hospital acquired infections
TRANSCRIPT
Hospital acquired infections : Molecular study and infection control guidlinesSupervisorsProf.Dr.Lotfy Abdel-Naby MahmoudProf. of Clinical PathologyFaculty of Medicine-Mansoura University
Dr.Wafaa Mohamed Mohamed ElemshatyAssociate Professor of Clinical PathologyFaculty of Medicine -Mansoura University
Essay By Abd Alla Ibrahim Ahmed Shady Resident in Clinical PathologyDepartmentFaculty of Medicine-Mansoura University
Acknowledgement
Thanks, first and last, now and forever to "ALLAH", for great care,
guidance, and help in every step of my life and for a l l the countless
gifts I have been offered. Of these gifts, those people who were assigned
to give me a precious hand to be able to fulfill this essay.
I would like to express my sincere gratitude, deepest thanks and
appreciation to Prof .Dr. Lotfy Abdel-Naby Mahmoud
, Professor of Clinical Pathology for his very kind supervision,
stimulating suggestions, continous encouragement, valuable advice and
endless effort in all the time of the research and writing of this essay.
I am deeply indebted to Dr . Wafaa Mohamed Mohamed
Elemshaty, Associate Professor of Clinical Pathology, Mansoura
University, for her kind cooperation, valuable aid, and advise, I am
deeply touched by their endless support and concern.
Abd Alla Ibrahim Ahmed Shady
2012
1Introduction and aim of work*
4
4
Review
Hospital acquired infections
Definitions
*
5Frequency of HAIs 8 Impact of HAIs
9 Routes of transmission
13 Predisposing factor for HAIs
1818
19
22
2425
27
2929
30
31
32
Common HAIs 1-Urinary Tract Infection
2- Respiratory tract infections
3-Surgical site infections
4- Burn infections
5- Blood stream infections
6-Device associated infection
7-Ocular Infections
8-Central Nervous System Infections
9-Ear & Nose infection
10-Gastrointestinal Tract Infections
11 -Obstetrics and neonatal Infections
35 Diagnosis of HAIs
41
4141
43
44
4648
49
52
5556
56
57
6264
65
67
68
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
70
74
Infection control Guidelines
1-Hand hygiene
80 2 -Environmental Decontamination &Cleaning
85 3-Nutrition 87 4-Surveillance & Outbreak control92 5-Preventing urinary tract infection and
urinary catheter infections
94 6-Preventing central venous line infection
and Blood stream infections 987-Preventing respiratory tract infections 1008-Control of infections related to surgery
and surgical equipment 104 9- Prevention of Gastrointestinal Tract
and waterborne hospital infections 106 10-Preventing burn infections 10911-Isolation and precautions 112Summary & Conclusion *118References*
1Arabic Summary *
Amplified fragment length polymorphismAFLP
Acquired immunodeficiency syndromeAIDS
Arbitrarily primed PCRAP-PCR
Catheter Related Blood stream infectionsCABSIs
Catheter-associated urinary tract infectionsCAUTIs
Clostridium difficile-associated diseaseCDAD
Centers for Disease Control and PreventionCDC
Contour-clamped homogenous electric fieldCHEF
Coagulase-negative staphylococciCo NS
Catheter Related Blood stream infectionsCRBSI
Central venous catheterCVC
Deoxynucleoside triphosphatesDNTPs
Group A β-hemolytic streptococci(Streptococcus pyogenes)
GABHS
Hospital acquired infectionHAI
Hospital Acquired PneumoniaHAP
Hepatitis B virusHBV
Health care associated infectionHCAI
Hepatitis C virusHCV
Healthcare workersHCWs
High Efficiency Particulate AirHEPA
High-intensity narrow-spectrum light environmental decontamination system
HINS-light EDS
Human immunodeficiency virusHIV
Heteroduplex Migration AnalysisHMA
Intensive care unitICU
Multidrug-resistant organismMDRO
Multidrug-resistant tuberculosisMDR-TB
Multi dose vialsMDV
Multi-locus sequence typingMLST
Multi Locus Variable copy Numbers of Tandem Repeats Analysis
MLVA
Methicillin-resistant Staphylococcus aureusMRSA
Mass spectrometryMS
Nosocomial infectionNI
Neonatal intensive care unitNICU
Needle stick and sharps injuryNSI
Polymerase chain reactionPCR
Pulsed-field gel electrophoresisPFGE
Postoperative endophthalmitisPOE
Post tympanostomy tube otorrheaPTTO
Repetitive element PCRRep-PCR
Restriction fragment length polymorphismsRFLP
Respiratory Syncetial VirusRSV
Severe acute respiratory syndromeSARS
Single-locus sequence typingSLST
Single Strand Conformation Polymorphism AnalysisSSCP
Surgical site infectionsSSIs
Urinary tract infectionsUTIs
Ventilator-associated pneumoniaVAP
Vancomycin-resistant enterococciVRE
World health organizationWHO
Figure 1 Studies on general HAIs rates from developing and developed countries 1995-2008
7
Figure 2 Recent evolution of bacterial strain identification for epidemiological purpose
40
Figure 3 Disk diffusion testing of a hyper b-lactamase producing Staphylococcus aureus.
43
Figure 4 Phage typing is the identification of bacterial species and strains by determining their susceptibility to various phages
45
Figure 5 Flow chart comparison of the different procedural steps used for various molecular typing techniques
47
Figure 6 Schematic diagram of Plasmid Analysis 49
Figure 7 Diagram of pulsed-field gel electrophoresis 51
Figure 8 Schematic diagram of Southern Blot Analysis-Ribotyping
54
Figure 9 Schematic diagram of Northern blotting 55
Figure 10 Schematic diagram of the amplified fragment length polymorphism (AFLP) technique
60
Figure 11 Schematic diagram of Restriction fragment length polymorphisms
61
Figure 12 Schematic diagram of DNA arrays 64
Figure 13 Overview of Raman procedure and spectrometer
66
Figure 14 Schematic diagram of hand washing 79
Table 1 Infection control program structure 71Table 2 Infection control program element 72Table 3 Non- pharmacological hospital infection
control strategies73
Table 4 Immediate control measures for outbreak management
91
Table 5 Colour coding for the disposal of clinical waste
97
Table 6 Major headings and some factors in prevention of surgical site infection
100
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
(French and Cheng , 1991) .They affect both developed and resource-
poor countries and constitute a significant burden both for the patient and
for the health care system (WHO, 2002) .
A prevalence survey was conducted, under the authority of the World
Heath Organization (WHO), in 55 hospitals from 14 countries
representing 4 regions (Europe, Eastern Mediterranean, Southeast Asia,
and Western Pacific).This survey revealed that an average of 8.7% of
hospitalized patients developed HAIs, with the highest frequencies of
such infections occurring among hospitals in the Eastern Mediterranean
and South east Asian regions “11.8%and10% respectively”(Talaat , et
al., 2006) .
Several risk factors for acquiring an infection have been commonly
cited, including the presence of underlying conditions (such as diabetes,
renal failure, or malignancies), long term hospitalizations, surgical
procedures, receipt of prior antimicrobial therapy, and the presence of
indwelling catheters (Singh et al., 2006) .
Understanding pathogen distribution and relatedness is essential for
determining the epidemiology of NI 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. This approach has begun to change over the past 2
decades, with the development and implementation of new technologies
based on DNA, or molecular, analysis (Arbeit , 1999 & Cockerill ,et
al.,2004 ) .
Most studies related to HAI were conducted in the developed countries
and demonstrated the efficacy of HAI surveillance and its significant
incidence concerning patient morbidity and mortality (Cooke , 2000 &
Barrett , 2002 & Gastmeier ,2006) .Conversely, in the developing
countries, few studies provide data of device associated infection rates
using the standardized definitions of HAI rates per 1000 days (Rosenthal
, 2006 & Leblebicioglu , 2007) .
More over, much of the recent research on NI has dealt with the
need for new antibiotics ,better antibiotic management and better
diagnostic techniques to detect infections earlier .Better drug treatment
and earlier infection diagnosis can certainly play a major role in reducing
morbidity and mortality from HAIs . However, there are many non
pharmacological interventions that can significantly reduce the incidence
of HAIs, but these are often overlooked in practice (Curtis, 2008) .
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.
Hospital acquired infections
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 (WHO, 2002) .
Endemic is the usual level or presence of an agent or disease in a
defined population during a given period. Epidemic is an unusual,
higher-than-expected level of infection or disease by an agent in a defined
population in a given period. This definition assumes previous knowledge
of the usual, or endemic, levels. Pandemic is an epidemic that spreads
over several countries or continents and affects many people
(Gordis ,2000).
An outbreak is defined as an unusual or unexpected increase of cases
of a known nosocomial infection or the emergence of cases of a new
infection. Outbreaks of nosocomial infection should be identified and
promptly investigated because of their importance in terms of morbidity,
costs and institutional image. Outbreak investigation may also lead to
sustained improvement in patient care practices (Gordis , 1996) .
Incidence rate is defined as the ratio of the number of new infections
or disease in a defined population in a given period to the number of
individuals at risk in the population. At risk is frequently defined as the
number of potentially exposed susceptibles. The rate is usually expressed
as numbers of new cases per thousands (1,000, 10,000, or 100,000) per
year . Prevalence rate is defined as the ratio of the number of individuals
measurably affected or diseased by an agent in a defined population at a
particular point in time, or over a specified time period, without regard to
when the process or disease began (Gordis ,2000).
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 (Starakis et al .,2002& Eriksen et al .,2005& Klevens et
al .,2007).
Compared with average prevalence of health-care-associated infection
in Europe (reported as 7.1 per 100 patients by the European Centre for
Disease Prevention and Control) and estimated incidence in the USA (4.5
per 100 patients ), prevalence of health-care-associated infection in
resource-limited settings is substantially higher, particularly in high-
quality studies (15.5 per 100 patients). The difference between
developing and developed countries is even more striking when
considering incidence of ICU-acquired infection (pooled density 47.9 per
1000 patient-days in developing countries), which is estimated to be 13.6
per 1000 patient-days in the USA (Klevens et al ., 2007).
Importantly, very high rates of health-care-associated infection in
neonatal and paediatric populations were noted not only in ICUs but also
in some paediatric wards and children’s hospitals. (Cavalcante et al .,
2006) . Previous findings indicate that surgical-site infection is both the
most frequently studied and the leading health-care-associated infection
hospital-wide in the developing world (Haynes et al ., 2009). In a nation
wide study undertaken in the USA, the cumulative incidence of surgical-
site infection was 2.6 per 100 surgical procedures; similarly, it was 2.9
per 100 surgical procedures in different European countries, and 1.6 per
100 procedures in Germany . ( Bhutta et al ., 2005).
Several studies have observed a high frequency of bloodstream
infections and high mortality among patients admitted to pediatric or
neonatal care units in Egypt. In a study of patients admitted to a pediatric
intensive care unit (ICU), investigators reported a high frequency of
sepsis and an overall mortality rate of 50%. Similarly, a study of 115
infants admitted to a neonatal intensive care unit (NICU) in a major
university hospital over 3-month period found that 77% had sepsis, with
hospital-acquired pathogens isolated from bloodstream, and mortality
rates exceeding 51%. The results of this investigation prompted a cross-
sectional survey of 22 NICUs, from various Ministry of Health hospitals
throughout Egypt, to assess the magnitude of this problem. Among the
180 infants admitted to these 22 units, 97 (54%) had a clinical diagnosis
of sepsis, and 86 (69%) had positive blood cultures at the time of the
survey (El-Nawawy , 2003). There are paucity of national reports accords
with findings of a survey done by WHO, in which only 23 of 147
developing countries (16%) reported a functioning national surveillance
system (figure 1) (WHO , 2008).
Figure 1 . Studies on general HAIs rates from developing and
developed countries 1995-2008 (WHO 2008).
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 . The increased length of stay for infected
patients is the greatest contributor to cost (Burke , 2004 &Klevens et
al .,2007& Edwards et al .,2007) .
The overall increase in the duration of hospitalization for patients
with surgical wound infections was 8.2 days, ranging from 3 days for
gynaecology to 9.9 for general surgery and 19.8 for orthopaedic surgery.
Prolonged stay not only increases direct costs to patients or payers but
also indirect costs due to lost work (Coello et al ., 1993). The increased
use of drugs, the need for isolation, and the use of additional laboratory
and other diagnostic studies also contribute to costs (Wenzel ,1995).
NI is the fourth commonest cause of in-hospital deaths, after
cardiovascular disease, cancers and community-acquired infections.
Lower respiratory tract and bloodstream infections were the main sites of
NI which contributed to death, surgical-site infections were the third most
common factor contributing to death. Conversely, urinary tract infections
rarely contribute to death, although some of them could be considered to
play a role in fatal outcomes, particularly when they were associated with
a bloodstream infection . Most cases of peritonitis (included in
gastrointestinal tract infections) were not healthcare-associated or of
iatrogenic origin, but were associated with underlying diseases, such as
cancer or immunocompromised status (Astagneau et al .,2001) .
Routes of transmission
For infection to take place, microorganisms must be transferred from
a reservoir to an acceptable entry site on a susceptible host in sufficient
numbers (the infecting dose) for multiplication of the agent to take place.
The infecting dose of a microorganism may depend in varying degrees on
the infectivity, pathogenicity, and virulence of the microorganism itself.
The entire transmission process constitutes the chain of infection. Within
the healthcare setting, the reservoir of an agent may include patients
themselves, healthcare workers, tap water, soap dispensers, mechanical
ventilators, intravenous devices and infusates, multidose vials, and other
factors in the environment (Johnson and Gerding ,1998). .
1-Direct 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) (Johnson and Gerding ,1998).
Droplet of saliva are expelled from respiratory tract by coughing .
sneezing and talking . These droplets may contain a small number of
pathogenic organism from respiratory tract . The large droplets (more
than 0.1 mm in diameter ) fall to the ground within a few seconds and are
not inhaled. Small droplet (less than 0.1 mm in diameter ) evaporate
rapidly and these very small particle , called droplet nuclei , can remain
airbrone for hours and be inhaled in the same way as small dust particles
and carried deep into alveoli of the lungs ( Goldmann et al , 1996).
Transmission of Neisseria meningitidis, group A streptococcus, or
the respiratory syncytial virus (an important cause of respiratory infection
in young children worldwide) by large respiratory droplets that travel
only a few feet is regarded as a special case of direct-contact
transmission. Vertical transmission of infection from mother to fetus is
another form of direct-contact transmission that may occur through the
placenta during pregnancy (e.g., HIV, rubella virus, hepatitis B virus, or
parvovirus) ,by direct contact of the infant with the birth canal during
childbirth (group B streptococci) or via breast milk (HIV) (Lennox et
al ,2004).
Numerous studies document the pivotal role of healthcare workers’
(HCWs) hands in the propagation of micro-organisms within the
healthcare environment and ultimately to patients (WHO,2006). Patients’
skin can be colonized by transient pathogens that are subsequently shed
onto surfaces in the immediate patient surroundings, thus leading to
environmental contamination.As a consequence, HCWs contaminate their
hands by touching the environment or patients’ skin during routine care
activities, sometimes even despite glove use (Pittet et al .,2006) .
Failure to perform appropriate hand hygiene is a leading cause of
health care associated infections and the spread of multidrug-resistant
organisms and contributes to outbreaks (CDC,2002). It has been shown
that organisms are capable of surviving on HCWs’ hands for at least
several minutes following contamination. (Karadeniz et al .,2001 ) .
2- Indirect transmission
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. Environmental conditions such as low temperature or
humidity appear to be crucial for the persistence of these organisms on
inanimate surfaces (Kramer et al ., 2006) .
The airborne transfer of droplet nuclei is the principal route of
transmission of Mycobacterium tuberculosis, varicella (chicken pox), and
measles ( Sepkowitz ,1996), while the transmission of Legionella spp.
through the air in droplet nuclei from cooling tower emissions, and from
environmental water sites, such as air-conditioning systems, central
humidifiers, and respiratory humidification devices, is another recent
important example of this type of spread ( CDC,1997).
Intrinsic contamination of commercially manufactured products, e.g.,
intravenous fluids, may also occur, e.g., as in 1970/1971 when 150 cases
of hospital-acquired bacteraemia were noticed during a large outbreak in
eight hospitals in the USA. Contamination of blood products and
heparine sodium chloride solutions may also occur . The most frequent
pathogens were Yersinia enterocolitica, and Serratia spp. for blood
products and Burkholderia cepacia and Enterobacter spp. for substances
other than blood products. (Vonberg and Gastmeier.,2006) . Another
frequent route of contamination is by the use of multi dose vials (MDV)
that had been manufactured for single use only (O’Grady et al .,2002 ).
