3-Subjects, Materials and Methods
Subjects, Materials and Methods
This work was carried out in Microbiology & Immunology
Department, Faculty of Medicine, Benha University in the period from
June 2013 to July 2014.
Subjects:
The current study was conducted on 100 strains of P.aeruginosa
isolated from 220 random clinical samples taken from patients attending
Benha University and Benha Teaching Hospitals.
Hospital departments included in this study were: ICU, neonatal
ICU, chest, general surgery, burn, urology, ophthalmology and ENT.
The examined clinical samples were; bronchoalveolar lavage
(BAL), sputum, wound swab, infected burn wound swab, urine, stool,
blood, ear and eye swabs.
The samples were taken from patients admitted to hospitals and
also from outpatient clinics.
The patients were 130 males and 90 females and their ages were
ranged from days to 80 years.
Full history was taken from each patient including: patient's name,
age, sex, duration of hospital stay, underlying diseases, antimicrobial
intake, and exposure to surgery.
This study was approved by Benha University ethical committee
and consent was obtained from all patients under study.
)81(
3-Subjects, Materials and Methods
Materials
A. Materials used for isolation and identification of P.
aeruginosa:
1.
Nutrient agar medium (Oxoid).
2.
Pseudomonas Cetrimide Agar (Oxoid):
Composition: (gm / liter)
-
Gelatin peptone 20 g/l.
-
Magnesium chloride 1.4 g/l.
-
Potassium sulfate 10.0 g/l.
-
Cetrimide 0.3 g/l.
-
Agar 13.6 g/l.
Preparation: according to manufacturer's recommendations: 45.3g
of Pseudomonas Cetrimide Agar was suspended in 1 litre of
distilled water. 10ml of glycerol was added and boiled to dissolve
completely. The medium was sterilized by autoclaving at 121°C
for 15 minutes, cooled to approximately 50°C and poured into
sterile Petri dishes.
3.
Peptone water (Oxoid): base for sugar media.
4.
Sugar fermentation media: 5 sugars were used (glucose, lactose,
maltose, mannite and sucrose) (Sigma). 1% sugar concentration was
added to pepton water. The sugar tube contains Andrade's indicator
which change to pink if acid is produced and an inverted small tube
(Durham's tube) to detect gas formation in its apex.
5.
Nutrient gelatin medium:
-
Nutrient broth 1 liter (Sigma).
-
Gelatin 120 g (Sigma).
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3-Subjects, Materials and Methods
6.
Gram's stain: It was used for microscopic examination of the
isolated colonies from primary culture plates.
7.
Oxidase detection test strips (Oxoid code BR0064A):
Impregnated with the oxidase reagent (tetramethyl-p-phenylene
diamine dihydrochloride) were used for detection of bacterial
cytochrome oxidase enzyme.
B. Materials used for antibiotic susceptibility testing:
1- Muller Hinton agar (Oxoid):
Composition: (gm / liter)
-Beef dehydrated infusion 300 g/l.
-Casein hydrolysate 17.5 g/l.
-Starch 1.5 g/l.
-Agar 10 g/l.
Preparation: according to manufacturer's recommendations: 38 g of
Muller Hinton Agar was suspended in 1 litre of distilled water. Then
boiled to dissolve the medium completely. The medium was sterilized
by autoclaving at 121°C for 15 minutes, cooled to 50°C and poured
into sterile petri dishes.
