RESEARCH
TITLE: Assessing the hospital environment as a potential
source of fungal nosocomial infections.
1.INTRODUCTION
Hospital environment is a potential source
of nosocomial pathogenic fungi due to overcrowding and poor
environmental sanitation, increase of immunocompromised people attending the
hospital for a visit or for other purposes. Patients are primarily admitted into hospital wards
for proper management of their ailments, but while on admission some patients
acquire other ailment than the one they were admitted for. These are called
hospital associated infections (nosocomial infections) which can result from
contact with a carrier directly or indirectly through inanimate objects or air.
Fungal nosocomial infections is the one of nosocomial infections which is most observed
in hospital areas these infections are aggravated by the reduced resistance of individual patients
(1)
The quality of indoor air in terms of fungi microbial
contamination in a given space at a given time period is said to be determined
by the quality of air entering the space, the number of occupants in the space,
number of patients in the hospital ward, their physical activities and
resultant aerosol generation, human traffic and the degree of ventilation.
Dust, which is a good vehicle of airborne contamination, may arise from human
activities, such as sweeping, movement, waving of handkerchief and bed making.
Sneezing has been described as the most vigorous mechanism of generating
millions of droplet into the environment, and. While the larger droplets fall
to the ground or on nearby objects, the smaller ones are rapidly evaporated to
their non-volatile residual forms and remain suspended as droplet nuclei. and
in this case they become infectious once they contain fungi.
Measures frequently taken in preventing fungal
nosocomial infections include effective use of antiseptics, disinfectants,
adequate cleaning, and isolation of patients with highly infectious diseases. Sanitation protocols regarding uniforms, equipment sterilization, washing, and other preventive measures. Thorough hand washing and/or use of alcohol rubs (disinfectants)by all medical personnel before and
after each patient contact is one of the most effective ways to combat
nosocomial infections
Nevertheless, less attention is paid to indoor air as
been a probable contributing factor to hospital acquired infections.
Ishida et al reported that airborne bacteria in the hospital
environment have been a major source of post-operative infection and a serious
problem in the Intensive Care Unit. Many of these isolates (bacteria and fungi )
are shown to be resistant to common antiseptics used in hospital, hence the
result into possible infections
The
organism that are more associated with fungal nosocomial infection include superficial
infections, these fungal infections affect the skin or
mucous membranes. Superficial fungal infections (e.g., yeast vaginitis, oral
thrush, and athlete’s foot) affect millions of people worldwide. Although
rarely life-threatening, they can have debilitating effects on a person's
quality of life and may in some cases spread to other people or become invasive
(systemic) and this is the case in Hospital environment. Most superficial
fungal infections are easily diagnosed and can be treated effectively. Other
organism are called systemic infections, these
occur when fungi get into the bloodstream and generally cause more serious
diseases. Systemic fungal infections may be caused either by an opportunistic
organism that attacks a person with a weakened immune system, or by an invasive
organism that is common in a specific geographic area, such as cocci and
histoplasma. Unlike superficial infections, systemic fungal infections can be
life-threatening. (3)
Infections are contagious and
represent a significant public health problem in many parts of the world. Dermatophytosis is not a reportable disease
but is a matter of concern because
of its contagiousness nature (4) correct identification of dermatophytes at the
species level is useful for differentiating between Dermatophytosis and
dermatomycosis (5), to control of environmental and animal sources of infection
and help for developing the preventive strategies
Conventional identification
techniques raises controversial issues like lack of
evidence to confirm the isolated pathogenic fungi types of species, Species-level
identification of these fungi classically relies on macro and micro
morphological features of the colonies on general and specific culture media and on some biochemical
and physiological complementary tests (6).