Nosocomial Infections are frequently caused by environmental
organisms and have been linked to a wide variety of contaminated
hospital equipment, suggesting that the risk of NI following contact with
equipment is high . Equipment used in the non-critical setting is less
likely to have standard cleaning protocols than equipment used in the
critical setting, making it more likely to carry large numbers of micro-
organisms. The majority of bacterial species isolated were those found in
normal skin and environmental flora; coagulase-negative staphylococci
were isolated most frequently ( Karadeniz et al .,2001).
The high levels of contamination ,on stethoscope membranes -
diagnostic ultrasound - stethoscope ear tips – stethoscopes - otoscopes
and auriscopes . A higher proportion of death was found in patients who
received insertions of arterial lines, chest tubes, central venous catheters,
urinary catheters, Port-A devices, and Swan-Ganz catheters. Likewise,
increased risk of mortality was evident in patients receiving hemodialysis,
endoscopic examination, total parenteral nutrition, and
immunosuppressive steroid or chemotherapy ( Sheng et al . ,2007) .
Vector-borne transmission by arthropods or other insects is the final
type of indirect transmission, and may be mechanical or biologic. In
mechanical vector-borne transmission, the agent does not multiply or
undergo physiologic changes in the vector; in biologic vector-borne
transmission, the agent is modified within the host before being
transmitted. In tropical countries with endemic vector-borne disease,
including dengue, yellow fever, and malaria, this type of transmission is
more important, requiring screening and other controls not required of
medical structures in colder climates (Daniel et al .,1992) .
Predisposing factors 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 , invasive procedures including central venous
or urinary catheter placements ,and antimicrobial misuse are another risk
factors (Wenzel , 2007 & Klevens et al .,2007) .
1-Microbial factor
The agents causing healthcare-associated infectious diseases are
microorganisms ranging in size and complexity from viruses and bacteria
to protozoa and helminths. Bacteria, fungi, and certain viruses have been
the agents most recognized and studied as causes of healthcare-associated
infections (Emori and Gaynes ,1993).
For transmission to take place, the microorganism must remain
viable in the environment until contact with the host has been sufficient to
allow infection. Reservoirs that allow the agent to survive or multiply
may be animate, as a healthcare worker carriage of staphylococci in the
anterior nares, or inanimate in the environment, as demonstrated by
Pseudomonas species or Legionella in air-conditioning humidification
systems (Alary ,1992), Clostridium difficile spores on patient surfaces, or
Serratia marcescens growing in contaminated soap or hand lotion
preparations (Archibald et al .,1997).
Certain intrinsic and genetically determined properties of a
microorganism are important for it to survive in the environment. These
include the ability to resist the effects of heat, drying, ultraviolet light,
and chemical agents, including antimicrobials; the ability to compete with
other microorganisms; and the ability to independently multiply in the
environment or to develop and multiply within another (vector) host
Cohen et al .,1991) .
Intrinsic factors important to the production of disease include
infectivity, pathogenicity, virulence, the infecting dose, the agent's ability
to produce toxins, its immunogenicity and ability to resist or overcome
the human immune defense system, its ability to replicate only in certain
types of cells, tissues, or hosts (vectors), its ability to persist or cause
chronic infection, and its interaction with other host mechanisms,
including the ability to cause immunosuppression (e.g., HIV) (Cohen et
al .,1991) .
Many patients receive antimicrobial drugs. Through selection and
exchange of genetic resistance elements, antibiotics promote the
emergence of multi drug resistant strains of bacteria; microorganisms in
the normal human flora sensitive to the given drug are suppressed, while
resistant strains persist and may become endemic in the hospital
(Ducel ,1995) .
2-Host factor
Normally, the patient has three principal defenses against infection:
physical defenses, nonspecific immune response and a specific immune
response. Changes in these defenses determine the patient’s susceptibility
to infection (Mele et al .,1998). The host immune defenses attempt to
prevent infection. Thus, any reduction in host defenses may allow
infection to take place with a smaller dose of microorganisms and/or at a
body site that is not usually susceptible to infection . Host factors
important to the development and severity of infection or disease may be
categorized as intrinsic or extrinsic (Scrimshaw ,1989) .
Intrinsic factors ; include the age at infection; birth weight; sex; race;
nutritional status ; comorbid conditions (including anatomic anomalies)
and diseases; genetically determined immune status; immunosuppression
associated with other infections, diseases, or therapy; vaccination or
immunization status; previous experience with this or similar agents; and
the psychologic state of the host ( Cohen et al .,1991 ) .
Comorbid condition also include malignancies, recent chemotherapy
or radiotherapy, chronic renal diseases, chronic heart diseases, chronic
liver diseases, and chronic lung diseases had higher proportion of deaths.
In contrast, patients with neurologic diseases had a lower risk for death
( Sheng et al .,2007) .
Very young children and the elderly have an increased risk of poor
clinical outcome than patients in other age groups (Pruitt ,2002). Obese
adults and those who have underlying medical conditions, such as
diabetes, have also been shown to have higher morbidity and mortality.
AIDS patients seem to have more complications because of infection,
delayed wound healing and increased mortality (McCampbell et
al .,2002& Memmel et al . ,2004).
Extrinsic factors include invasive medical or surgical procedures;
medical devices, such as intravenous catheters , mechanical ventilators or
flexible bronchoscope . The epidemiologist discovered important failures
in processing and storage of the flexible bronchoscope . In this report,
cross infection was also a significant risk factor in the development of
infection, probably via the hands of healthcare workers (McNeil et
al .,2001& Zawacki et al .,2004).
3-Environmental factor
The potential for contaminated environmental surfaces to contribute
to transmission of healthcare-associated pathogens depends on a number
of factors, including the ability of pathogens to remain viable on a variety
of dry environmental surfaces, the frequency with which they
contaminate surfaces commonly touched by patients and healthcare
workers, and whether or not levels of contamination are sufficiently high
to result in transmission to patients (Duckworth and Jordens ,1990).
Pathogens such as Methicillin-resistant Staphylococcus aureus ,
Vancomycin-resistant enterococci and C. difficile have the ability to
remain viable on dry surfaces for days, weeks or even months. The
proportion of hospital surfaces contaminated with MRSA has varied
considerably in published reports, ranging from 1% to 27% of surfaces in
patient rooms on regular hospital wards, and from a few percent to 64%
of surfaces in burn units with MRSA patients. MRSA was recovered most
frequently from bedside rails (100% of those cultured), followed by blood
pressure cuffs (88%), television remote control devices (75%), bedside
tables and toilet seats (63% each), toilet rails and dressers (50% each),
door handles (38%) and intravenous pumps (25%) (Bhalla et al .,2004 &
Johnston et al .,2006).
Device-associated infections , such as catheter-associated urinary tract
infections (CAUTIs), central line-associated blood stream infections
(CABSIs), and ventilator-associated pneumonia(VAP) pose the greatest
threat to patient safety in intensive care units(ICUs). Multivariate analysis
revealed that ICU location, major teaching hospitals, university hospitals,
the type of ICU, the number of hospital beds, and beds per infection .
CABSI rates were higher in medical ICUs and VAP rates were greater in
surgical ICUs (Tambyah et al ., 2002).
Special units for intensive medical or surgical care for extensive
burns, trauma, transplantation, and cancer chemotherapy frequently house
patients with little resistance to infection and infectious diseases (Pittet et
al .,1998). In these patients, reduced inocula of pathogens are required to
cause infection, infection may take place at unusual sites, and usually
nonpathogenic agents may cause serious disease and death. Frequent
opportunistic infections in these patients require repeated, broad, and
extended therapy with multiple antimicrobials, leading to increasingly
resistant resident microbial populations (Goldmann et al .,1996 &
Archibald et al .,1997) .
Common HAIs
Urinary tract, respiratory tract, surgical site and bloodstream
infections are currently recognized as the major nosocomial infections.
Surveillance for these infections is well developed. However, it is
becoming increasingly clear that gastroenteritis outbreaks are also a
major burden on the health services of industrialized nations (Chadwick
et al.,2000) .
1-Urinary Tract Infection
Hospital-acquired urinary tract infections (UTIs) are the most
frequent nosocomial infections and are responsible for 20—30% of
nosocomial infections in medical or surgical intensive care units (ICUs) .
Patients aged over 50 years, diabetic patients, or patients who are
immunocompromised are more likely to develope a hospital-acquired
UTI (Mojtahedzadeh et al ., 2008) .
Urinary catheters are characterized by site of insertion (e.g., urethral,
suprapubic, or nephrostomy) and by duration of use (e.g., intermittent or
indwelling). Modern catheters are typically manufactured of latex rubber,
silicone- or Teflon-coated latex rubber, or solid silicone, and come in a
variety of types and sizes (Rosser et al .,1999) .
For the diagnosis of catheter-associated urinary tract infection
(CAUTI), the patient must meet 1 of 2 criteria. The first criterion is when
a patient with a urinary catheter has 1 or more of the following symptoms
with no other recognized cause: fever (temperature > 38°C), urgency, or
suprapubic tenderness or when the urine culture is positive for 105 colony
forming units per mL or more, with no more than 2 microorganisms
isolated. The second criterion is when a patient with a urinary catheter
has at least 2 of the following criteria with no other recognized cause
positive dipstick analysis for leukocyte esterase or nitrate, pyuria (≥ 10
leukocytes per mL of urine), organisms seen on Gram stain, physician
diagnosis of urinary tract infection, or physician initiates appropriate
therapy for a urinary tract infection ( Madani et al .,2009) .
The overall rates of CAUTIs in the 4 ICUs in Alexandria University
hospitals (15.7/1000 catheter-days) were higher than the US rates
described by the National Nosocomial Infections Study (3.9/1000
catheter-days) and the overall CAUTI rates described in 8 Latin
American countries as presented by the International Infection Control
Consortium (8.9/1000 catheter-days) (Talaat et al .,2009).
Several microbial agents have been found to be responsible for ICU-
acquired UTIs such as Escherichia coli, Pseudomonas spp, Proteus
mirabilis, Klebsiella spp, Enterobacter spp, Staphylococcus spp,
Enterococcus faecalis, Candida spp, and Enterococcus spp. E. coli is the
leading pathogen, implicated in 24% of all nosocomial UTIs . K.
pneumoniae is the fifth leading cause of nosocomial UTIs and is
recovered from 8% of cases. Enterobacter spp. and P. mirabilis are
ranked sixth, and are recovered from 5% of cases (Laupland et
al .,2005& Wagenlehner et al .,2006) .
2- Respiratory tract infections
Hospital Acquired Pneumonia (HAP) is defined as an inflammatory
condition of the lung parenchyma caused by infectious agents not present
or incubating at the time of hospital admission; that develop more than
48h after admission (Tablan et al .,2003). Ventilator-Associated
Pneumonia (VAP), on the other hand, is a subset of HAP and includes all
patients receiving mechanical ventilation at the time of infection. VAP
occurs almost exclusively in the ICU and represents approximately 86%
of all ICU HAP (Rotstein et al ., 2008).
HAP is the second most common nosocomial infection with a crude
overall rate of 6.1 per 1000 discharges . By comparison, the infection rate
for nosocomial urinary tract infection, the most common hospital-
acquired infection, is 11 per 1000 discharges. The incidence of HAP
varies depending on the hospital environment. Death from bacteremic
HAP occurred in 20% of patients within one week of their first positive
blood culture, and Pseudomonas aeruginosa bacteremia was associated
with the highest mortality rate (45%) (Rotstein et al ., 2008).
Ventilator-associated pneumonia is the diagnosis in a mechanically
ventilated patient with a chest radiograph that shows new or progressive
infiltrates, consolidation, cavitation, or pleural effusion. The patient must
also have at least 1 of the following criteria: new onset of purulent
sputum or change in character of sputum; organism cultured from blood;
or isolation of an etiologic agent from a specimen obtained by tracheal
aspirate, bronchial brushing , bronchoalveolar lavage, or biopsy ( Madani
et al .,2009). In general, the bacteriology of patients with HAP or VAP
is similar, although Stenotrophomonas maltophilia and Acinetobacter
species are found predominantly in VAP (Cross and Campbell .,2001&
Hanes et al .,2002&Vincent ,2004) .
Due to the predominance of certain virulent pathogens in HAP and
VAP, the concept of ‘core’ pathogens was developed . Core pathogens
should be considered as potential causes of HAP or VAP in all patients.
Core pathogens include Streptococcus pneumoniae, Streptococcus
species, Haemophilus influenzae, Enterobacteriaceae such as Escherichia
coli, Klebsiella species, Enterobacter species, Proteus species and
Serratia marcescens, as well as methicillin-susceptible Staphylococcus
aureus . Unusual pathogens such as Aspergillus species, Candida species,
Legionella pneumophila, Pneumocystis jiroveci (previously Pneumocystis
carinii), Nocardia species and viruses such as cytomegalovirus are causes
of HAP and VAP in patients who are immunosuppressed (Lynch ,
2001& Diaz et al .,2003).
In studies using expectorated sputum or endotracheal secretions
from intubated patients, up to 30% of the pathogens involved in
nosocomial pneumonia are Enterobacteriaceae . Enterobacter spp. and K.
pneumoniae are the third and fourth (11% and 8%) leading causes of
nosocomial pneumonia after S. aureus and P. aeruginosa (19% and 17%)
(CDC,1997) .
Multidrug resistant P. aeruginosa isolates were defined as isolates
demonstrating resistance to antimicrobials from 2 or more different
classes. P. aeruginosa and other nonfermentative Gram-negative bacilli
are resistant to many common antibiotics, including first- and second-
generation cephalosporins. Only advanced-generation cephalosporins,
extended-spectrum penicillins, carbapenems, aminoglycosides, and
fluoroquinolones generally offer useful activity (Jones et
al .,1997&Visalli et al .,1998& Quinn ,1998).
Mycobacteria distinct from the Mycobacterium tuberculosis complex
(M. tuberculosis, Mycobacterium microti, Mycobacterium bovis and
Mycobacterium africanum) and Mycobacterium leprae occasionally
cause disease in humans. Referred to by various names such as ‘atypical
mycobacteria’, ‘mycobacteria other than M. tuberculosis complex’ and
‘non tuberculous mycobacteria’, these mycobacteria have been
responsible for increasing worldwide reports of hospital outbreaks and
isolated cases of atypical or non-tuberculous mycobacterial infections
( Kotach ,2004).M. tuberculosis is by far the most frequent and most
important pathogen in this complex . Pulmonary TB is the most common
form of the disease, and the most important from the perspective of
hospital infection control (Huard et al .,2003).
The infectiousness of a TB patient correlates with the number of
microorganisms expelled into the air; this correlates with the site of
disease (pulmonary, laryngeal, tracheal, or endobronchial TB being the
most infectious), the presence of cough (or performance of cough
inducing procedures), the presence of acid-fast bacilli on sputum smears,
the presence of cavitation on chest radiograph, the duration of adequate
chemotherapy, and the ability or willingness of the patient to cover his or
her mouth when coughing (Kelly et al .,2004).
Multidrug-resistant TB (MDR-TB), defined as disease caused by
strains with resistance to at least isoniazid and rifampin,Transmissibility
continues for at least one week after the start of TB treatment, and this
period may be even longer for those with extensive disease or MDR-TB.
The ‘potential time of transmission’ of TB within the hospital is defined
as the infectious time span of pulmonary TB inpatients from date of
admission to seven days after starting anti-TB therapy (Phillips et
al .,2004&Andrews et al .,2007).
3-Surgical site infections
Surgical site infections(SSIs) are defined as infections occurring
within 30 days after a surgical operation (or within one year if an implant
is left in place after the procedure) and affecting either the incision or
deep tissue at the operation site. These infections may be superficial or
deep incisional infections (Owens and Stoessel ,2008 ) .
The increasing use of minimally invasive (laparoscopic) surgery has
resulted in a decrease in the incidence of SSIs. For example, in patients
undergoing cholecystectomy, the SSI rate following laparoscopic
procedures has been reported to be 1.1%, compared with 4% following
open procedures. Similarly, in patients with acute appendicitis, the SSI
rate has been reported to be 2% with minimally invasive procedures and
8% with open procedures (Boni et al . ,2006).
Possible reasons for the lower incidence of SSIs with minimally
invasive procedures include the smaller incision, earlier mobilization,
better preservation of immune system function, and decreased use of
central venous catheters . SSIs impose a substantial clinical burden.
Patients with SSIs are more likely to require readmission to hospital or
ICU treatment, and are at higher risk of death, than those without such
infections (Boni et al .,2006).
In most SSIs, the responsible pathogens originate from the patient’s
endogenous flora. The most commonly isolated organisms are S. aureus,
coagulase-negative staphylococci, Enterococcus spp. and Escherichia
coli; however, the pathogens isolated depend on the procedure . An
increasing number of SSIs are attributable to antibiotic-resistant
pathogens such as meticillin-resistant S. aureus (MRSA) or Candida
albicans. This development may reflect the increasing number of severely
ill or immunocompromised surgical patients, and the widespread use of
broad-spectrum antibiotics (Dohmen ,2006).