2- Antibiotic discs (Bioanalyse) with antimicrobial content matching
Clinical
Laboratory
Standards Institute
recommendations
(CLSI) (Clinical Laboratory Standards Institute, 2012): The discs
were stored at 2-8°C in an airtight container with a desiccant supplied
by the manufacturer. The used antibiotic discs are listed in table (3-1)
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3-Subjects, Materials and Methods
Table (3-1): Antibiotic discs that were used in the study
Antibiotic
Symbol
Disc
content
Sensitive
(mm)
Intermediat
e
(mm)
Resistant
(mm)
1
Amikacin
AK
30 ug
≥17
15-16
≤14
2
Gentamycin
CN
10 ug
≥15
13-14
≤12
3
Tobramycin
TOB
10 ug
≥15
13-14
≤12
4
Piperacillin+tazo
TPZ
100/10
≥21
15-20
≤14
bactam
ug
5
Meropenem
MEM
10 ug
≥19
16-18
≤15
6
Imepinem
IMP
10 ug
≥19
16-18
≤15
7
Ceftazidime
CAZ
30 ug
≥18
15-17
≤14
8
Cefoperazone
CEP
10 ug
≥21
16-20
≤15
9
Cefotriaxone
CRO
30 ug
≥21
14-20
≤13
10
Cefotaxime
CTX
30 ug
≥23
15-22
≤14
11
Cefepime
FEB
30 ug
≥18
15-17
≤14
12
Aztreonam
ATM
30 ug
≥22
16-21
≤15
13
Amoxycillin/
AMC
30 ug
≥18
14-17
≤13
clavulanic
2:1
acid
14
Ciprofloxacin
CIP
5 ug
≥21
16-20
≤15
15
Polymixin B
PB
10 ug
≥12
-
≤11
16
Colistin
CT
10 ug
≥11
-
≤10
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3-Subjects, Materials and Methods
3- Sterile cotton swabs.
4- McFarland 0.5 turbidity standard:
McFarland 0.5 standard was prepared as following (Cheesbrough,
2006):
1. A 1% v/v solution of sulfuric acid is prepared by adding 1ml of
concentrated sulfuric acid to 99 ml of water and mixing.
2. A 1% w/v solution of barium chloride is prepared by dissolving 0.5
g of dihydrate barium chloride (BaCl2.2H2O) in 50 ml of distilled
water.
3. 0.6 ml of the barium chloride solution is added to 99.4 ml of the
sulfuric acid solution.
4. A small volume of the turbid solution is transferred to a screwcaped tube of the same type as used for preparing the test inoculum.
5. McFarland 0.5 standard tubes are stored in the dark at room
temperature. They can be stable for 6 months.
C. Materials used for phenotypic detection of MβL
production:
1- Micropipette.
2- Filter papers (Whatman Filter paper).
3- Di sodium EDTA ( 0.5 M EDTA )(Hi-Media, India) solution was
prepared by dissolving 18.61 g of EDTA in 100 ml of distilled water and
adjusting pH 8.0 by using NaOH (Yong et al., 2002).
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3-Subjects, Materials and Methods
D. Materials used for phenotypic detection of MβLs:
1- DNA extraction: Thermo Scientific GeneJET Genomic DNA
Purification Kit #K0721
Table (3-2): Components of the DNA extraction kit.
Proteinase K Solution
1.2 ml
RNase A Solution
1 ml
Digestion Solution
11 ml
24 ml
Lysis Solution
Wash Buffer I (concentrated)
10 ml
10 ml
Wash Buffer II (concentrated)
Elution Buffer (10 mM Tris-Cl, pH
9.0, 0.5 mM EDTA)
GeneJET Genomic DNA
Purification Columns pre-assembled
with
Collection Tubes
Collection Tubes
30 ml
50
50
2- DNA amplification
a- Master mix (Thermo Scientific, EU Lithuania) Maxima Hot Start
PCR Master Mix (2X) #K1051
Table (3-3): components of Maxima hot start PCR master mix
#K1051
100 rxns of 50 μl
Component
Maxima Hot Start
PCR Master Mix (2X)
Water, nuclease-free
2x1.25 ml
2x1.25 ml
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3-Subjects, Materials and Methods
Maxima Hot Start PCR Master Mix (2X) composition:
Maxima Hot Start Taq DNA polymerase is supplied in 2X
Hot Start PCR buffer, 400 μM dATP, 400 μM dGTP, 400 μM
dCTP, 400 μM dTTP and 4 mM Mg2+.
b- Primers (Biosearch technologies, USA) (Ellington, 2007)
Table (3-4): Sequence of primers that were used in this study.
