the serotype
specific plasmid

34. 8 -DNA arrays

35. 9 -Pyrosequencing
Pyrosequencing is an alternative sequencing method
that generates short sequence read lengths of less than
200 base pairs. It does not require labeled
nucleotides, capillary electrophoresis, or post-reaction
purification. In pyrosequencing, or sequencing by
synthesis, the sequence is read as the nucleotides are
incorporated. The chemistry differs from modified
Sanger sequencing in that it uses a combination of
enzymes, including DNA polymerase, ATP
sulfurylase, luciferase, and apyrase, along with
adenosine 5' phosphosulfate and luciferin substrates

36. 10 –Spectroscopy

37. 11-Proteomics and metabolomics
Clinical proteomics is a branch of classical proteomics
focusing on the identification of clinically relevant
protein expression . The full spectrum of usually
small-molecular size metabolites in a biological
system is called the metabolome, and the discipline
studying the full composition of the metabolome is
called metabolomics.

38. 12- Nucleotide sequence- based analysis
 Single-locus sequence typing Sequence data for
specific loci (genes for virulence, pathogenicity, drug
resistance, etc.) from different strains of the same
species have revealed variability in a specific gene,
such as single-nucleotide polymorphisms and areas
with repetitive sequence that demonstrate potential
for epidemiologic application
 Multi-locus sequence typing
Multi-locus sequence typing (MLST) is a powerful
molecular tool used for characterizing relationships
among bacterial isolates for epidemiological purposes

39. Infection control Guidelines
IC activities are still developing in many health
institutions in Egypt. The national infection control
program was started in 2003 by the Ministry of
Health and Population. The national IC strategic plan
entailed instituting IC programs in all hospitals in
Egypt by 2010 . The components of an infection
control program are drawn from regulatory
requirements, current nursing home practices, and
extrapolations from hospital programs. The limited
resources affect the type and extent of programs
developed .

43. Conclusion
 The evaluation of hospital-associated infections will
continue to rely on clinical infection surveillance as
the first step to understanding disease epidemiology
and management of infections.
 Molecular testing will continue to be an essential tool,
for tracing of the source of infection .
 Outbreak Control . A system for detection,
investigation, and control of epidemic infectious
diseases is an important component of infection
control program.

44.  Isolation—An isolation and precautions
system to reduce the risk of transmission of
infectious agents
 Continuing education in infection prevention
and control ,Resident health program ,
Employee health program , Disease reporting
to public health authorities , Facility
management, including environmental control,
waste management, product evaluation and
disinfection, sterilization and asepsis are
integrated component of infection control
program.

45. Recommendations
 Proper hand washing
 Better nutrition
 Housing patients in separate rooms
 Sufficient numbers of nursing staff
 Coated urinary and CVCs
 Lower overall antibiotic use which will reduce risk
of antibiotic-resistant organisms and improve
efficacy of antibiotics given to patients who acquire
nosocomial infections.
 Molecular technique can be very effective in tracing
the spread of nosocomial infection .