Pathogens may also originate from preoperative infections at sites
remote from the operative site, particularly in patients undergoing
insertion of a prosthesis or other implant. In addition to the patient’s
endogenous flora, SSI pathogens may originate from exogenous sources
such as members of the surgical team, the operating theatre environment,
and instruments and materials brought within the sterile field during the
procedure. Such pathogens are predominantly aerobes, particularly Gram-
positive organisms such as staphylococci and streptococci (Owens and
Stoessel ,2008 ).
4-Burn infections
Burns are one of the most common and devastating forms of trauma.
Patients with serious thermal injury require immediate specialized care to
minimize morbidity and mortality. Burn wound infections are one of the
most important and potentially serious complications that occur in the
acute period following injury (Appelgren et al .,2002).
Risk factors identified in patients colonized with drug-resistant
organisms include prior use of third-generation cephalosporins and
antibiotics active against anaerobes, critically ill patients with severe
underlying disease or immunosuppression and prolonged hospital stay
(Tredget et al .,2004).
Sources of organisms may be endogenous (patient’s own normal flora)
or exogenous (environmental or from health care personnel). Organisms
associated with infection in burn patients include Gram-positive, Gram-
negative and yeast or fungal organisms . Organisms of particular concern
include MRSA , enterococci, group A B-hemolytic streptococcus, Gram-
negative rods such as Pseudomonas aeruginosa and Escherichia coli
(Tredget et al .,1992).
The major streptococcal species encountered are group A β-
hemolytic streptococci (Streptococcus pyogenes) (GABHS), group Bα -
hemolytic streptococci (Streptococcus agalactiae) and Streptococcus
pneumoniae . GABHS were important pathogens in burn units before the
introduction of routine penicillin prophylaxis for patients with thermal
injuries. They continue to cause episodic burn wound infections and
occasional outbreaks (Stanley et al .,1997).
Fungal organisms, especially Candida (yeast) species and molds such
as aspergillus, mucor and rhizopus, have been associated with serious
infections in burn patients . Candida colonization seems to be primarily
from endogenous sources, whereas molds are ubiquitous in the
environment and can be found in air handling and ventilation systems,
plants and soil ( Rafla and Tredget , 2010).
5-Blood stream infections
Bloodstream infections are an important cause of morbidity and
mortality in immunocompromised population .In patients with
haematological disorders, infection may lead to delayed administration of
chemotherapy, prolonged hospitalisation, and additional costs associated
with directed antimicrobial therapy ( Cherif et al .,2003).
Staphylococcus aureus bacteraemia is not uncommon in patients with
haematological malignancy. One 10-year study estimated that 7% of all
bacteraemic episodes in neutropenic patients with malignancy were
caused by this organism . As with non-neutropenic patients, S. aureus
bacteraemia is associated with skin and soft-tissue infections, or with
intravascular devices ( Gonzalez-Barca et al .,2001).
The Coagulase-negative staphylococci (CoNS) are responsible for
the largest proportion of central venous catheter-related bloodstream
infections in inpatients with post-operative endophthalmitis , prosthetic
valve endocarditis , native valve endocarditis , shunt-related central
nervous system infections , pneumonia, osteomyelitis and wound
infection (O’Grady et al .,2002 ).
Coagulase-negative staphylococci are frequently identified as skin
commensals in this population, and are often regarded as a contaminant
when isolated in blood cultures ( Favre et al .,2005). However CoNS
may also be responsible for clinically significant nosocomial bacteraemia,
especially in relation to intravascular devices and underlying
immunocompromised states (Beekmann et al .,2005). This group of
organisms is the single largest cause of bloodstream and central venous
catheter (CVC)-related bloodstream infections (Velasco et al .,2004&
Ortega et al .,2005 ).
Methicillin-resistant Staphylococcus aureus (MRSA) has been
recognised as an important and universal hospital-acquired pathogen
causing endemic and epidemic infections in healthcare centres
worldwide. MRSA bacteraemia is usually hospital acquired, has a high
incidence in ICU, and is commonly associated with intravenous devices.
Several different genotypes of MRSA rather than a specific type are
associated with invasive infections in a hospital . Mortality associated
with MRSA bacteraemia has been reported to be significantly higher than
Methicillin-susptible Staphylococcus aureus (MSSA) bacteraemia
(Whitby et al .,2001).
Streptococcus pneumoniae bacteraemia may be associated with
pneumonia or CVC-related infection. Risk factors for pneumococcal
bloodstream infection include autologous transplantation with total body
irradiation conditioning, allogeneic bone marrow transplantation , and
chronic Graft versus host disease , with significant mortality
(approximately 20%) in both early and late transplant periods (Engelhard
et al .,2002).
Outbreaks of Pseudomonas spp. bacteraemia have been reported in
relation to disinfectant fluids. Pseudomonal bloodstream infections may
be associated with high mortality ( Vianelli et al .,2006& Siebor et
al .,2007).
In adult Bone marrow transplant recipients, anaerobic bloodstream
infections have been found to be associated with severe mucositis.
Anaerobic bacteraemia in the haematology population has also been
associated with neutropenic enterocolitis ( Lark et al .,2001). In patients
with haematological malignancy, colonization with Candida and previous
glycopeptide therapy have been associated with increased risk for
development of candidaemia (Worth and Slavin , 2009).
Candida species were isolated as a causative agent in 10—15% of all
nosocomial infections, 70—80% of all nosocomial fungal infections and
of 8—10% of nosocomial bloodstream infections within the last 10—15
years. Candidemia is the most frequently encountered clinical form of
invasive candidiasis (Diekema et al .,2004& Puzniak et al .,2004) .
C. albicans (40—60%), C. glabrata, C. tropicalis and C.
parapsilosis are the most frequently encountered causative agents in
candidemia (Viudes et al .,2002&Pfaller et al .,2006). Cutaneous lesions
may develop in patients with candidemia, especially those with acute
leukemia. Although these lesions may be extremely variable in number
and appearance, they are usually described as firm, erythematous, raised
nodules (Darmstadt et al .,2000).
The use of total parenteral nutrition , candida colonization (or
candiduria) and surgery (especially abdominal surgery) were the most
frequently encountered independent risk factors for candidemia.
However, the type of ICU, the severity of the underlying diseases and
antibiotic use policies can also have an important effect on the risk factors
found in all ICU candidemia studies (Gürcüog et al .,2010).
6-Device associated infection
Central venous catheter-associated blood stream infection is
laboratory-confirmed when a patient with a CVC has a recognized
pathogen that is isolated from 1 or more percutaneous blood cultures after
48 hours of vascular catheterization and is not related to an infection at
another site. The patient also has at least 1 of the following signs or
symptoms: fever (temperature > 38°C), chills, or hypotension. With skin
commensals for example, Diphtheroids, Bacillus spp., Propionibacterium
spp., Coagulase negative staphylococci, or micrococci) ( Madani et
al .,2009).
There are two main pathways leading to catheter related bloodstream
infections . The first is contact between skin surface organisms either at
the time of insertion or thereafter, leading to migration of organisms
down, and colonization of, the external catheter surface. It is thought to
be the dominant mechanism associated with short-term catheters. The
second involves transfer of organisms to the catheter hubs from patient
skin or healthcare workers’ hands, usually leading to colonisation of the
internal catheter surface, and is more common in long-term
catheters(Maki et al .,1997).
In developing and transitional economy countries, nosocomial
transmission of hepatitis C virus (HCV) through the re-use of
contaminated or inadequately sterilised syringes and needles used in
medical, paramedical and dental procedures remains a major source of
HCV infection and puts the public in these areas at high-risk (Simonsen
et al .,1999& Kane et al .,1999).
In Egypt, treatment of endemic schistosomiasis in mass programmes
(discontinued in the 1980s) that frequently used unsterilised needles and
syringes has lead to a national HCV prevalence of more than 14%, with
rates of 20–30% in young male adults (Meky et al .,2006).HCV
nosocomial transmission continues to be a problem in many countries
because of ongoing reuse of unsterilised needles and syringes in health
care facilities and programmes .Chronic HCV infection is common in
patients on chronic haemodialysis, with prevalences of 10–33% (Medhat
et al .,2002& Stoszek et al .,2006).
Intravascular catheters may not only serve as the portal of entry
for Candida species and be an important primary source of candidemia
but also provide a secondary site of attachment for Candida species that
invade the bloodstream from other sites, most frequently the
gastrointestinal tract (Walsh et al ., 1993) .
7-Ocular Infections
Postoperative endophthalmitis (POE) is defined as a severe
inflammation involving both the anterior and posterior segments of the
eye secondary to an infectious agent. Bacterial and fungal
endophthalmitis following penetrating keratoplasty is unusual, but the
frequency is likely higher than the rate of endophthalmitis following
cataract surgery or pars plana vitrectomy (Taban et al .,2005&Wilhelmus
and Hassan ,2007).
Complications of POE may be devastating. Despite appropriate
therapy, POE results in severe visual loss in at least 30% of patients, and
retinal detachment in 8–10% of patients ( Doft et al .,2000). Blindness
secondary to POE has been reported in up to 18% of patients (Yamada et
al .,2002). Common pathogens associated with POE are Staphylococcus
aureus (24%), coagulase negative Staphylococci (23%), Pseudomonas
aeruginosa (13%), Streptococcal species (8%), and Escherichia coli
(7%). Extrinsic contamination of a new intravenous anesthetic agent
without a preservative was also responsible for an outbreak of post
surgical C. albicans fungemia and endophthalmitis ( David et al .,2004) .
8-Central Nervous System Infections
Bacterial meningitis is a significant problem among hospitalized
patients. Risk factors for nosocomial meningitis were categorized as:
(1) a history of neurosurgery, CSF leakage or recent head trauma
(2) a distant focus of infection (e.g. otitis/sinusitis or pneumonia) or
(3) an immunocompromised state (Weisfelt et al ., 2007).
Isolated micro-organisms were classified as cutaneous
(Staphylococcus aureus, coagulase-negative Staphylococci,
Propionibacterium acnes), or non cuteaneous (Enterobacteriaceae,
Pseudomonas aeruginosa, Acinetobacter spp., Streptococci,
Pneumococci, enterococci)( Korinek et al .,2006).Factors associated with
a nosocomial Acinetobacter sp. meningitis include intraventricular
haemorrhage and invasive neurosurgical procedures (Wang et al .,2005).
9-Ear & Nose infection
Posttympanostomy tube otorrhea (PTTO), defined as active otorrhea
from the middle ear cavity through the patient tympanostomy tube, is the
most common complication of tube insertion. PTTO can be classified
according to time of its onset, with early PTTO occurring within 2 weeks
of tube insertion, and late (delayed) PTTO occurring after 2 weeks. PTTO
persisting for over 8 weeks can be classified as chronic PTTO. the
incidence of early PTTO has been reported to range from 5 to 38%. A
recent meta analysis found that late PTTO occurred in 26% of patients,
transient PTTO in 16%, and chronic PTTO in 3.8% ( Jung et al .,2009).
PTTO was Previously found to be caused by the same strains of
bacteria that give rise to acute otitis media , most typically Haemophilus
influenzae, Moraxella catarrhalis and Streptococcus pneumoniae.
Recently, however, atypical strains, including Staphylococcus aureus and
Pseudomonas aeruginosa, have also been isolated from PTTO. These
include antibiotic-resistant strains, including MRSA ( Coticchia and
Dohar ,2005).
Sinusitis, one of the complications of intubation or mechanical
ventilation, can lead to severe sepsis in critically ill patients. Several
studies have shown that prolonged naso-gastric or naso-tracheal
intubation, prolonged mechanical ventilation, administration of high-dose
corticosteroids and prolonged antibiotic therapy are risk factors for
sinusitis . In addition traumatic head injury may have a higher risk in
terms of maxillary sinusitis than mechanically ventilation, because of the
possible existence of bleeding in maxillary sinuses (Cengiz et al .,2009).
10-Gastrointestinal Tract Infections
Nosocomial outbreaks of gastroenteritis are a major disruption to
health services in many countries. Noroviruses are the predominant
pathogen detected in such outbreaks (Billgren et al .,2002& Green et
al .,2002&Lopman et al .,2004).Nosocomial Rotavirus gastroenteritis
(RVGE) is a major component of hospital-acquired infections in children
(Gleizes et al .,2006& Waisbourd-Zinman et al .,2009).
Prolonged hospitalisation, age above 65 years, antibiotic usage,
underlying medical conditions, neoplastic disease, gastrointestinal
surgery, nasogastric tubes, and gastrointestinal disorders including
inflammatory bowel diseases are the most important risk factors for the
development of nosocomial C. difficile-associated disease ( CDAD)
(McFarland et al .,2007& Issa et al .,2007) .
Studies have found that the prevalence of asymptomatic colonization
with C. difficile is 7%–26% among adult inpatients in acute care facilities
and is 5%– 7% among elderly patients in long-term care facilities. Other
studies, however, indicate that the prevalence of asymptomatic
colonization may be more on the order of 20%–50% in facilities where
clostridium difficile infection is endemic. The risk of colonization
increases at a steady rate during hospitalization, suggesting a cumulative
daily risk of exposure to C. difficile spores in the healthcare setting
( Rivera and Woods , 2003& Riggs et al .,2007) .
The disease has been reported in children aged from 5 days to 17
years. Risk factors for children include disrupted normal microflora of the
gastrointestinal tract (antibiotic-associated and non-antibiotic associated),
age, immune response, diet, underlying conditions, concurrent infections,
and cancer (McFarland et al .,2000) .
Ciprofloxacin and other oral quinolones are active against many
intestinal bacteria and are highly concentrated in the stool. Patients with
CDAD are at least five times more likely to have been exposed to
ciprofloxacin than control patients (Angel et al ., 2004).
Over the last two decades, candida has emerged as an important
healthcare-associated pathogen in the world .There are few studies on the
source of candidaemia. The gastrointestinal tract has been considered the
main endogenous reservoir of Candida spp., but there is increasing
evidence of exogenous acquision (Nucci and Anaissie ,2001& Aragao et
al .,2001).
11- Obstetrics and neonatal Infections
Pregnant women are more susceptible to certain infections due to
reduced cell-mediated immunity and raised corticosteroid levels. The
onset of life-threatening sepsis in pregnant women can be insidious, with
rapid clinical deterioration, and pyrexia is not always present. One reason
that the prognosis is still more favourable than the nonobstetric
population, is that the source of infection is usually the pelvis, and
potentially more amenable to intervention (Farmer et al .,2005).
Conditions associated with an increased risk of sepsis are prolonged
rupture of membranes, emergency Cesarean Section , instrumentation of
the genital tract, and retained products of conception or placenta.
Endotoxin producing aerobic Gram negative bacilli are the most common
cause (60–80%), but sometimes Gram-positive bacteria, mixed (usually
anaerobes such as Bacteroides or Clostridium) or fungal infections are
implicated (Nelson-Piercy ,2006).
Septic shock with Disseminated Intravascular Coagulopathy is an
ominous sign if it develop . Sepsis is defined as Systemic inflammatory
response syndrome secondary to an infection, and severe sepsis when
there are features of organ dysfunction such as hypotension or oliguria.
Septic shock has developed when hypotension persists despite adequate
fluid resuscitation. Sepsis is the leading cause of multiple organ failure,
acute renal failure, and Acute Respiratory Distress Syndrome , and carries
a mortality of 40–60% (James et al .,2005) .
Vaginal flora is a dynamic ecosystem, with some differences between
vaginal and cervical flora. Anaerobic bacteria usually outnumber aerobes,
with anaerobic and facultative lactobacilli predominating. Other
anaerobes include Peptostreptococcus species, Bacteroides species, and
Prevotella species. The aerobic gram-positive flora include coagulase-
negative staphylococci, with varying amounts of streptococci,
enterococci, and Staphylococcus aureus, and the gram-negative flora
include Escherichia coli, Gardnerella vaginalis, Enterobacter species,
Klebsiella pneumoniae, and Proteus mirabilis . Both Mycoplasma and
Ureaplasma are also found in the vagina. Vaginal flora may change
during pregnancy. Some studies suggested that lactobacilli increase in
pregnancy and that other anaerobes decrease . Antibiotics also change the
flora, and the use of multiple doses of cephalosporins for prophylaxis has
been reported to increase enterococci and perhaps Enterobacter
species .C. albicans is a part of the normal microbial flora of the human
respiratory, enteric, and female genital tracts (Casey and Cox ,1997).