IMP family
Imp-F
5'-GGA ATA GAG TGG CTT AAY TCT C-3'
Imp-R
5'-CCA AAC YAC TAS GTT ATC T-3'
VIM family
Vim-F
5'-GAT GGT GTT TGG TCG CAT A-3'
Vim-R
GIM-1
Gim-F
Gim-R
SPM-1
Spm-F
Spm-R
5'-CGA ATG CGC AGC ACC AG-3'
5'-TCG ACA CAC CTT GGT CTG AA-3'
5'-AAC TTC CAA CTT TGC CAT GC-3'
5'-AAA ATC TGG GTA CGC AAA CG-3'
5'-ACA TTA TCC GCT GGA ACA GG-3'
SIM-1
Sim-F
5'-TAC AAG GGA TTC GGC ATC G-3'
Sim-R
5'-TAA TGG CCT GTT CCC ATG TG-3'
3- DNA detection by agarose gel electrophoresis:
Reagents:
1) 40x TAE (Tris acetate EDTA) buffer #V4281. (Promega,
U.S.A)
2) Ethedium bromide solution (10mg/ml). (Promega, U.S.A)
3) Agarose. (Hopkins and Williams, England)
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3-Subjects, Materials and Methods
4) PCR marker (gene ruler 100 bp DNA ladder) (Fermentas) was
used that contained the following bands: 100, 200, 300, 400, 500,
600, 700, 800, 900 and 1000 bp.
Additional materials and equipment required:
 PCR thermal cycler (G-storm, United kingdom).
 Ultraspeed centrifuge (sigma, Germany).
 Thermomixer (peqlab TS 100, Germany).
 Agarose gel electrophoresis apparatus: (Biometra, Germany).
 Ultraviolet transilluminator: (Biometra ,Germany).
 Vortex (Shelton scientific, USA).
 Shaking water bath (Koterman, Germany).
 Ethanol (96-100%) (Adwic, Egypt).
 Pipettes and pipetts tips.
 Microcentrifuge tubes (1.5 ml).
 Disposable gloves.
)88(
3-Subjects, Materials and Methods
Methods
A- Isolation and identification of P. aeruginosa:
Cultivation of samples:
Sputum, BAL, wound exudate, burn wound exudates,urine, stool,
ear swabs, and eye swabs were cultured directly on Pseudomonas
Cetrimide Agar.
Blood samples were inoculated into blood culture bottles (Egyptian
Diagnostic Media) then incubated at 37 °C for 7–14 days. Subcultures
were done every 48 h on Pseudomonas Cetrimide Agar.
The plates were incubated at 37°C for 1-2 days. Bacteria growing
on the Pseudomonas Cetrimide Agar were further identified as follow:
Identification of the isolated strains:
1- Characteristic Colony morphology and exopigments production:
Demonstration of the presence of the pyocyanin is absolute
confirmation of a strain as P. aeruginosa as the major diagnostic test
(Govan, 1996).
Nutrient agar slopes, were inoculated with the isolates, incubated at
37°C for 24 hours, and then at room temperature for one week. After
incubation, they were examined for diffusible pigments. Pyocyanin is
blue green and non-fluorescent (Govan, 1996).
When pyocyanin production is absent or doubtful, P. aeruginosa,
was characterized by other biochemical reactions according to table (35).
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3-Subjects, Materials and Methods
Table (3-5): Distinguishing characters of clinically relevant
Pseudomonas species (Govan, 1996)
Pyocyanin Fluorescein
P. aeruginosa
P. cepacia
P. pickettii
P. stutzeri
P. fluorescens
P. putida
+
-
+
+
+
Growth
at 42°C
Gelatinase
+
+
+
±
-
+
+
+
-
Maltose
Lactose
(oxidative) (oxidative)
+
+
+
-
+
+
-
2- Microscopic examination of Gram stained film:
Gram-stained films were made from the resulting colonies and
examined microscopically. P. aeruginosa appears as Gram negative bacilli.
3- Oxidase test:
Oxidase test was performed using Oxidase Detection Strips (Oxoid).
-
The colony to be tested is touched by the Oxidase Detection Strip.
-
The strip is observed for up to 5 seconds.
-
A deep blue/violet colour indicates a positive reaction.
4- Examination of flourescein production: (Govan, 1996)
Fluorescein production is determined by visual examination of the
plates under ultraviolet transilluminator. A positive result is the
observance of a greenish-yellow fluorescent pigment in the agar.
5- Growth at 42 °C: (Cheesbrough, 2006)
Slants of nutrient agar were inoculated with the isolates, incubated at
42°C for 24-48 hours and examined for the presence of growth. Growth at
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3-Subjects, Materials and Methods
42°C differentiates P. aeruginosa from the less commonly isolated
pseudomonads, P. putida and P. fluorescens.