However, in many circumstances
phenotypic characteristics overlap
between species, and many isolates have
atypical nature in primary isolation thus attempt for final identification is time consuming
and requires expertised personnel on macroscopical
properties (7). By development of PCR
technology, a wide variety of molecular techniques such as RAPD-PCR, Nested-PCR, PCR-RFLP, PCR-EIA, Real-time PCR and microarray
technology were employed as possible alternatives for routine identification of fungi including
dermatophytes (7).At the present study, the ITS1-5.8S-ITS2 fragment of ribosomal DNA gene (rDNA) in
the Dermatophyte species will be used as
a reliable marker for species
identification. We will retrieve the reliable sequences of internal transcribed
spacers (ITS) regions from GenBank, then omputationally (in-silico) and practically
subjected them to a polymerase chain reaction-restriction enzyme (PCR-RE) assay
for identifying nearly all pathogenic Dermatophyte species. Additionally, we will
amplify and digest the DNA target in some reference Dermatophyte strains to confirm
the method. We will prepare a relatively
perfect restriction fragment length polymorphism (RFLP) barcode by using only a
single enzyme and believe that it could be useful for clinical and
epidemiological aims .
Therefore this study will be to aimed to presents the findings about the possible source of
nosocomial fungal infections in hospital environment . it will also investigate the quality of indoor air of different wards
and units of a chosen Hospital, to
ascertain their contribution to infection rate in the hospital. It will also
provide a baseline information on the quality of indoor air, hospital wards which
before was not available.
2. STATEMENT OF THE RESEARCH PROBLEM
It has been
proved that in hospitals pathogenic
fungi identifications can
facilitate tracing the sources of
infections in hospitalized
patients and then get raid on them ,get in control of them .
Previous studies has shown that hospitalized patient are
more likely to be affected by any pathogenic fungi even if in its mildest
infection stage
There is still more to assess about hospital environment,
like where is the highly infected environment that can raise contamination of
the immunocompromised people
And also how hospitalized people get infected by
nosocomial fungal infections and who can possibly be responsible.
3. OBJECTIVES
3.1. GENERAL OBJECTIVE
Assessing the hospital environment as a potential
source of fungal nosocomial infections.
3.2. SPECIFIC OBJECTIVES
- To
determine molecular relatedness of
isolates for epidemiologic investigation;
- To
determine the relationship of infected areas and fungal
infection rate in the Hospital;
- To
determine the diversity and distribution areas of Pathogenic fungi in Hospital
4. SIGNIFICANCE OF THE
STUDY
Why assessing hospital environment as a source of nosocomial fungal
infections?
Patients are primarily admitted for proper management instead they
acquire nosocomial fungi infections
Identification of the infected
hospital areas by fungi would be advantageous and will help in getting raid of
the nosocomial fungi infections
Assessing hospitals environment
will help the doctors to advice the patients and to educated the world how to
behave when at the hospitals
5. LITERATURE REVIEW
6. STUDY AREA
7. HYPOTHESES/RESEARCH QUESTIONS
Ø Can molecular relatedness
of isolates for epidemiologic investigation be determined?
Ø Can infected areas contribute
to fungal infections rate in hospital?
Ø Can the diversity and
distribution areas of Pathogenic fungi in Hospitals be determined?