Group B streptococcus (GBS) is the leading cause of neonatal sepsis
and meningitis. Early-onset (EO) GBS disease is usually defined as
infection presenting in the first 6 days of life (some definitions use the
first 2, 3 or 5 days as the cut-off) and accounts for approximately 60–70%
of all GBS disease in the first 3 months of life. Maternal carriage of GBS
in the gastrointestinal and/or genital tracts is a prerequisite for EO
disease, vertical transmission occurring prior to or during birth. An
estimated 20–30% of pregnant women are colonized by GBS(Jones et
al .,2006), 50% of babies become colonized perinatally, and 1% becomes
infected. Disease occurs rapidly, being evident at birth or within 12 hours
in over 90% of cases, and presenting with overwhelming sepsis (60%) or
pneumonia (25%) (Heath et al .,2004).
Late-onset disease appears to have a different pathophysiology. It is
predominantly caused by serotype III, is acquired perinatally,
nosocomially, or from community sources, and in up to 50% of cases
presents with meningitis (Weisner et al .,2004).GBS disease is associated
with significant morbidity and mortality. GBS meningitis leaves half of
those infected with long-term neurodevelopmental effects at 5-year
follow-up. Neurodevelopmental impairment is also associated with
clinical (i.e. culture-negative) neonatal sepsis (Stoll et al .,2004).
Another GBS disease that is difficult to quantify is prematurity. The
association between the two is complex, but GBS is recognized as one of
the infections that may cause prematurity. The incidence of invasive
disease is certainly higher among preterm infants than among those born
at term (Malek et al .,1996). An important contributory factor may be the
lack of maternal antibody, as materno-fetal transport of IgG is inversely
related to gestational age. Thus, preterm infants born to GBS-colonized
mothers will have low or absent concentrations of protective maternal
antibody. Premature rupture of membranes (i.e. rupture before
spontaneous onset of labour) is strongly associated with early-onset GBS
disease (Oddie and Embleton ,2002).
Diagnosis of HAIs
Sampling
Examples of biological materials that are analyzed in clinical
laboratories include whole blood , serum , plasma , urine , feces , saliva ,
spinal and other body fluid . All these specimens also used for molecular
testing . Specimens devoid of patient cells are useful in detection of
infectious agents (CLSI .,2006) .
1-Blood
Special blood-collecting equipment is used . Sterile collecting
bottles containing the proper nutrient broth media , blood collecting sets
with needles and tubing that allow the blood to flow into the collecting
bottle and the proper skin-cleaning supplies are necessary to ensure a
properly collected blood specimen for culture . Special care must be taken
to clean the venipuncture site carefully before puncture to avoid possible
contamination of blood sample with skin contaminants . One method uses
an initial cleaning with a 70% solution of alcohol to remove dirts and
lipids . A circular motion moving from puncture site out is used . A 1-
10% povidine-iodine solution is used , followed by an alcohol rinse
(CLSI ., 2003).
2-Cerebospinal fluid
Physician collects Cerebospinal fluid (CSF) through a lumbar
puncture. Rapid handling of CSF samples in the laboratory is important
because of serious nature of meningitis and organism in meningitis are
sensitive to temperature change ,so refrigeration not done . CSF is
typically placed in sterile tubes . The tubes are sent to laboratory
immediately for testing , including culture and Gram stain (Finn ,2007) .
3-Stool
Stool specimens contain large numbers of bacteria (normal flora) ,
and a stool specimen is usually cultured only to isolate certain types of
pathogenic enteric organism . Stool specimen should be cultured within 2
hours . If this cannot be done , transport media special for stool samples
can be used . Swabs of the rectal area can be used for infant , but this is
not the preferred method of collection for other age groups (Finn,2007) .
4-Sputum
When a specimen of sputum is collected , the patient must cooperate
fully to ensure that a proper specimen is obtained . Sputum is usually
collected in the morning and it should be sent to the laboratory and
processed immediately . Deep coughing will usually bring up a good
sputum specimen . It is necessary to avoid collecting saliva . A wide
mouthed sterile container is best used for collecting this type of specimen.
An acceptable sputum specimen that free from contamination with saliva
can be Gram-stained and checked microscopically for the presence of
squamous epithelial cells . Finding an average of more than 10 squamous
epithelial cells per low power field indicate that the specimen is saliva
and is not acceptable specimen to culture ( Muarry et al ., 2003) .
5-Swab of various fluids
Swabs are used to collect cultures from various openings of the body
, such as the nose ,throat , mouth , vagina , anus and wound . These swabs
must be collected carefully and placed in the proper transport media
before they are taken to the laboratory for processing . If swabs are not
properly handled , the micro-organism may dry out , or their numbers
may be insufficient for culture (Finn , 2007) .
6-Urine
The collection of urine for microbiological studies also requires the
cooperation . A clean –catch midstream sample , usually the first morning
specimen , is suitable for culture , provided care has been taken to clean
the uretheral area before the collection . A sterile container must be used
for the collection of the urine for the culture . When the patient is too ill
or cannot void properly , aspecimen is obtained by catheterization . After
collection , specimens should be sent to the laboratory for the immidate
processing or refrigeration , or a preservative can be used to maintain
bacterial counts (CLSI ., 2001) .
Methods :
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. This approach has begun to change over the past 2 decades, with
the development and implementation of new technologies based on DNA,
or molecular, analysis. These DNA-based molecular methodologies ,
include pulsed-field gel electrophoresis (PFGE) and other restriction-
based methods, plasmid analysis, and PCR-based typing methods. The
incorporation of molecular methods for typing of nosocomial patho-gens
has assisted in efforts to obtain a more fundamental assessment of strain
interrelationship (Cockerill and Smith, 2004).
A number of nosocomial infections are endemic, epidemic or
sporadic infections. Most nosocomial infections are endemic and are the
focus of most infection control efforts. The earliest methods that were
used to identify and type organisms were based upon their phenotypic
characteristics. This is followed by a series of genotypic methods (Figure
2) , one of the most widely utilized techniques is biotyping, or the
differentiation of strains based on properties such as differences in
biochemical reactions, morphology, and environmental tolerances (Singh
et al . ,2006 ).
There are a number of important attributes for successful typing
schemes : the methodologies should be standardized, sensitive, specific,
objective, and subject to critical appraisal. All typing systems can be
characterized in terms of typeability, reproducibility, discriminatory
power, ease of performance and interpretation, and cost (in terms of time
and money) . Typeability refers to the ability of a technique to assign an
unambiguous result (type) to each isolate. Non typeable isolates are more
common with phenotypic methods but can also occur with genotypic
methods. 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 . In general, phenotypic methods have lower discriminatory power
than genotypic methods (Olive and Bean ,1999).
Figure 2. Recent evolution of bacterial strain identification for
epidemiological purpose ( Belkum , 2007)
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 often lacks discriminatory power
because of variations in gene expression and random mutations that may
alter biologic properties of microorganisms. 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 (Struelens ,2002) .
2-Antimicrobial susceptibility testing
Strains defined by this method should always be confirmed by
genomic typing, because unrelated clones can undergo evolutionary
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. Disk
diffusion methods are not used as commonly as they once were because
of the lack of automation for testing. Microdilution testing provides a
quantitative measure of the minimum bactericidal concentration (MIC),
which is defined as the lowest concentration of the antimicrobial agent
that inhibits the growth of the organism (Barenfanger et al .,1999).
Bactericidal activity can be measured by one of three methods:
(1) calculation of the minimum bactericidal concentration;
(2) performance of time-kill studies; or
(3) serum bactericidal assay . The minimum bactericidal concentration is
obtained by subculturing the tubes (macrodilution) or wells
(microdilution) that do not demonstrate growth at 24 hours and is defined
as the lowest concentration of antibiotic at which a 99.9% reduction of
viable organisms occurs.A bactericidal drug achieves this within two
dilutions of the MIC (Bates et al .,1991).
Agar dilution is a well-established technique for obtaining
quantitative susceptibility results and is the reference method commonly
performed in Europe. Mueller-Hinton agar (MHA) is the recommended
medium for the testing of most commonly encountered aerobic and
facultative anaerobic bacteria and is standardized such that calcium and
magnesium supplementation is not indicated. For predictable diffusion,
the depth of the agar should be between 3 and 4 mm. The recommended
final inoculum is 104 CFU per spot. The method is labor-intensive and
costly, but is recommended for fastidious organisms that do not grow
well in broth media (Neisseria gonorrhoeae, anaerobes)( Jorgensen and
Turnidge , 2007).
The disk diffusion method has been standardized for the testing of
common, rapidly growing organisms and allows for a qualitative
categorization of isolates as susceptible, intermediate, or resistant. An
antibiotic-impregnated filter paper disk is placed on the surface of an agar
plate inoculated with a ‘‘lawn’’ of organism at a known turbidity (0.5
McFarland). The inoculum may be prepared by either log phase growth
or direct suspension from colonies on the agar plate. As with dilution
methods, direct suspension is preferred for fastidious organisms and for
detection of methicillin resistance in staphylococci. The antibiotic
diffuses through the agar almost instantaneously after placement on the
agar and creates a zone of inhibition that can be measured in millimeters
(edge to edge, including the disk) (Figure 3) ( Jorgensen and
Ferraro ,2000) .Interpretation of the test is based on the inverse
correlation of the zone diameter with MIC for each combination of
antimicrobial and organism (CLSI,2008).
Figure 3. Disk diffusion testing of a hyper b-lactamase producing
Staphylococcus aureus. The zone of inhibition (clear halo) around the
cefoxitin disk (FOX) is sharp. In contrast, the zone of inhibition around
the oxacillin disk (OX) is blurred by the presence of individual colonies
(Malhotra-Kumar et al .,2008).
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 ( Reis et
al .,2000) .
There are a number of different ways in which serotyping can be
performed; each varies in the way in which the antibody-antigen reactions
are detected. Often direct antibody-antigen agglutination is used, in which
a bacterial cell suspension is mixed with panels of antibodies. Based upon
agglutination profiles, the serotype is determined. Additionally, for
organisms such as S. pneumoniae the quellung test is used, in which test
antibodies bind to the corresponding capsular antigens and induce
swelling of the capsule, which can be observed with microscopy.
Limitations of serotyping include a lack of availability of certain antisera
and problems with standardization of different methods(Babl et
al .,2001).
4-Bacteriophage and bacteriocin typing
Bacteriophage and bacteriocin typing as epidemiologic tools are
limited to bacteria. Bacteriophage (phage) (figure 4) 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 ( Hopkins et al .,2004).
Bacteriophage typing has some drawbacks due to a lack of
widespread availability of biologically active phages and the technical
difficulty of performing the technique, but the method has been applied to
a number of bacteria associated with nosocomial infections, such as S.
aureus, P. aeruginosa and Salmonella species. Additionally strains can be
typed based on their susceptibility to a set of heterogeneous substances
(generally proteins) that are produced by other bacteria. These inhibitory
compounds, or bacteriocins, often limit the growth of closely related
species . Bacteriocin typing has had limited utility because of drawbacks
similar to those of phage typing, but it has been used for typing P.
aeruginosa . Additionally, an analogous approach has been developed for
Candida species (particularly C. albicans) (Singh et al .,2006 ).
Figure 4 . Phage typing is the identification of bacterial species and
strains by determining their susceptibility to various phages
(pearson , 2004) .
B-Genotypic methods.
Molecular techniques can be very effective in tracing the spread of
nososcomial infections due to genetically related pathogens, which would
allow infection control personnel to more rationally identify potential
sources of pathogens and aid infectious disease physicians in the
development of treatment regimens to manage patients affected by related
organisms. Therefore, the use of molecular tests is essential in many
circumstances for establishing disease epidemiology, which leads to
improved patient health and economic benefits through the reduction of
nosocomial infections (figure 5) (Singh et al .,2006 ).
The isolates involved in a nosocomial outbreak are genetically
related and thus originate from the same strain. Therefore, the use of
strain typing in infection control decisions is based on several
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 ).
Figure 5. Flow chart comparison of the different procedural steps
used for various molecular typing techniques (Chang and
Chui ,1998).
1- Plasmid Analysis
Among genotyping methods, plasmid analysis is essential for the
epidemiologic analysis of HAIs with multiple antibiotic- resistant
organisms and for tracing dissemination of mobile antibiotic-resistance
genes. Plasmid typing was the first molecular method to be used as a
bacterial typing tool . Plasmids are self-replicating, often-transferable
extrachromosomal DNA elements in the prokaryote cytoplasm. Typing is
performed through the isolation of plasmid DNA and comparison of the
numbers and sizes of the plasmids by agarose gel
electrophoresis(Eisgruber et al .,1995).
Some bacteria have large plasmids (Figure 6) in the range of 100 to
150 kb, making their separation difficult; for these strains, the addition of
a restriction endonuclease digestion step following plasmid isolation will
often aid in typing because multiple fragments are generated, which
makes interpretation of strain relatedness more feasible. Plasmid
restriction is also commonly used for the analysis of staphylococci and
enterococci, whose plasmids are typically less than 50 kb in size. The
inclusion of restriction enzyme analysis increases the discriminatory
power of plasmid analysis (Liu et al .,1996).
Plasmids are not generally helpful in differentiation between endemic
and epidemic strains, because plasmids are often mobile
extrachromosomal DNA fragments that can be acquired and deleted. A
consequence of this plasmid mobility is that epidemiologically related
isolates can exhibit different plasmid profiles. Many plasmids carry
antibiotic resistance determinants that are contained within mobile
genetic elements (transposons) that can move in or out of plasmids and
the chromosome, allowing for the DNA composition of a plasmid
potentially to change rapidly (Figure 6) (Feil et al ., 2003).
A transposon epidemic is suggested when isolates from different
species, or isolates of the same species with differing PFGE and profiles
plasmid contents, have similar resistance genes. Further analysis by PCR
and DNA sequencing of transposon content by insertion sequence
evaluation have been useful to establish the potential of a transposon
epidemic (Thal et al .,1998).
Figure 6 ; Schematic diagram of Plasmid Analysis (Access
Excellence , 2009)
2- Pulsed-field gel electrophoresis
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 (Figure 7) . 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. More recently, restriction
enzymes that cleave chromosomal DNA less frequently (<30) recognition
sites have been utilized for analysis. The resulting DNA fragments are too
large to be separated by conventional agarose gel electrophoresis . A
number of alternative methods, generally classified as PFGE, are capable
of separating these large DNA fragments (Stephenson , 2004 & Roberts
et al ., 2010). It has excellent discriminatory power and is broadly
applicable to bacteria and yeasts. Consensus rules are available for
interpretation of patterns in outbreak investigations ( Struelens et al .,
2001).
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 (Figure 7) 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 (Goering , 2010).
Field inversion gel electrophoresis utilizes a conventional
electrophoresis chamber in which the orientation of the electric field is
periodically inverted by 180o. CHEF uses a more complex electrophoresis
chamber with multiple electrodes to achieve highly efficient electric field
conditions for separation; typically the electrophoresis apparatus reorients
the DNA molecules by switching the electric fields at 120o angles. CHEF
has been used to evaluate the spread of various antimicrobial resistant
bacteria. The finding of isolates that have identical or related restriction
endonuclease patterns suggests spread from single strains (Finney, 1993).
Figure 7 . Diagram of pulsed-field gel electrophoresis (Davidson ,
2002) .
Ideally, the PFGE isolates representing an outbreak strain will be
indistinguishable from each other and distinctly different from those of
epidemiologically unrelated strains. If this occurs, the outbreak is
relatively easy to identify. Alternatively, random genetic events, such as
point mutations or insertions and deletions of DNA, that can alter the
restriction profile obtained during the course of an outbreak can occur
(Quintiliani and Courvalin, 1996& Thal et al .,1997).
The purpose of interpretative criteria is to establish a guide for
distinguishing true differences in strains from random genetic
polymorphisms that may occur over the time of a given nosocomial
outbreak. Appropriate interpretative criteria provide consistent, objective
guidelines for correlating restriction pattern variations observed between
individual isolates and the putative outbreak strain and provide an
estimate of the likelihood that the isolate is part of the outbreak
( Goering, 1998).
This correlation focuses on the number of genetic events required to
generate the observed pattern variation. Because only a small portion of
the organism’s genetic component is undergoing analysis, isolates that
give identical results are classified as “indistinguishable,” not “identical.”
With the detection of two genetic variation events by differences in
fragment patterns compared to the outbreak strain, the determination of
relatedness to an outbreak falls into a gray zone. The results may indicate
that these isolates are related (especially if isolates were collected over a
long period of time, such as 3 to 6 months), but there is also a possibility
that strains are unrelated and not part of the outbreak (Struelens , 1996 &
Tenover et al .,1997).
3-Southern Blot Analysis-Ribotyping
The bacterial DNA is digested using a frequent cutting restriction
enzyme, the DNA fragments are separated by agarose gel electrophoresis,
and then the fragments are transferred (blotted) onto a nitrocellulose or
nylon membrane. Next, a labeled (colorimetric or radioactive) piece of
homologous DNA is used to probe the membrane. Under the appropriate
conditions, the probe hybridizes to a complementary base pair, and the
banding patterns are resolved through the detection of the probe label.