6- Examination of motility: (Collee et al., 1996)
The isolate was grown in nutrient broth for 16 hours and examined
for motility by the hanging drop technique.
7- Gelatin liquefaction test: (Collee et al., 1996)
A stab cultures were made in the nutrient gelatin test tubes, they
were incubated at 37°C for 24 hours. After incubation the nutrient
gelatin cultures were removed and held at 4°C for 30 min before reading
the results. Positive result is indicated by liquefaction of gelatin.
8- Sugar utilization tests: (Collee et al., 1996)
The isolate was inoculated in sugar test tubes; pink colour indicate
acid production and gas formation is detected in the apex of Durham's
tube
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B- Antimicrobial susceptibility
Antimicrobial susceptibility testing was performed for sixteen
different antibiotics by the Kirby–Bauer disc diffusion method
(Cheesbrough, 2006)
1.
Using a sterile wire loop, 3-5 well-isolated colonies of similar
appearance to the test organism were emulsified in 3-4 ml of
sterile physiological saline.
2.
The turbidity of the suspension was adjusted to match to the 0.5
McFarland
turbidity
standard.
The
standard
was
mixed
immediately before use.
3.
Using a sterile cotton swab, a plate of Mueller Hinton agar was
inoculated: After dipping the swab in the suspension, the excess
fluid was removed by pressing and rotating the swab against the
side of the tube above the level of the suspension. Then the swab
was streaked evenly over the surface of the medium in three
directions, rotating the plate approximately 60° to ensure even
distribution.
4.
Petri dishes with the lid in place were left for 3-5 minutes to allow
the surface of the agar to dry.
5.
Using sterile forceps, the 16 antimicrobial discs were evenly
distributed. The discs should be about 15mm from the edge of the
plate and no closer than about 25 mm from disc to disc.
6.
The plates were incubated aerobically at 35 °C for 16-18 h.
7.
After overnight incubation, using a ruler on the underside of the
plate, the diameter of each zone of inhibition was measured in
mm.
8.
The
diameters
were
'Intermediate/Moderately
interpreted
sensitive',
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as
'Resistant',
'Sensitive
(Susceptible)
3-Subjects, Materials and Methods
according to CLSI published diameters table (3-1) (Clinical
Laboratory Standards Institute, 2012).
C- phenotypic detection of MβL production:
1. IMP-EDTA CDT:
The IMP-EDTA CDT was performed as described by Yong et
al., (2002). Test organisms were inoculated on Mueller Hinton agar
plates. Two (10 μg) imipenem discs were placed on the plate, and
appropriate amounts of 10 μL of EDTA solution were added to one of
them. The inhibition zones of the imipenem and imipenem-EDTA discs
were compared after 16 to 18 hours of incubation aerobically at 35°C. In
the CDT, if the increase in inhibition zone with the imipenem and EDTA
disc was ≥ 7 mm than the imipenem disc alone, it was considered as MβL
positive.
2. IMP-EDTA DDST:
The IMP-EDTA DDST was performed as described by Lee et al.,
(2003). Test organisms were inoculated on to plates with Mueller Hinton
agar. An imipenem 10 μg disc was placed 20 mm (centre to centre) from
a blank disc containing 10 μL of 0.5 M EDTA. Enhancement of the zone
of inhibition in the area between imipenem and the EDTA disc in
comparison with the zone of inhibition on the far side of the drug was
interpreted as a positive result (Lee et al., 2003)
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Preparation of EDTA paper discs:
Using an ordinary office two-hole puncher, paper discs with
approximate diameter of 6.3mm., were punched out one by one from a
sheet of paper. Precautions were taken to avoid overlapping of holes.
Since the paper discs had a tendency to curl after punching, these were
flattened by spreading them in a single-layer on a clean smooth surface
then pressed by rolling a bottle repeatedly. The discs were placed in vials
then autoclaved for 15 minutes and allowed to cool.
Impregnation of disc
The sterile discs were placed in sterile petri dishes (150 x 20mm)
approximately 5mm apart. Using a mechanical pipettor with a fixed
volume delivery of 10 ul, the discs were loaded with EDTA solution
taking precautions that the tip was in light contact with the disc.
Drying and storage
Without covering the petri dishes, the discs were allowed to dry in
a clean incubator at 35°C for 2-3 hours.