8. MATERIALS AND METHODS
8.1 MATERIALS
ü Blunt scalpel
ü sterile petriplate
ü white envelopes
ü Sabouraud’s dextrose Agar (Difco) (SDA)
ü Chromo agar Candida
ü Glass bead
ü of
lysis buffer (100 mM Tris-Hcl pH
8, 10
mM EDTA, 100
mM NaCl, 1%
sodium dodecyl sulfate
(SDS), 2% triton
X-100
ü phenol
chloroform-isoamyl alcohol (25:24:1)
8.2. METHODS
Samples
will be collected from the air
,hospital ward surfaces where patients have access to . The specimens will
be placed in clean white envelopes with
each areas code labeled and later they will be
transferred to a sterile petriplate, we will also collect sample from
indoor and outdoor environment (including
air, medical devices, clothes, catheters
and ) of the Hospital. We are
expecting to get a lot of fungi
isolates (including yeasts and
moulds) which will be
collected from clinical
specimens consisting of urine,
surgical wounds, bedsores,
sputum and others,
and some locations
where Fungi are considered as
normal flora such as skin,
oral and nasal cavities
Isolation medium:
The fungi will be allowed to grow on Sabouraud’s
dextrose Agar (Difco) (SDA) with chloramphenicol (Chloramphenicol inhibits the
growth of bacterial contaminants) and bromocresol purple milk glucose agar (Ellis
et al.,2007)
Yeast, Fungi, Moulds isolates
will be sub cultured
on Chromo agar Candida,
incubated at 35oC for
2-4 days and
harvested yeast, fungi, moulds
colonies will be stored at -20°C
DNA
extraction:
Further Genomic DNA will be extracted using
glass bead disruption. Briefly, 300 mg
of 0.5 mm
diameter glass beads, 300µg of lysis buffer (100 mM Tris-Hcl pH 8, 10
mM EDTA, 100
mM NaCl, 1%
sodium dodecyl sulfate
(SDS), 2% triton
X-100) and 300
µl of phenol
chloroform-isoamyl alcohol (25:24:1)
will be added to
about 1-5 mm of
a fresh yeast
colony in a
1.5 ml tube. The samples will be shaked vigorously for 5 min, centrifuged
for 5 min at 5000
rpm, and the
supernatant will be transferred
to a fresh tube. The supernatant will be extracted again
with chloroform and
DNA will be precipitated by adding
the equal volume
of isopropanol and
0.1 volume of
3 M sodium
acetate (pH 5.2).
The solution will be
vortexed and incubated
for 10 min
at -20°C and
centrifuged for 15
min at 12000
rpm
Restriction
fragment length polymorphism( RFLP) analysis :
Fungal-specific universal primer pairs will
be used to amplify internal transcribed
spacer 1 ( ITS1)—5.8SS- ITS2 regions of rDNA in the tested fungi, the
amplification reaction will be performed
in a final volume of 25 µl 1 containing 1µl of extracted genomic DNA ( about
20mg),1,25 U of Taq DNA polymerase,0.3Mm
of each deoxynucleoside triphosphate mix( dATP,dTTP,dGTP,dCTP) 0.4µM of
each of
forward ITS1 ( 5`-TCC GTA GGT GAA CCT GCG G-3`) and reverse ITS4 (5`-TCC
TCC GCT TAT TGA TAT GC-3`) primers, 1.5Mm of MgCl2 and 2,5µL OF 10X PCR buffer.
PCR will be carried out in a thermal
cycler with the following
temperature profile : 1 cycle of
5 min at 95 OC,followed by 35 cycles of 1 min at 94 OC, 1
min at 56 OC and 1 min at 72 OC
and a final extension step at 72 OC
for 10 min. PCR products
will be
analyzed in 2% agarose gel with 0.5X
Tris - Borate-EDTA buffer, and it will b e stained with ethidium bromide and be visualized in UV
light.
Digestion with
restriction enzymes (endonucleases):
Ten microliters of each PCR product will be separately
digested with 10U of
HinfI and HaeIII restriction enzymes
at 37 OC overnight(The amplified products were subjected to
digestion with MvaI Fast digest (Fermentas Life Sci-ences, Lithuania) for 10
min at 37ºC. The reaction mixture contained 10 μl of PCR amplicons, 0.5 μl of the enzyme, 1.5 μl of 10X buffer
and 3 µl of water to a final volume of 15 µl.). Restriction fragment will be analyzed in 2.5% agarose gel
electrophoresis in 0.5 X Tris Borate-EDTA buffer for about 2.5 h ATT 80V and visualized by staining with
0.5 µg Ml-1 of ethidium bromide.
Data analysis
The gels will be visualized
using UV light system and we will record
photographically, then we will
compare with the profiles which will be
obtained in silico analysis.
Data of the isolates which
will show growth on SDA will be analysed using SPSSTM The results
will be then presented in descriptive
statistics using frequency tables, cross tabulation and bar charts.
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