The discriminatory power of this method is related to the copy numbers
of the targeted genetic elements in the bacterial genome and their
distribution among the restriction fragments following electrophoresis.
Variations in the number and sizes of fragments detected are used to type
the microorganisms (Singh et al , 2006).
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 methods ; however, a variety of
organisms have been studied using this method . Most bacterial species
have several ribosomal operons per chromosome and produce ribotype
patterns of 5–15 bands. Ribotyping exhibits excellent reproducibility but
only moderate discriminatory power for most bacterial species. An
automated ribotyping system is commercially available (figure 8) (
Maiden et al ., 1998).
Figure 8 . schematic diagram of Southern Blot Analysis (pearson ,
2004) .
4-Northern blotting
Northern blotting is a technique that uses RNA rather than DNA .
RNA is transferred from the gel after electrophoresis onto a solid support
followed by hybridization with specific labeled probe . Because RNA
molecules have defined lengths and are much shorter than genomic
DNA ,it is not necessary to cleave RNA before
electrophoresis .However , because of the secondary structure of RNA , it
is necessary to perform electrophoresis under denaturing condition,
usually with formaldehyde or fomamide buffer in agarose gels. RNA
extracted from cells consists primarily of ribosomal and transfer
RNA .The mRNA comprises only 1% to 2% of total cellular RNA . After
electrophoresis and staining ,intact RNA reveals two clearly visible bands
of ribosomal RNA (figure 9) (Benson et al .,2002).
Figure 9 . Schematic diagram of Northern blotting (Alberts etal .,
2008).
5-Heteroduplex Migration Analysis
Heteroduplex migration analysis reveals the presence of mutation by
the altered electrophoretic mobility of a dsDNA fragment that contains
one or more mismatched bases (heteroduplex ) versus one that is
perfectly matched ( homoduplex ). 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 0 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 .
Heteroduplexes usually tend to migrate more slowly than
homoduplexes during electrophoresis . Whereas mutant alleles are often
present as heterozygotes in a clinical specimen , homozygous mutation
require mixing with wild –type DNA for the mutation to be detected
(Schouten et al .,2002).
6-Single Strand Conformation Polymorphism Analysis
Single strand conformation polymorphism analysis is a technique
used to scan for unknown variants in nucleic acid sequence . 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 . If
the sequence of a reference sample differs from that of the fragment being
tested , even by only a single nucleotide , often at least one of the strands,
if not both , will adopt different conformations and exhibit a unique
banding pattern . Results are visualized by silver staining of the gel or by
fluorescent detection using labeled primers during PCR (Reed and
Wittwer , 2004) .
7-Typing Methods Using PCR
a-Multiplex PCR
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. 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 (Focucault et
al .,2005). 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, especially as
the number of primer pairs in the reaction mixture increases (Francois et
al .,2004).
b-Nested PCR
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 is used to
amplify a target sequence , 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; if the primary amplification was
nonspecific, the secondary amplification would not occur . A major
drawback of nested PCR is that the reaction vessel needs to be opened in
order to add the second primer set, increasing the potential for
contamination of the work environment with amplified DNA (Eribe and
Olsen , 2000).
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 (Struelens et al,2004).
This simple and rapid technique can be used for strain typing of bacteria,
fungi and protozoa. AP-PCR typing exhibits variable discriminatory
power according to the number and sequence of arbitrary primers; its
performance is limited by a poor reproducibility and the lack of
consensus rules for interpretation of results. 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
(Louie et al .,1996).
Amplification will continue if two of the primers bind in close enough
proximity to one another on the complementary strands to allow synthesis
of the DNA fragment. Although the method is much faster than many of
the other typing methods for nosocomial pathogens, it is much more
susceptible to technical variation than most other methods. Slight
variations in the reaction conditions or reagents can lead to difficulty in
reproducibility of results and to differences in the band patterns
generated. Therefore, trying to make comparisons among potential
outbreak strains can be very problematic. When tightly controlled, AP-
PCR can provide a high level of discrimination, especially when multiple
amplifications with different primers are performed (Samore et
al .,1996).
d- Amplified fragment length polymorphism
Amplified fragment length polymorphism (AFLP) is a typing
method that utilizes a combination of restriction enzyme digestion and
PCR . In the AFLP procedure,( Figure 10) the DNA is digested with two
different restriction endonucleases, usually chosen so that one cuts more
frequently than the other. This restriction strategy generates a large
number of fragments. In order to make the interpretation of the results
more feasible, only a specific subset is used for isolate comparison. The
subset is generated by linking adapter sequences to the ends of the
restriction fragments extending the length of the known end sequences.
PCR primers are designed to hybridize to the adapter sequence, the
remaining restriction site sequence, and an additional one or two
nucleotides of the unknown template sequence (Neeleman et al .,2004).
The addition of each nucleotide, chosen at random, to the end of the
primer reduces the number of fragments that will be amplified by a factor
of four. Following PCR, the reaction products are separated by gel
electrophoresis and their banding patterns are resolved. ( Szczuka and
Kaznowski , 2004& Whatmore et al .,2005).
Figure 10 . Schematic diagram of the amplified fragment length
polymorphism (AFLP) technique. (Jiang et al ., 2000) .
e- Restriction fragment length polymorphisms
As alternative, several PCR-based typing methods have been
developed. In PCR-RFLP typing, a polymorphic DNA target sequence is
PCR-amplified and digested with restriction endonucleases, separated by
electrophoresis, and isolates are compared by RFLP pattern(Figure 11).
This simple technique is well reproducible, with moderate discrimination.
Genome RFLP analysis can use different types of probes, including
ribosomal RNA or cloned ribosomal DNA sequences. The latter
application, called ribotyping, is the most universal RFLP typing strategy
(Leeuwen et al .,2002).
Figure 11 . Schematic diagram of Restriction fragment length polymorphisms (Freeman , 2002) .
f-Repetitive element PCR
Repetitive element PCR (rep-PCR) typing targets spacer fragments
lying between repeat motifs of the genome. It shows moderate
discriminatory power but a better reproducibility than AP-PCR analysis.
These PCR typing methods are often used as a first-pass method to assess
the clonality of organisms during outbreak investigations. 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 (Leeuwen et al .,2002) .
The number of repetitive elements and their respective genomic
locations are used to genotype isolates and to differentiate highly related
strains. In rep-PCR, primers are designed to amplify regions between
repetitive sequences, resulting in products of various lengths and
sequences. Electrophoretic resolution of these fragments generates a
profile, or “fingerprint,” that contains multiple bands of different sizes.
The profiles are compared to a library database, and cluster analysis is
performed to identify matching or similar patterns. (Clarridge,2004).
g- Multi Locus Variable copy Numbers of Tandem Repeats Analysis
Multi Locus Variable copy Numbers of Tandem Repeats (VNTR)
Analysis (MLVA) is a method for high -resolution typing of microbial
isolates based upon VNTR) . 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 . MLVA has high
discriminatory power within clonal species and appears to be more rapid
and more amenable to standardization than pulse-field gel electrophoresis
for both surveillance and outbreak investigations (Torpdahl et al .,2007) .
8 -DNA arrays
Conventional DNA micro arrays consist of nucleic acid probes
deposited on a planar glass surface. The surface is usually coated with
chemically reactive groups (epoxy, poly-L-Lysine or aldehyde) to ensure
efficient binding of nucleotidic probes on the surface. To assess the
presence of target genes, nucleic acid samples are labeled, either
chemically or by an enzymatic reaction. Labeled samples are then
hybridized onto the array, and washed using different stringency buffers.
The remaining signal resulting from specific interactions between probes
and target nucleic acids is measured using a confocal micro array scanner.
Only probes hybridized to a labeled target will yield signal, thus revealing
the presence of the cognate nucleic acid motif in the sample (Wang et al .
, 2003).
High-density DNA micro arrays have been used in a broad variety of
applications such as transcriptomics, comparative genome hybridization
(CGH), resequencing, drug discovery, microbial community
characterization or single nucleotide polymorphism (SNP) analysis. The
field of application depends primarily on the strategy and the marker
genes used to design the probes. A variety of genes (virulence factors,
phylogenetic markers, antibiotic resistance genes, etc.) have been
employed on microbial diagnostic microarrays, depending on the
question raised by the researcher. Microarrays have thus been
instrumental in the detection of known pathogens as well as in the
discovery of novel infectious agents, such as severe acute respiratory
distress syndrome (SARS) (Lin et al ., 2007).
Comparative genome hybridization, called comparative phylo-
genomics by some, is the technology used to assess differences among
bacterial strains by hybridizing labeled genomic DNA fragments to
synthetic DNA arrays. The components of these arrays share homology
with all of the genes identified to be present in the organism on the basis
of the species’ genome sequence . Binary probe typing systems detect
polymorphism in the bacterial genome by solid-phase hybridisation of
total DNA with a panel of sequence-variant specific probes immobilized
on a solid substrate . This highly reproducible method is currently
developed on DNA micro-array technology (Figure 12). These
approaches are suitable to combine genotyping with resistance and
virulence gene profiling, thereby providing increased information for the
epidemiologic monitoring of pathogens. ( Stabler , 2006).
These methods identify the genome complements of strains for
which no genome sequence is available . The outcome of such analyses
can lead to the development of high-throughput and sensitive typing
methods by selecting for only those genes that show differential absence
or presence in a black-and-white strain-specific fashion . For example,
recent studies with S. aureus revealed that approximately 20% of the
staphylococcal genome consists of core-variable and variable sequences
Obviously, the later category of nucleic acid sequences is best suited for
identification of potentially useful typing targets (Lindsay ,2006).
Figure 12 . Schematic diagram of DNA arrays (Affymetrix .,2002) .
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. The
polymerase incorporates nucleotides (deoxynucleoside triphosphates
[dNTPs]) as they are added one at a time to extend the complementary
strand from the primer sequence. Incorporation of the nucleotide is
accompanied by eqimolar release of pyrophosphate, which is
subsequently converted to ATP by ATP sulfurylase (Ronaghi , 2001) .
ATP is used to drive the luciferase mediated conversion of luciferin
to oxyluciferin. This reaction produces light, and a charge-coupled-device
camera detects the chemiluminescent signal. The sequence data are
interpreted as a peak in the pyrogram, and the peak height is proportional
to the number of incorporated nucleotides. Unincorporated nucleotides
and ATP are degraded by apyrase, and the entire process begins again
with the addition of a different dNTP. Today, with increased automation
and decreasing costs of DNA sequencing technology, determination of
nucleotide sequence is emerging as the reference method for comparing
microbial and virus types based on localised genomic polymorphism. It is
the most accurate and informative method. Moreover, sequence data are
completely portable between laboratories. For typing bacterial pathogens
of nosocomial importance, several methods are used (Tavanti et
al .,2003) .
10 -Spectroscopy
Various types of mass spectrometry (MS) are being developed to
facilitate bacterial genotyping. MS was initially focused toward species
identification, also in complex genera, such as the staphylococci .The
mass spectra of bacterial whole cells can be measured, compared, and put
into frameworks of molecular diversity. Alternatively, sequence-variable
PCR products or RNA transcripts there of can be analyzed by MS, which
can be very helpful in the generation of strain-specific MS fingerprints
(Carbonnelle , 2007).
Using this technique,( Figure 13 ) pure bacterial isolates are mixed
with a chemical matrix, spotted onto a metal plate, and then ionized by a
laser. Ionized molecules corresponding to fragmented bacterial peptides
are released from the matrix (desorption) and move in an electric field
toward a detector. The “time of flight“ corresponds to the time required
for charged ions to reach the detector and depends on the mass to charge
(m/z) ratio of the individual molecules. The resulting signal profiles of
unknown samples provide proteomic patterns, or “spectral fingerprints,”
that can be used for identification when compared to a database. The
spectra are specific to genera, species, or even strains of bacteria.
Although the spectral profiles correspond to peptides derived from
multiple bacterial proteins, the majority of signals are due to highly
abundant proteins, such as ribosomes (Sauer and Kliem , 2010 ).
Figure 13. Overview of Raman procedure and spectrometer. Biomass
from a bacterial culture (a) on Trypticase soy agar (TSA) medium is
collected using a inoculation loop and suspended in of demineralized
water (b). After a brief centrifugation step to remove air bubbles, the wet
pellet is transferred onto a fused silica slide (c), where it is allowed to dry
(a typical slide holds 24 samples). The slide with the dried biomass is
placed in the measurement stage (d), where the samples are illuminated
with laser light (e). The Raman signal generated is collected along the
same optical path and separated from the laser light using an optical filter
(f) that only reflects light of a higher wavelength than the laser. The laser
light is passed through. The wavelength of the Raman signal is dispersed
on an optical grating (g) and collected using a near-infrared-optimized
charge-coupled device detector (h). The Raman spectra are gathered,
stored, and analyzed on a personal computer (i) (Scholtes-Timmerman et
al .,2009).
11 -Proteomics and metabolomics
Proteomics combines all technologies that can be used for protein
separation and molecular characterization. Large numbers of individual
proteins expressed in living organisms can thus be identified down to the
amino acid sequence; such panels of proteins generate strains pecific
polypeptide fingerprints. Clinical proteomics is a branch of classical
proteomics focusing on the identification of clinically relevant protein
expression . Major efforts to standardize this technology are ongoing,
which may be instrumental in the introduction of proteomics in bacterial
typing (Mischak , 2007 ).
Metabolites are the intermediate or final products of chemical
reactions occurring in living creatures. 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. This discipline assesses the
intracellular complexity of compounds, such as amino acids, glucosides,
hormones, and others. Since there is extensive variability in the intra-
cellular presence and concentration of small molecules, the specific
composition of a strain’s metabolites when grown under defined
conditions can be highly characteristic (Raman ,2005).
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 (Koreen et al .,2004). 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 . Although it is applicable
only to S. aureus, spa typing appears to be very robust, with benefits in
throughput, ease of use, and interpretation that tend to balance a lower
level of epidemiologic discrimination than that of established genotypic
methods such as PFGE ( Belkum et al .,2006).
* Multi-locus sequence typing
Multi-locus sequence typing (MLST) is a powerful molecular tool
used for characterizing relationships among bacterial isolates for
epidemiological purposes. MLST utilizes sequence analysis of internal
fragments (~500 bp) for six or seven housekeeping genes. Multiple genes
are used because each gene alone does not provide adequate
discriminatory power . Gene targets selected for MLST analysis should
not be under selective evolutionary pressure (i.e., virulence genes),
present in multiple copies, subject to recombination, or closely linked
with other genes (Gevers , 2005).
When designing primers for MLST studies, it is important to note
that there are no sets of primers that are universally applicable to all
bacteria, and alternative sets of primers may need to be designed for each
gene, since there may be sequence variation at the primer-annealing site
among different strains of the same species (Maiden, 2006).
Although MLST generates an important frame work for the
assessment of microbial (non)clonality and ecological spread
(Ogden ,2007). Derived, in principle, from multilocus enzyme
electrophoresis, MLST utilizes a larger, and potentially more
representative, portion of the genome than SLST. MLST compares the
nucleotide sequences of internal 400- to 500-bp regions of a series of
housekeeping genes (typically seven or more) which are present in all
isolates of a particular species. For each gene fragment, genetic
polymorphisms in sequences are considered distinct alleles. Each isolate
is defined by the alleles at each of the sequenced housekeeping loci,
which together comprise the allelic profile or sequence type. Because
there are many potential alleles at each of the loci, it is unlikely that
identical allelic profiles will occur by chance. Thus, isolates with the
same allelic profile are assigned as members of the same clone (Lemee et
al .,2004).
MLST was originally employed to identify hypervirulent lineages of
Neisseria meningitides .However, the approach has now been applied to a
variety of other pathogens, including S. aureus and enterococci , for the
assignment of S. pneumoniae strains to major hypervirulent, penicillin-
resistant, and multiple-antibiotic resistant clones and to a large number of
other organisms (Stampone et al .,2005).
Infection control Guidelines
Healthcare-associated infection affects hundreds of millions of
people world wide and is a major global issue for patient safety. It
complicates between 5 and 10% of admissions in acute care hospitals in
industrialized countries. In developing countries, the risk is two to twenty
times higher and the proportion of infected patients frequently exceeds
25% . 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 (Yassin etal .,
2003).
The structure and components of an infection control program are
shown in Tables 1, 2 and 3 . Several authors have discussed the
components of an infection control program in the Long-term care
facilities (LTCF) . Most authors feel that an infection control program
should include some form of surveillance for infections, an epidemic
control program, education of employees in infection control methods,
policy and procedure formation and review, an employee health program,
a resident health program, and monitoring of resident care practices. The
program also may be involved in quality improvement, patient safety,
environmental review, antibiotic monitoring, product review and
evaluation, resident safety, prepareness planning, and reporting of
diseases to public health authorities (Ouslander et al.,2006) .