After drying, 50 to 100 discs were placed in small sterile air-tight
labeled containers. The discs were stored in a freezer at -20°C. Unopened
containers were removed from the freezer 1 or 2 hours before use to
equilibrate to room temperature before this is opened to minimize the
amount of condensation that may occur when warm room air reaches the
cold containers (Antonio-Velmonte et al., 1988).
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3-Subjects, Materials and Methods
D. Genotypic detection of MβL genes by using Multiplex
PCR :
A- Genomic DNA extraction:The procedure was carried out according to manufacturer instructions as
follows:
Gram-Negative Bacteria Genomic DNA Purification Protocol:
1. Ethanol (96-100%) was added to the concentrated wash Buffer (WB) I
and concentrated WB II prior to first use to prepare the working solution
according to the following table:
Table (3-6): Preparation of Wash Buffers
WB I
WB II
10 ml
10 ml
Ethanol (96-100%)
30 ml
30 ml
Total volume
40 ml
40 ml
Concentrated wash
solution
2. Bacterial cells (2x109) were harvested in a 1.5 or 2 ml microcentrifuge
tube by centrifugation for 10 min at 5000 x g. The supernatant was
discarded.
3. The pellet was resuspended in 180 μl of Digestion Solution. Proteinase
K Solution 20 μl was added and mixed thoroughly by vortexing to obtain
a uniform suspension.
4. The samples were incubated at 56°C in the thermomixer until the cells
were completely lysed ∼30 min.
5. RNase A Solution 20 μl was added, mixed by vortexing and the
mixture was incubated for 10 min at room temperature.
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6. Lysis Solution 200 μl was added to the sample then Mixed thoroughly
by vortexing for about 15 seconds until a homogeneous mixture was
obtained.
7. 400 μl of 50% ethanol was added and mixed by pipetting or vortexing.
8. The prepared lysate was transferred to a GeneJET Genomic DNA
Purification Column inserted in a collection tube. The column was
centrifuged for 1 min at 6000 x g. The collection tube containing the
flow-through solution was discarded. The GeneJET Genomic DNA
Purification Column was placed into a new 2 ml collection tube.
9. 500 μl of WB I was added, and then the mixture was centrifuged for 1
min at 8000 x g. The flow-through was discarded and the purification
column was placed back into the collection tube.
10. 500 μl of WB II was added to the GeneJET Genomic DNA
Purification Column and then was Centrifuged for 3 min at maximum
speed (≥12000 x g).
Optional. If residual solution was seen in the purification column, the
collection tube was emptied and the column was re-spined for 1 min. at
maximum speed.
The collection tube containing the flow-through solution was discarded
and the GeneJET Genomic DNA Purification Column was transferred to
a sterile 1.5 ml microcentrifuge tube.
11. 200 μl of elution Buffer was added to the center of the GeneJET
Genomic DNA Purification Column membrane to elute genomic DNA,
Incubated for 2 min at room temperature and centrifuged for 1 min at
8000 x g.
For maximum DNA yield, the elution step was repeated with
additional 200 μl of Elution Buffer.
12. The purification column was discarded and the purified DNA was
used immediately in downstream applications or stored at -20°C.
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B- DNA amplification:
The procedure was carried out according to manufacturer's instructions as
follows:
1. Maxima Hot Start PCR Master Mix (2X) was gently vortexed after
thawing.
2. Two PCR reactions were made for each sample. Reaction 1 (M1) was
containing three primers (SIM-1, SPM-1, and GIM-1) while reaction 2
(M2) was containing two primers (IMP and VIM).
3. M1 PCR tube was containing the following components for each 50
μl reaction:
Table (3-7): components of M1 PCR tube.
Maxima Hot Start PCR
Master Mix (2X)
Each forward primer
(SIM-1, SPM-1, and
GIM-1)
Each reverse primer
(SIM-1, SPM-1, and
GIM-1)
25 μl
1 μl (total 3 μl)
1 μl (total 3 μl)
5 μl
Template DNA
Water, nuclease-free
14 μl
Total volume
50 μl
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3-Subjects, Materials and Methods
M2 PCR tube was containing the following components for each 50 μl
reaction:
Table (3-8): components of M2 PCR tube.