Table 1 ; Infection control program structure .
(ICP) Infection control professional . (ICC) infection control committee
(Philip et al ., 2008).
Table 2 ; Infection control program element .
(LTCFs) Long-term care facilities . (PI) Professionals in Infection
Control (Philip et al ., 2008).
Table 3 ; Non- pharmacological hospital infection control strategies
(Curtis et al ., 2008)
1-Hand hygiene
The extreme importance of hand washing has been known since at
least 1847, when Dr Ignaz Semmelweis discovered that washing hands
before performing obstetric exams on pregnant women reduced
childbirth-related infectious mortality from more than 10% to less than
1% (Bjerke ,2004). However, rates of hand washing among healthcare
providers usually range from only about 20 to 50% per hospital patient
encounter, although some studies have reported hand-washing rates as
high as 81%. Viable pathogens are often found on hands of healthcare
providers (Rosenthal et al .,2005).
The skin is an inhospitable environment for most micro-organism as
it is dry , acidic and poor in nutrients. However , some micro-organism
have adapted to these conditions and exist in stable population known as
the resident or normal flora . These organism live in deep crevices in the
skin , in hair follicle and sebaceous glands . The micro-organism present
in largest numbers are Gram –positive bacteria , mainly coagulase
negative staphylococci, micrococci and coryneform . Viruses are also
easily acquired (Sattar et al .,2002).
The ‘‘My Five Moments for Hand Hygiene’’ program included in the
WHO guidelines document deserves emphasis because of its success rate
in 400 hospitals worldwide in 2006 to 2008. The evidence-based program
is easy to follow and reminds the health care worker to practice good
hand hygiene:
1. before touching a patient
2. before a clean/aseptic procedure
3. after body fluid exposure risk
4. after touching a patient
5. after touching patient surroundings .
Indications for hand washing and hand antisepsis:
* When hands are visibly dirty or contaminated with proteinaceous
material or are visibly soiled with blood or other body fluids, wash hands
with either a non antimicrobial soap and water or an antimicrobial soap
and water.
- Proper hand washing technique is essential for best results:
Wet hands with water
Apply enough soap to cover all surfaces
Rub hands palm to palm
Right palm over left dorsum with interlaced finger and vice versa
Palm to palm with fingers interlaced
Backs of fingers to opposing palms with fingers interlocked
Rotational rubbing of left thumb clasped in right palm and vice
versa
Rotational rubbing, backwards and forwards with clasped fingers
of right hand in left palm and vice versa
Rinse hands with water
Dry thoroughly with a single-use towel
Use towel to turn off faucet/tap
Duration of entire procedure: 40 to 60 sec and your hands are safe.
- Paper towels, warm air dryers, and cloth towels were no different in the
efficiency to dry wet hands.
- When using towels, pat dry instead of rubbing dry to avoid cracking
(Lederer et al .,2009) .
* If hands are not visibly soiled, use an alcohol-based hand rub for
routinely decontaminating hands or wash hands with an antimicrobial
soap and water.
- Proper hand rubbing with alcohol-based hand rub is:
Apply a palm full of the product in a cupped hand and cover all
surfaces
Rub hands palm to palm
Right palm over left dorsum with interlaced finger and vice versa
Palm to palm with fingers interlaced
Backs of fingers to opposing palms with fingers interlocked
Rotational rubbing of left thumb clasped in right palm and vice
versa
Rotational rubbing, backwards and forwards with clasped fingers
of right hand in left palm and vice versa
Duration of the entire procedure: 20 to 30 sec and, once dry, your
hands are safe (figure 14) (WHO , 2009) .
It is estimated that hand washing with plain soap for 30 second (s)
removes most soil and dirt, eliminates about 90% of transient hand flora
but a low percentage of resident hand flora. Hand washing for 15 s with a
soap containing chlorhexidine or triclosan removes most soil and dirt and
about 99.9% of transient flora and about 50% of resident flora. Hand
rubbing for 15 s with an alcohol-based gel does not remove soil or dirt,
but kills about 99.9% of transient flora and about 99% of resident flora
(Simon ,2004).
Alcohol-based hand-washing solutions are generally considered to be
more effective than soap and water. Compared with plain soap and water,
some studies have reported significantly lower rates of nosocomial
infections when alcohol-based solutions or chlorhexidine- or triclosan-
based hand-washing agents are used (Brown et al .,2003).
Many healthcare providers prefer using alcohol based solutions
instead of soap and water, and compliance rates are generally higher
when alcohol- based hand-washing solutions are used. Use of alcohol-
based cleaners saves time and these generally abrade and irritate the skin
less than antiseptic soaps. However, some people complain that alcohol-
based cleaners dry out and crack their skin. Hospitals and healthcare
providers may want to experiment with several alcohol or chlorhexidine-
based hand cleaners. Soap and water may still have to be used in cases
when hands are visibly soiled. In that case, staff and visitors should wash
hands carefully for at least 15 s with soap and water (Widmer ,2000).
Gloves are used to prevent contamination of healthcare personnel
hands when;
1(anticipating direct contact with blood or body fluids, mucous
membranes, non intact skin and other potentially infectious material.
2 (having direct contact with patients who are colonized or infected with
pathogens transmitted by the contact route e.g., vancomycin-resistant
enterococci (VRE), meticillin-resistant Staphylococcus aureus
) MRSA ,(Respiratory Syncential Virus (RSV) .
3(handling or touching visibly or potentially contaminated patient care
equipment and environmental surfaces(CDC ,2002) .
Gloves can protect both patients and healthcare personnel from
exposure to infectious material that may be carried on hands . The extent
to which gloves will protect healthcare personnel from transmission of
blood borne pathogens (e.g., HIV, HBV, HCV) following a needle stick or
other pucture that penetrates the glove barrier has not been determined.
Although gloves may reduce the volume of blood on the external surface
of a sharp by 46 to 86% , the residual blood in the lumen of a hollow bore
needle would not be affected; therefore, the effect on transmission risk is
unknown (Duckro et al .,2005) .
It is not certain what type of glove provides the best protection for
infection control. Some studies have suggested that latex gloves are
somewhat better in preventing penetration of water and virus than vinyl
gloves. However, about 3 to 16% of healthcare workers are sensitive to
latex and sometimes experience severe respiratory reactions to it. If latex
gloves are used in the healthcare setting, only the powder-free gloves
should be used since these release much lower levels of latex allergens
than the powdered latex gloves. Nitrile gloves also have good barrier
penetration but are more expensive and heavier than either latex or vinyl
gloves (Yip ,2004) .
Shoe and head covers are often recommended for use in areas
containing immunocompromised or surgical patients. Although bacterial
pathogens have been collected from shoes, research on the use of shoe
covers and/or separate hospital shoes and spread of pathogens has been
meager (Santos et al ., 2005) .
Proper cleaning techniques and proper cleaning chemicals can also
significantly reduce hospital pathogen levels and risk of nosocomial
infections . Terminal room cleaning after patient discharge was able to
adequately clean only a mean of 49% of the standardised surfaces,
including less than 30% for toilet hand holds, bedpan cleaners, room door
knobs and bathroom light switches. It is recommended that hospitals
monitor performance of cleaning personnel and provide feedback and
training as needed to optimise cleaning effectiveness (Carling et
al .,2008).
Figure 14 . Schematic diagram of hand wahing (WHO , 2007).
2-Environmental Decontamination &Cleaning
Greater awareness of the hospital environment as a source of
nosocomial pathogens has led to calls for enhanced investment in more
effective conventional cleaning, as well as encouraging the development
of a range of new cleaning and decontamination technologies(Casey et
al .,2010) . Sites that are frequently touched by hands are thought to
provide the greatest risk for patients, and those situated right beside
patients provide the biggest risk of all. The responsibility for cleaning
near-patient hand-touch sites does not always rest with the ward cleaners
(White et al .,2008) .
The microbial pathogens that cause HAI have two special properties:
first, they are recognized as hospital pathogens; second, they have an
innate ability to survive on surfaces in the hospital environment for long
periods of time . They include organisms such as MRSA, Clostridium
difficile, VRE , Acinetobacter spp. and norovirus (Dancer , 2008).
The potential for contaminated environmental surfaces to contribute
to transmission of healthcare-associated pathogens depends on a number
of factors, including the ability of pathogens to remain viable on a variety
of dry environmental surfaces, the frequency with which they
contaminate surfaces commonly touched by patients and healthcare
workers, and whether or not levels of contamination are sufficiently high
to result in transmission to patients . Any isolation of pathogens, or
pathogen indicators, causes concern and warrants immediate action. By
contrast, environmental surface sampling in hospitals usually only takes
place in response to an outbreak and then only if the infection control
team responsible has the motive, means and interest to initiate
environmental screening (Dancer , 2009) .
Disinfection is one of the cornerstones of infection prevention and
control, defined as the antimicrobial reduction of micro-organisms to a
level specified as appropriate. This definition is intentionally broad to
cover a variety of applications, including medical/ surgical device
reprocessing, liquid/gas treatment and general environmental surface
disinfection . It encompasses other processes, such as pasteurization,
sanitization, antisepsis, fumigation and preservation. Disinfection
methods can be classified as being physical or chemical in antimicrobial
activity . Physical methods include radiation and heat, while chemical
methods are based on the use of biocides such as alcohols, aldehydes,
halogens and quaternary ammonium compounds (McDonnell and
Burke ., 2011) .
Given the range of disinfection methods available and their clinical
applications, classification systems are used to aid healthcare workers to
choose the correct method to safely reduce patient risks. One such system
is the Spaulding classification for surgical or medical devices, which has
been in use since 1957.Spaulding defined the minimum levels of
disinfection to be employed according to the infection risk associated
with a device when used with a patient. Critical devices present the
highest risk as they enter a normally ‘sterile’ area of the body, such as the
bloodstream. Sterilization of these devices is recommended . Sterilization
is distinct from but encompasses disinfection, being defined as a process
used to render a surface or product free from viable micro-organisms,
including bacterial spores. A sterile device is free from viable organisms,
while disinfected devices or surfaces can only be presumed to have
reduced microbial levels. Typical sterilization processes use steam,
ethylene oxide, liquid peracetic acid and hydrogen peroxide gas (Song et
al .,2009) .
Semi-critical devices pose a lower risk as they may only contact
mucous membranes or non-intact (broken) skin. In the past, many of
these devices (such as flexible endoscopes) could not be sterilized in a
reasonable time frame for practical clinical use. The compromise was to
recommend high-level disinfection, thereby inactivating most pathogenic
micro-organisms such as viruses, bacteria (including mycobacteria),
fungi and, if possible, bacterial spores (in these cases, generally requiring
longer exposure times). High-level disinfectants, such as those based on
heat (hot water for some devices), glutaraldehyde, ortho-phthaldehyde ,
hydrogen peroxide and peracetic acid, could provide rapid turn around
times for these devices (Antonnucci et al .,2008) .
Non-critical devices present the lowest risk to patients, as they may
only contact intact skin. In these cases, low- or intermediate level
disinfection is often recommended, encompassing certain types of viruses
[especially enveloped viruses such as influenza and human
immunodeficiency virus (HIV)], most bacteria and some fungi.
Intermediate-level disinfectants should also provide efficacy against a
broader group of viruses (non-enveloped) and some mycobacteria.
Examples include alcohol-, aldehyde-, phenolic- and quaternary
ammonium- compound-based disinfectants (Beguma et al .,2009) .
* Laboratory-acquired bacterial infection
Laboratory-acquired bacterial infection is a documented occupational
hazard for staff working in microbiology laboratories. Bacterial
contamination of door handles, telephones and computer keyboards has
been demonstrated in the clinical setting but there are few comparable
data for microbiology laboratories. Two studies documented the presence
of environmental contamination with vancomycin-resistant enterococci
but did not look at hand acquisition ( Ng et al ., 2011).
Other than enterococci , staphylococci are common laboratory
isolates and documented to survive well on environmental surfaces in a
clinical setting; Enterobacteriaceae and P. aeruginosa also represent
potential bacterial pathogens. Further indirect evidence of laboratory
environmental contamination with bacteria comes from studies reporting
evidence of cross-contamination of specimens with enterococci and
Salmonella spp. It is demonstrated that the density of commensal bacteria
on the hands of laboratory workers was increased by the use of jewellery
or wrist watches (Dancer ,2008& Lappe et al .,2009) .
Some studies demonstrate that the use of gloves is protective against
hand acquisition of MRSA by laboratory technicians. MRSA is the
commonest bacterial pathogen isolated from laboratory surfaces,
particularly from frequently handled surfaces such as telephone keypads
and computer keyboards. Hand washing at the end of work sessions was
effective at removing pathogenic bacteria (Lappe et al .,2009) .
The primary method of recovery of bacteria from surfaces was the
swab-elution method without enrichment, and this method is subject to
various limitations, including the underestimation of bacteria
density .Some studies prove to perform enrichment of environmental
swabs so that an estimation of relative bacterial density from different
surfaces could be performed. Similarly, it would not have detected
contamination of hands with low numbers of pathogenic bacteria, as the
lower limit of detection was 200 cfu for each tested hand (Landers et
al .,2010) .
* Air filtration and air handling
High-Efficiency Particulate Air (HEPA) filtration is relatively
inexpensive and probably should be used for all hospital rooms. Various
studies have found that the HEPA filtration in hospitals can significantly
reduce airborne levels and/or infection rates for several aerosolized
pathogens. Many studies have reported that the HEPA filters in patient
rooms can significantly reduce both airborne aspergillus concentrations
and rates of human aspergillus infections .Use of HEPA filters has been
found to significantly reduce airborne levels of MRSA and P. aeruginosa
in hospitals and reduce airborne concentrations of droplet nuclei (which
transport tuberculosis) by 90% (Boswell et al .,2006).
Hospital air filtration system filters air at 60 air changes per hour and
uses a ‘cold plasma’ system to destroy microbes. Early tests have
indicated that such a system has a more than 99% single-pass efficiency
in destroying bacteria, viruses and moulds such as Aspergillus (Poirot et
al .,2007). UltraViolet (UV) light machines in rooms or in ventilation
systems can effectively kill mycobacteria, legionella and many viruses,
but UV light is not effective in killing many species of bacteria and
moulds (Leung and Chan , 2006).
3-Nutrition
Better nutrition can also play a critical role in reducing nosocomial
infections. Malnutrition is very common in hospitalised patients.
Malnutrition was measured by such parameters as weight loss ( body
mass index, grip strength) respiratory function, nutritional intake and
blood levels of albumin, and prealbumin. Many nutrients play a key role
in maintaining immunity including protein, omega-3 fatty acids, vitamins
A, B6, B12, C, D, and E; selenium, zinc, copper and iron. Most of these
nutrients become depleted following acute illness. Malnutrition is a major
risk factor for infection (Wintergerst et al .,2007) .
The liquid nutrients provide a favourable medium in which bacteria
can grow and are easily contaminated during assembly and manipulation
of the administration sets . Many types of bacteria ,including
Salmonella ,Klebsiella , Enterobacter , E.coli and S.aureus have been
found in high concentrations in enteral feeds . These may cause
gastroenteritis and, through colonization of the gut , may also result in
septicaemia and pneumonia (Howell , 2002) .
Manipulation of enteral feeding systems increases the risk of feed
contamination . Administration sets should be designed to require
minimal manipulation and recessed connection may help to reduce the
risk of contamination . Hands must be washed before handling enteral
feeds or the feed administration systems and a rigorous no-touch
technique must be used when assembling the administration sets and
handling the feed . The use of clean ,disposale gloves has been
recommended to minimize the risk of contamination (pellowe et
al .,2003).
Sterile ready-to-use feeds should be administered over a maximum
of 24h . The feeding tube should be flushed with fresh tape water before
and after aspiration , feed changing or drug administration to minimize
the risk of micro-organism adhering to the internal surface and the
administration reservoir and tubing should discarded after a maximum of
24 h in use (Howell, 2002) .
4-Surveillance & Outbreak control
Infection surveillance may either include all residents in a facility or
be targeted at specific subpopulations. Although facility-wide
surveillance is useful for calculating baseline rates and detecting
outbreaks, a more focused analysis could include examination of
infection rates in residents who are at risk for certain kinds of infection
(such as aspiration pneumonia in residents receiving tube feedings or
bloodstream infection among residents with indwelling vascular
catheters). These surveillance data are used primarily to guide control
activities, to plan educational programs, and to detect epidemics, but
surveillance also may detect infections that require therapeutic action
(McGeer et al .,1991).
Surveillance requires objective, valid definitions of infections. Most
hospital surveillance definitions are based on the National Nosocomial
Infections Surveillance System (NNIS) criteria, Prevalence studies detect
the number of existing (old and new) cases in a population at a given
time, whereas incidence studies find new cases during a defined time
period. The latter is preferred because more concurrent information can
be collected by an incidence study if data are collected with regularity
(Satterfield ,1993).
Surveillance systems must be extremely flexible; effective infection
control teams will not use a “one size fits all” approach for surveillance.
Some hospitals should focus on patients at high risk, such as those
hospitalized in intensive care and neonatal units (Pottinger et al .,1997).
After defining the priorities of the institution, the focus could also be on
specific problems, such as bacteremia or surgical site infection. Focusing
on neonatal bacteremia pays high because extrinsic contamination of IV
fluids seems to be a common problem in many settings (Avila-Figueroa
et al .,2000).
Automated surveillance (AS) is the process of obtaining useful
information from infection control data through the systematic
application of medical informatics and computer science technologies.
(Wright , 2008) .
* Surveillance Methods
1- Case-finding Issues
First, should infections be sought by passive or active means . In
passive surveillance, persons who do not have a primary surveillance
role, that is, persons other than ICPs , are relied on for identification and
reporting of infections. Active surveillance is the process of vigorously
looking for nosocomial infections using trained personnel, nearly always
ICPs. ICPs seek out nosocomial infections by using various data sources
to accumulate information and decide whether or not a nosocomial
infection has occurred (Bates et al ., 2003) .
Second, should infection detection be patient- or laboratory-based .
Patient-based surveillance includes counting nosocomial infections,
assessing risk factors, and monitoring patient care procedures and
practices for adherence to infection control principles. It requires ward
rounds and discussions with caregivers. In laboratory-based surveillance,
detection is based solely on the findings of laboratory studies of clinical
specimens. Third, should infections be detected prospectively or
retrospectively . Prospective surveillance refers to monitoring patients
while they are still hospitalized and, for SSIs, includes the postdischarge
period. Retrospective surveillance uses chart review after patient
discharge as the sole means of identifying infections (Burke , 2004).
2- Incidence Versus Prevalence in Hospital-Wide Surveillance
Incidence surveillance is continual monitoring of all patients for
new nosocomial infections of all kinds on all wards. It has also been
termed ongoing, total, housewide, or comprehensive surveillance .
Hospital-wide surveillance has the advantage of providing a global view
of what is happening in the hospital so that potential clusters of infection
or antibiotic resistance can be detected anywhere . The advantage of
prevalence surveillance is that it is a rapid inexpensive way to estimate
the magnitude of nosocomial infection problems in a hospital . There are
two major disadvantages of prevalence surveillance. First, in small
hospitals, the number of patients surveyed is insufficient to detect
important differences among patient populations . Second, patients' risk
of infection is overestimated with the prevalence rate, which is calculated
as the number of active infections on the day of the visit divided by the
number of beds visited (Gaynes et al ., 2001) .
3- Targeted Surveillance
These strategies focused or targeted efforts on certain areas in the
hospitals (e.g., ICUs), patient groups (e.g., surgical patients), or infection
sites (e.g., bloodstream infections). These targeted efforts have become
increasingly common in this decade not only because of their positive
impact on resource management but because they have the potential for
yielding more meaningful results than hospital-wide surveillance. A
disadvantage of these limited strategies is that clusters of infection in
areas not under surveillance may be missed ( Emori et al ., 1998).
4- Objective/Priority-Directed Surveillance
Accordingly, SSIs and pneumonias would be allocated the most
surveillance resources (one half and one third, respectively), with much
less for bloodstream and urinary tract infections. Objectives for
surveillance should be evaluated annually and adjusted as necessary. The
obvious advantage of this method is that specific measurable objectives
are set and attainment is carefully evaluated. Therefore, ICP time and
effort are directed in a very productive manner. A potential disadvantage
is undetected outbreaks, although some studies recommended that the
ICP train other hospital staff to be alert for and report unusual clustering
(Leape , 2002).
5- Limited Periodic Surveillance
This method is a combination of hospital-wide and site-specific
targeted surveillance. Some studies used total surveillance for 1 month
per quarter and targeted bloodstream infection surveillance during the
other 8 months. Although the potential for missing clusters is less than for
targeted methods, it still exists during two thirds of the year (Platt ,
2002).
6- Postdischarge Surveillance
Because of the shorter postoperative stay, it is estimated that as
many as 50% of SSIs may be missed if a formal postdischarge
surveillance system is not in place . Significant methodologic problems
with postdischarge surveillance include reliance on physicians to return
information on patients to the ICP in a timely manner, patients' inability
to accurately diagnose infection , and determination of how to handle
patients lost to follow-up (Sands et al ., 1999) .
* Outbreak control
In fact, we are living in a worldwide pandemic in many hospitals
where sophisticated protocols are performed with no attention to the
risks and no control programs; under such conditions, a substantial
proportion of NIs occur as outbreaks . Hospitals having outbreaks (Table
4) as main causes of NIs must implement programs focused on their
prevention as a first step towards consolidation of their programs
(Ostrosky-Zeichner et al .,2000).
Table 4 ; Immediate control measures for outbreak management
(Fletcher ,1996).
Type of transmission suspected Suggested action
Cross-transmission (transmission between Patient isolation and barrier
individuals) precautions determined by
infectious agent(s)
Hand transmission Improvements in
Hand washing; cohorting
Airborne agent Patient isolation with
appropriate ventilation
Waterborne agent Checking of water supply
and all liquid containers
Use of disposable devices
Food borne agent Elimination of the food at
Risk
5-Prevention of urinary tract and urinary catheter
infections
About 80 to 95% of hospital-acquired urinary tract infections
originate from urinary catheters. Guide for CAUTIs outlines interventions
for avoiding and/or minimizing the duration of indwelling urinary
catheter use, including the following:
(1) Using catheters only when medically necessary
(2) assessing patients daily for the need for catheterization and
documenting a continued need
(3) using reminder systems for health care workers aimed at removing
catheters
(4) using external catheters in men when feasible
(5) considering intermittent catheterization instead of indwelling catheter
insertion
(6) promptly removing unnecessary urinary catheters. About 15% of
urinary HAIs have been linked to improper hand washing and poor
aseptic techniques in cleaning the urinary meatus area and inserting and
maintaining the urinary catheters (Rebmann and Linda., 2010).
Other interventions aimed at preventing CAUTIs focus on
maintaining a sterile, closed drainage system to prevent microorganism
colonization of the catheter. Examples include ;
(1) using aseptic technique during catheter insertion
(2) allowing only trained health care professionals to insert urinary
catheters
(3) securing catheters to prevent movement and urethral traction
(4) keeping the drainage bag below the level of the bladder
(5) changing the indwelling catheter or urinary drainage bag only when
necessary (ie, do not routinely change the catheter or drainage system at
arbitrary intervals)
(6) Other recommended measures include avoiding irrigation unless the
catheter is obstructed
(7) scanning the bladder for residual urine amounts
(8) administering anti infective therapy only when an infection is
suspected rather than after colonization ( CDC .,2009).
Bacteria enter the drainage system in the drainage bag or at the
junction between the catheter and the drainage bag . These bacteria reach
the bladder along the tubing after a few days . The drainage system
should not be opened to take specimens which should instead be obtained
aseptically from sampling port with a needle . The drainage bag should be
emptied when necessary to avoid reflux of urine . A clean pair of gloves
should be worn for emptying the drainage bag and discarded on
completion of the procedure . When the bag is emptied ,care should be
taken to ensure that micro-organism are not introduced on to the tap by
contact with a contaminated container or other surface . Containers
should be decontaminated in a bedpan washer or autoclaved after each
use (Pratt et al ., 2001).
Another CAUTI prevention intervention is the use of antimicrobial-
or silver-coated catheters. The use silver-tipped catheters (both short-
and long-term users) was associated with a 57% reduction in urinary tract
infections (Rupp et al .,2004).
6-Prevention of central venous line and Blood stream
infections
The prevalence of Catheter Related Blood stream infections
( CRBSI) increases linearly with the duration of catheterization. Thus, the
continued need for CVCs , and other short-term invasive devices, should
be assessed each day and the devices should be removed as soon as they
are no longer required for patient care (Mermel , 2007).
Use of maximal barrier precautions for CVC insertion (i.e. use of
long-sleeved gown, sterile gloves, mask, cap and large sterile sheet drape)
reduces catheter-related bloodstream infections compared to minimal
(sterile gloves and small drape) precautions and should be the standard of
care. Placement of CVCs in the femoral vein compared with the
subclavian vein increases the risk of CVC colonization , with increased
risk of CRBSI. Femoral CVC placement also increases the risk of
thrombosis compared with subclavian vein insertion . Thus, CVC
placement in femoral veins should be avoided and if such placement is
necessary in an emergent situation, such catheters should be replaced in
another anatomical site as soon as it can be safely done (Rijnders et
al .,2002).
A meta-analysis of prospective, randomized studies has found that
use of chlorhexidine-containing antiseptics for preparing the catheter
insertion site reduces risk of CRBSI compared with povidone iodine and
the former antiseptics should be the standard of care for cleaning catheter
insertion sites.Use of a chlorhexidine containing dressing directly over
the CVC insertion site has been demonstrated to reduce the risk of
catheter colonization in paediatric patients , especially in neonates.
However, this dressing can cause skin irritation in very low birth-weight
neonates. Thus, such dressings should be considered for use over CVC
insertion sites in paediatric patients, apart from very low birth-weight
neonates, and considered for high-risk adult patient populations (Levy et
al .,2005).
Doubling the number of hours per ICU shift in which float nurses
work instead of nurses who regularly staff ICUs leads to a nearly 4-fold
independent increased risk of primary bloodstream infections, most of
which are due to CRBSI (Edwards et al .,2003).
One prospective, randomized trial found that continuous infusion of
heparin (100 units/kg/d) in haematology/oncology patients with a CVC in
place reduced the incidence density of CRBSI compared to control
patients continuously infused with saline .The incidence of CVC-related
deep venous thrombosis was similarly reduced (Abd el kefi et
al .,2005 ) .
Use of vancomycin-containing catheter lock solutions has been shown
to reduce the incidence of CRBSI .Other antimicrobial agents have also
been used in catheter lock solutions to prevent CRBSI. Concerns about
the potential risk of antibiotic resistance have limited the widespread use
of this preventative strategy. Nevertheless, certain high-risk patient
populations, such as patients who require a long term CVC but have
limited vascular access and/or recurrent CRBSI despite other
interventions, are good candidates for such an intervention (Safdar and
Maki , 2006).
Needleless catheter connectors have come into widespread use in
some countries in an effort to reduce sharps injuries . A prospective,
randomized trial found that use of a needleless catheter connector was
independently associated with reduced catheter colonization. Despite
these results, a more recent study found that the incidence of CRBSI
increased more than 3-fold in a pediatric ICU when one type of
needleless catheter connector was substituted by another type.
(Maragakis et al .,2006).
* Safe Disposal Of Sharp Instrument and Waste
Injection drug use remains the single most important vector for the
spread of blood-borne disease in the United States, accounting for
approximately one third of all AIDS cases and 60% of new hepatitis C
infections. Access to sterile syringes helps prevent the spread of blood-
borne infections among injection drug users (IDUs) ( Jarlais et al .,2005
& Talaat et al .,2006).
Clinical wastes predominantly comprise wound dressings and swabs
together with infusion and irrigation equipment, catheters, blades,
syringes and needles. Also included are tissue and postmortem wastes,
waste from clinical laboratories, sanitary wastes including incontinence
pads and nappies etc., and waste pharmaceuticals. Most wastes will be
disposed to yellow bags or rigid yellow bins, including sharps bins where
appropriate, and removed from clinical areas for destruction by high
temperature incineration, or made safe using one of a number of alternate
treatment technologies including autoclave, microwave or hot oil auger
treatments (Table 5) (Rushbrook et al .,1999) .
Table 5 ; Colour coding for the disposal of clinical waste (Health
Services Advisory Committee , 1999 ) .
Colour of bag Type of waste .
Black Household waste ,treated clinical waste
(e.g .paper ,food ,flower ,etc)
Yellow Clinical waste (e.g. material contaminated
with blood or body fluid ,human or
animal tissue )
Yellow sharps container Needle syringes ,broken glass and any
other contaminated sharp item
Blue or transparent with Waste for autoclaving (e.g. pathology
blue inscription specimens)
Yellow black stripes Non-infectious human waste (e.g. sanitary
towels , incontinence pad )
Multihazardous waste includes waste that is infectious and that
contains radionuclides and/or hazardous chemicals. An example is waste
contaminated with blood or body fluids and with a chemotherapy drug.
Multihazardous waste is best managed and treated separately from other
infectious waste . Low-level radioactive infectious waste (e.g. swabs,
syringes for diagnostic or therapeutic use) may be collected in yellow
bags or containers for infectious waste if these are destined for
incineration. It should be noted that mercury thermometers are not
infectious waste, and they should not be classified and managed as such.
All unwanted or broken mercury thermometers should be managed and
disposed of as hazardous chemical waste. They should never be placed in
sharps containers (Denys , 2000) .
7-Preventing respiratory tract infections
Preventive strategies to reduce the colonization of the upper
respiratory tract are the selective decontamination of the digestive tract
(SDD), oropharyngeal decontamination and combinations of these with
or without the use of systemic antibiotics. Oropharyngeal
decontamination can be achieved using topical antibiotics, which may
increase the risk of antibiotic resistance, or using topical antiseptics.
Though decolonization of the oropharynx using topical antimicrobial
agents like chlorhexidine (CHX) should prove useful in the prevention of
nosocomial respiratory tract infection, scientific evidence to recommend
routine oral decolonization is lacking, as several randomized clinical
trials have failed to demonstrate a significant reduction in the incidence
of respiratory tract infections in the CHX group (Fourrier et al .,2005).
Supine patient positioning facilitates aspiration; semirecumbent
positioning decreases it . Infection in patients in the supine position was
associated with the simultaneous administration of enteral nutrition and
an increased risk of aspiration of gastric contents (Tablan et al .,2004).
Gastroesophagal reflux occurs less frequently in the semirecumbent
position. Thus, it is recommended that intubated patients should be
managed in a semirecumbent position, particularly during feeding .
Kinetic beds or continuous lateral rotational therapy is a technique using
a continuous movement of the bed along its longitudinal axis within a
certain range (–40° to +40°) . This movement improves secretion
drainage, and thus may decrease the risk of VAP (Collard et al .,2003 &
Dodek et al .,2004).
The use of endotracheal tubes bypasses the natural defense
mechanisms of the upper respiratory tract and impairs the host’s
capability to fend off infection . Endotracheal tubes also favour the
development of bacterial biofilms that may contribute to the occurrence
of VAP. Noninvasive positive pressure ventilation (NIV) using a face
mask is an alternative to intubation (Esteban et al .,2004). There are two
types of suctioning catheters: open single-use and closed multiuse. There
is no significant difference in the incidence of VAP. A closed suction
catheter changed between each patient is more advantageous in terms of
cost and maintenance (Hubmayr et al .,2002).
The CDC guideline for prevention of pneumonia is oriented toward
acute care hospitals , including respiratory therapy equipment, suctioning
techniques, tracheostomy care, prevention of aspiration with enteral
feedings, and immunizations. Examples of relevant recommendations
include hand hygiene after contact with respiratory secretions, wearing
gloves for suctioning, elevating the head of the bed 30 to 45 degrees
during tube feeding and for at least 1 hour after to decrease aspiration,
and vaccination of high-risk residents with pneumococcal vaccine
( CDC ,1997).
The evidence for the efficacy of pneumococcal vaccine in high-risk
populations, including the elderly population, is debated. However, the
vaccine is safe, relatively inexpensive, and recommended for routine use
in individuals over the age of 65 years (Watson et al .,2002).
Bacterial colonization of condensates in ventilatory circuits plays a
role in the pathogenesis of VAP (Cook et al .,1998). Frequent changes of
ventilator circuits should theoretically lower the risk of initial bacterial
colonization .The upper airways filter, heat and moisten the inspired air
so that it reaches the lower airways at body temperature with added
water. In mechanically ventilated patients the ventilator tube by passes
the upper airways and inspired gases require artificial conditioning by
heating and humdification to prevent mucosal injury and ventilator-
associated pneumonia (VAP) (Kollef et al .,1995).
8-Control of infections related to surgery and surgical
equipment
Surgical site infections (SSIs) are among the most common and
serious complications for patients who undergo operative procedures.
SSIs account for 14% to 17% of all hospital-acquired infections and 38%
of nosocomial infections in surgical patients. The Centers for Disease
Control and Prevention (CDC) (Table 6) estimates that SSIs complicate
approximately 5% of the nearly 30 million surgeries performed each year
(John et al .,2010).
Table 6 ; Major headings in prevention of surgical site infection
(National Institute for Health and Clinical Excellence ,2008) .