Maxima Hot Start PCR
Master Mix (2X)
25μl
Each forward primer
(IMP-VIM)
Each reverse primer
(IMP-VIM)
1 μl (total 2 μl)
Template DNA
1 μl (total 2 μl)
5 μl
Water, nuclease-free
16 μl
Total volume
50 μl
4. The samples were gently vortexed and spinned down.
5. The thermal cycler contains a heated lid, so no need to overlay the
reaction mixture with mineral oil.
6. PCR was performed using the recommended thermal cycling
conditions outlined below:
Table (3-9): the program used in the thermal cycler (Ellington, 2007)
Step
Temperature (°C)
Time
95°C
4 min
Denaturation
95°C
30 s
Annealing
46°C
30 s
Extension
72°C
45 s
Final extension
72°C
10 min
Initial
denaturation
)98(
Number
of cycles
1
45
1
3-Subjects, Materials and Methods
7. PCR products were separated by 2% agarose gel electrophoresis.
Agarose gel was stained by ethedium bromide and visualized by
ultraviolet transilluminator.
C- DNA detection by agarose gel electrophoresis:
The procedure was carried out according to (Yilmaz et al., 2012).
1) The comb was placed across the rim of the casting tray to form sample
wells. The height of the teeth was adjusted so that they penetrate the gel,
about 1mm of gel was left between the teeth and remaining plate.
2) The working solution of TAE 1x buffer was prepared as follows:
 25ml of 40x TAE buffer
 975ml distilled water
3) The agarose solution with concentration of 2% was prepared as
follows:
 1.5gm agarose.
 75 ml TAE buffer was added.
 The agarose was melted in water bath, then left to cool down to
reach 60°C.
 2μl ethedium bromide was added.
4) The liquid agarose was poured into the casting tray, air bubbles were
avoided. Then the agarose was set into a gel for 30 minutes.
5) The comb was removed carefully from the edges of the casting tray.
6) The casting tray containing the agarose gel was put into the
electrophoresis buffer tank and the gel was covered with 200 ml TAE
buffer.
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7) The DNA samples were prepared as follow:
-15 μl of DNA were loaded into the gel (no need for addition of
dye as master mix contains a dye for monitoring electrophoresis
progress).
-15 μl of DNA ladder was loaded directly into the gel as the ladder
is ready to use (it is premixed with the 6X Orange DNA Loading
Dye).
8) The lid was placed on the unit so that the cathode was nearest the
sample wells, since the nucleic acid samples migrated towards the anode.
9) The power supply was connected. The length of the run was 40
minutes, at 80 volt and 300 mAmp.
10) The DNA was observed by fluorescence of ethedium bromide.
 The gel was transferred to the UV transilluminator.
 DNA bands were observed by using ultraviolet light at wave length
312 nm in a dark room.
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STATISTICAL ANALYSIS
The data were recorded, tabulated, coded then analyzed using the
computer program SPSS (Statistical package for social science) version 16
to obtain:
Descriptive data:
Descriptive statistics were calculated for the data in the form of
Number and percent.
Analytical statistics:
A- In the statistical comparison between the different groups, the
significance of difference was tested using one of the following tests:1- Inter-group comparison of categorical data was performed by
using chi square test (X2-value),
2- Z test for proportions is used to compare one outcome in two
different groups.
P value <0.05 is considered statistically significant, P value <0.001
is considered highly significant in all analyses and P value ˃ 0.05 is
considered insignificant.
B- The sensitivity and specificity of IMP EDTA CDT and IMP EDTA
DDST in comparison with PCR were examined using receiver operating
curve (ROC curve) analysis to determine the best diagnostic method.
C- Kappa test: The measure of the level of agreement between two
categorical measures.
Value of kappa Strength of agreement
<0.20 Poor
0.21–0.40 Fair
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0.41–0.60 Moderate
0.61–0.80 Good
0.81–1.00 Very good
D- Positive predictive values (PPV): the proportion of people with a
positive test who have the disease.
E- Negative predictive values (NPV): the proportion of people with a
negative test who are free of disease
F- Odds ratio (OR), confiedence interval (CI) are used to assess risk
factors.
Odds: the ratio of the number of times an event happens to the
number of times it does not happen in a group of patients.
Odds ratio: the odds of an event happening in one group divided by
the odds in a different group.
CI: the range around a study’s result within which we would expect
the true value to lie.
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