Preoperative strategies
About 2 to 5% of all surgical patients develop a significant infection
at the wound site . Higher rates of surgical infections are associated with
operations of two or more hours, a contaminated or dirty procedure, or
inadequate scrubbing procedures (Cheadle ,2006). Preoperative
strategies focus on controlling patient related risk factors and appropriate
hand/forearm antisepsis for surgical team members . Pre-existing
infections at sites remote from the operation site should be identified and
treated, and if practicable elective surgery should be delayed until such
infections have resolved. Obese patients should be encouraged to lose
weight before surgical operations and smokers should be encouraged to
stop smoking (although such lifestyle modifications may be unrealistic
for many patients) (Mangram et al .,1999).
On the night before the operation, the patient can wash or shower
with an antiseptic agent, and immediately before the operation the skin
should be adequately cleaned with an antiseptic solution. However, hair
removal should be avoided unless it is likely to interfere with the
operation. If hair removal is necessary, clippers should be used rather
than shaving, since there is evidence that shaving can result in
microscopic skin cuts that can act as foci for subsequent colonisation and
infection (Tanner et al .,2006).
Short courses of antimicrobial prophylaxis are widely used to reduce
SSI risk. The aim of this approach is not to sterilise tissue, but to reduce
intraoperative contamination to levels where it does not overwhelm the
patient’s defences. Antimicrobial prophylaxis is primarily indicated in
elective procedures in which skin incisions are closed in the operating
theatre. The choice of agent should be based on the pathogens most
commonly associated with the procedure being performed (Mangram et
al .,1999) .
In practice, broad-spectrum beta-lactam agents (particularly
cephalosporins) are most widely used, with an agent such as
metronidazole being added if necessary to provide cover against
anaerobes; vancomycin is not recommended for routine prophylaxis . The
first dose should be timed to ensure that bactericidal concentrations are
achieved in serum and tissue at the time of the incision, and these
concentrations should then be maintained for up to a few hours after
wound closure in the operating theatre. Recently, a statement for
urological surgery incorporated important principles of appropriate
surgical prophylaxis and offered sensible options in terms of the choice of
antibiotics for a variety of urological procedures. (Nicolas et al .,2007).
Perioperative strategies
The CDC guidelines emphasise the importance of good surgical
technique and aseptic precautions for the prevention of SSIs. Good
surgical technique requires attention to the maintenance of haemostasis,
removal of devitalised tissue and foreign bodies as completely as
possible, and elimination of dead space at the surgical site. Gloves,
facemasks, caps, gowns and sterile drapes should be used to minimise
transmission of potential pathogens to the wound. Surgical instruments
should be adequately sterilised according to published guidelines; flash
sterilization should be reserved only for instruments intended for
immediate use . It should be noted that despite precautions such as these,
some contamination of the surgical site is inevitable because some
endogenous bacteria remain even after excellent preoperative preparation
of the site (Owens and Stoessel , 2008 ).
Surgical personnel should undertake a thorough surgical scrub before
donning surgical gowns and gloves. Personnel who are colonised or
infected with potential pathogens should be encouraged to report their
condition, and procedures developed to prevent transmission of
pathogens from colonised personnel to the patient . Hand hygiene is
regarded as one of the key components in any infection prevention
strategy (Humphreys ,2009).
Warming the patient before or during surgery has also been shown to
significantly reduce rates of surgical infection .Warming may reduce
surgical infection rates by improving blood circulation and immune
function in the surgical areas (Melling et al .,2001).
Tissue hypoxia leads to necrosis and is often followed by infection.
High blood glucose levels, such that occur in patients with diabetes
mellitus for example, are also associated with increased risks of infection.
It is logical to assume that normalizing these will assist in the prevention
of SSI, and recent studies have supported this . It is believed that the
mechanism of action is by more effective neutrophil killing of potential
pathogens (Belda et al .,2005).
Postoperative strategies
The risk of SSI can persist for up to 30 days after a surgical operation
or for as long as one year after an operation in which the patient is given
an implant; indeed, a significant proportion (12 to 84%) of SSIs are first
detected after the patient has been discharged from hospital . The CDC
guidelines recommend that incisions that have been closed by primary
intention should be protected by sterile dressings for 24 48 h, and that
personnel should use sterile technique when changing dressings on any
kind of skin incision . However, two recent systematic reviews and meta-
analyses strongly suggest that prophylactic intranasal mupirocin
significantly reduces the rate of postoperative infections including that
caused by MRSA and meticillin-susceptible S. aureus (MSSA) ( Rijen et
al ., 2008) .
One of the major advances in surgical practice in recent decades has
been the development of laparoscopic or minimally invasive surgery.
This offers the potential for reducing infection as the incision site is much
smaller, but it is unclear whether all other practices and the environment
setting associated with open surgery should be replicated (Smyth et
al .,2005). Meta-analysis of laparoscopic compared with open repair of a
perforated peptic ulcer suggests that the risk of postoperative SSI is
reduced when carried out laparoscopically, and similar findings have
been associated with colon surgery (Poon et al .,2009).
9-Prevention of Gastrointestinal Tract and
waterborne hospital infections
A number of interventions have been proven effective in reducing
rates of hospital waterborne infections. Numerous studies have found that
replacing tap water with sterile water for drinking, bathing and
procedures can significantly reduce rates of many hospital infections
including crytosporidium, legionella, aeromonas and stenotrophomonas.
Sterile sponges can be used for bathing. Boiling and water filtration in
hospital water systems can also sterilise water, but these systems need to
be monitored closely because many problems can develop which cause
these systems to fail . Daily cleaning of patient shower areas with a
detergent and phenolic compound has been shown to significantly
decrease airborne levels of moulds including aspergillus (Anaisie et
al .,2002).
Heating water to more than 50 0 C has been shown to significantly
reduce levels of Legionella spp. in storage tanks and hospital water
systems; however, water heating alone will not usually eliminate all
legionella in a contaminated hospital water system. Some studies have
found that the UV-light water treatment can greatly reduce levels of
legionella in hospital water systems (Modol et al .,2007). Copper silver-
based ionisation systems can also significantly reduce waterborne
concentrations of legionella, moulds and Gram-negative bacteria such as
P. aeruginosa and Actinetobacter baumannii. Routine surveillance of
hospital water supplies for legionella is highly recommended in cases of
confirmed legionella infections; however, it is controversial as to whether
such routine testing is needed in hospitals with no legionella infection
history (Huang et al .,2008).
All water leaks and water damage should be repaired and remediated
within 24 h to prevent growth of pathogenic bacteria and moulds.
Hospitals should avoid using indoor decorative fountains since they
encourage legionella and the splashing water facilitates ready
aerosolisation of the organism (O’Neill and Humphreys ,2005).
10-Preventing burn infections
Prevention of burn wound infection involves assessment of the
wound at each dressing change for changes in the character, odor or
amount of wound drainage. Strict aseptic technique should be used when
handling the open wound and dressing materials and frequency of
dressing should be based on wound condition. If the wound has necrotic
material present, a debriding dressing should be chosen, whereas a
protective dressing is preferable for clean healing wounds. Treatment of
an existing wound infection includes considering changes to the topical
agent being used along with changing the frequency of the dressing
changes. In those cases where invasive infection is present, surgical
excision of the infected wound and appropriate systemic antimicrobial
therapy may be required (Weber et al .,2002).
The open burn wound increases the environmental contamination
present around the patient, which is the major difference in burn versus
non burn patients. The larger the burn size the more vulnerable it is to
contamination (Weber et al .,1998).
Patients with larger burn injuries (>25–30% Total Body Surface Area
(TBSA) are also immuno compromised, because of the larger size of their
injury , loss of physical defenses and need for invasive devices . These
patients also represent a significant risk for contamination of their
surrounding environment with multiple resistant organisms . For these
reasons, it is recommended that patients with larger burn injuries be
isolated in private rooms or other enclosed bed spaces (Kates et
al .,1991).
Special attention is also required for patients with smaller burn
injuries who are colonized or infected with multiple drug-resistant
organisms (i.e., methicillin-resistant S aureus, vancomycin-resistant
enterococci, multiple drug-resistant gram negative organisms). This is
especially true for patients with wound drainage that cannot be
adequately contained in dry, occlusive dressings or pediatric patients who
cannot comply with hand washing or other precautions. Patients
transferred to the burn unit after treatment in another hospital should also
be included in this group until the results of their admission cultures are
known. These patients are frequently colonized with resistant organisms
and may serve as an unsuspected reservoir for transmission to other
patients unless they are isolated. Isolation for this group of patients
generally includes placement in a private room and contact precautions,
with the addition of droplet precautions in some circumstances (Weber et
al .,1993).
Patients colonized with multiple drug resistant organisms must
frequently have their need for isolation balanced with their need for
rehabilitation and psychosocial needs. In general, if the patient’s dressing
cannot be occlusive, the patient should not be taken to the rehabilitation
department for therapy when other patients are present in the same area.
If rehabilitation needs cannot be met in the patient’s room, then sufficient
time should be scheduled in the rehabilitation department to allow for the
patient’s treatment followed by thorough cleaning of all equipment and
surfaces before the area is used by other patients. The rehabilitation
therapy staff should wear appropriate attire during therapy (Rutala and
Weber , 2004).
Pediatric burn patients should also have policies restricting the
presence of non-washable toys, such as stuffed animals and cloth objects.
These can harbor large numbers of bacteria and are difficult to disinfect.
Routine cleaning, disposal of waste and gathering of soiled linen is
essential to reduce the load of organisms and ensure that the unit is as
clean as possible. Routine environmental surveillance culturing is
generally limited to the hydrotherapy room and common treatment room
used in burn wound care; however, its scope needs to be extended
(Rutala and Weber , 2004).
Plants and flowers should not be allowed in units with burn patients
because they harbor gram-negative organisms, such as Pseudomonas
species, other enteric gram-negative organisms and fungi. Many of these
organisms are intrinsically resistant to multiple antibiotics, which may
serve as reservoirs to colonize the burn wound .Modern burn centers have
a contained perimeter that is designed to minimize unnecessary traffic of
healthcare workers and visitors through the unit . Cross-contamination is
further diminished within the burn unit by housing burn patients in
individual nursing units composed of individual isolation rooms, each
with its own laminar airflow (Herndon and Spies ,2001).
11-Isolation and precautions
Isolation precautions are an important aspect of hospital infection
control programs and are particularly important in pediatric settings given
the high admission rates for viral respiratory (VRI) and gastrointestinal
(VGI) infections. Factors such as diapering of patients and inability of
young patients to adhere to proper respiratory etiquette help facilitate
transmission of infectious pathogens. Education and periodic evaluation
of adherence to precautions are recommended administrative controls to
optimize isolation practices in hospitals (Page ,2005).
Although the neonatal intensive care unit (NICU) had the lowest rate
of isolation overall, it had the highest rate of isolation for health care-
associated infections/ multidrug-resistant organism (MDRO)
colonization, with colonizations with MDROs accounting for the majority
of isolations. The hematology/oncology/ hematopoeitic stem cell
transplantation ward followed, with a high rate of patients isolated for
health care associated infections/MDRO colonization, in this case,
primarily contact isolation for gastrointestinal infection including VGI
and Clostridium difficile infection. Isolation for MDROs accounted for
12.3% of patients in isolation. Few studies have reported on actual
compliance with isolation precautions (Chatterjee et al .,2004) .
Options for patient placement include single patient rooms, two
patient rooms, and multi-bed wards. Of these, single patient rooms are
prefered when there is a concern about transmission of an infectious
agent (Cepeda et al .,2005) .
In the absence of obvious infectious diseases that require specified
airborne infection isolation rooms (e.g., tuberculosis, SARS, chickenpox),
the risk of transmission of infectious agents is not always considered
when making placement decisions. When there are only a limited number
of single-patient rooms, it is prudent to prioritize them for those patients
who have conditions that facilitate transmission of infectious material to
other patients (e.g., draining wounds, stool incontinence, uncontained
secretions) and for those who are at increased risk of acquisition and
adverse outcomes resulting from HAI (e.g., immunosuppression, open
wounds, indwelling catheters, anticipated prolonged length of stay, total
dependence on HCWs for activities of daily living)(Krause et al .,2003) .
During a suspected or proven outbreak caused by a pathogen whose
reservoir is the gastrointestinal tract, use of single patient rooms with
private bathrooms limits opportunities for transmission, especially when
the colonized or infected patient has poor personal hygiene habits, fecal
incontinence, or cannot be expected to assist in maintaining procedures
that prevent transmission of microorganisms (e.g., infants, children, and
patients with altered mental status or developmental delay) (Bridges et
al .,2003) .
High-intensity narrow-spectrum light (HINS-light) is a new light-
based disinfection method that has been shown to inactivate a wide range
of bacterial pathogens including those that are commonly associated with
hospital-acquired infections. The HINS-light method utilises a narrow
bandwidth of high-intensity visible violet light, with peak output at 405
nm. Inactivation of bacteria by exposure to high-intensity 405 nm light is
thought to be associated with the photo-excitation of molecules such as
porphyrins within the bacteria, a process that results in the production of
highly reactive compounds such as singlet oxygen which are strongly
bactericidal (Maclean et al .,2009) .
Although HINS-light EDS (high-intensity narrow spectrum light
environmental decontamination system) has wide spectrum bactericidal
activity, it is concentrated on its effectiveness in reducing mainly
staphylococcal bacteria. Staphylococcus aureus is one of the most
commonly isolated pathogens from infected burns patients .In addition,
the inanimate environment surrounding burns patients has been shown to
be a reservoir for pathogens including MRSA, and various studies have
shown an association between S. aureus dispersal from burns patients and
the size of the burn wound (Dettenkofer and Spencer ,2007) .
* Special interventions for control of tuberculosis
Tuberculosis (TB) remains a serious health problem in both the
developed and developing world. Recent CDC guidelines have
recommended a number of administrative, engineering and personal
protection measures to control TB spread in healthcare settings.
Recommended administrative controls include TB testing for all patients
at risk of TB, implementing a written TB control plan in the hospital and
housing infected patients in separate rooms. All rooms housing TB
patients should have at least 12 outdoor air changes per hour (ACH) ,
have a negative pressure of at least 0.01 inch water, and the rooms of
patients with actual or suspected TB should be checked visually with tests
such as smoke tests. HEPA air filters in patient rooms and UV irradiation
in the ventilation systems or upper part of rooms is also strongly
recommended to reduce airborne TB levels. HEPA masks or other
respiratory protection need to be worn by healthcare workers and visitors
to rooms of infectious TB patients. Proper cleaning and disinfecting of
instruments used by TB patients are also essential (Humphreys ,2007) .
Summary & Conclusion
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 .
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 .
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 .The increased length of stay for infected
patients is the greatest contributor to cost .
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) .
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. Environmental conditions such as low temperature or
humidity appear to be crucial for the persistence of these organisms on
inanimate surfaces .
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 .
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 .
Analysis of nosocomial pathogens has relied on a comparison of
phenotypic characteristics such as biotypes, serotypes, bacteriophage or
bacteriocin types, and antimicrobial susceptibility profiles. This approach
has begun to change over the past 2 decades, with the development and
implementation of new technologies based on DNA, or molecular
analysis. These DNA-based molecular methodologies, include pulsed-
field gel electrophoresis (PFGE) and other restriction-based methods,
plasmid analysis, and PCR-based typing methods.
There are a number important attributes for successful typing
schemes: the methodologies should be standardized, sensitive, specific,
objective, and subject to critical appraisal. All typing systems can be
characterized in terms of typeability, reproducibility, discriminatory
power, ease of performance and interpretation, and cost (in terms of time
and money) . The use of strain typing in infection control decisions is
based on several 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 .
Molecular techniques can be very effective in tracing the spread of
nososcomial infections due to genetically related pathogens, which would
allow infection control personnel to more rationally identify potential
sources of pathogens and aid infectious disease physicians in the
development of treatment regimens to manage patients affected by related
organisms. Therefore, the use of molecular tests is essential in many
circumstances for establishing disease epidemiology, which leads to
improved patient health and economic benefits through the reduction of
nosocomial infections .
Infection control (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 . The infection control program
should include some form of surveillance for infections, an epidemic
control program, education of employees in infection control methods,
policy and procedure formation and review, an employee health program,
a resident health program, and monitoring of resident care practices. The
program also may be involved in quality improvement, patient safety,
environmental review, antibiotic monitoring, product review and
evaluation, resident safety, prepareness planning, and reporting of
diseases to public health authorities .
Conclusion
There are issues of concern about the emergence of nosocomial
infections, and the increase in morbidity, mortality, and costs
associated with these infections will drive the need for refinement
of molecular approaches to aid in the diagnosis and epidemiologic
analysis of nosocomial infections.
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.
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.
Recommendations
Many non-pharmacological interventions have been shown to
significantly reduce rates of HAIs, but are often overlooked in clinical
practice so this article recommend ;
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 .
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