CHAPTER 3: METHODOLOGY

Cross Sectional Study Design

This study was carried out with a purpose of evaluating the haematological and
biochemical changes and antioxidants levels in the plasma of the student patients of typhoid
fever. Following flow chart diagram shows the cross-sectional study design for the assessment of
biochemical and hematological biomarkers among student patients of typhoid fever.

Figure 3.1 Flow Chart Diagram for Study Design

Context
Khushab is a District. It is situated in Punjab. Punjab is the province of Pakistan. It covers
a range of 6,511 square kilometers. The district comprises of

 3 Tehsils
 1 Sub Tehsil
 Four chief towns

Its administrative capital is Jauharabad. The district is named after the historical city of
Khushab sited within its boundaries. Rendering to the 1998 census, the population was 905,711
with 24.76% living in urban areas. Khushab comprises of agricultural lowland plains, lakes, and
hills. Parts of the Thal desert touch the district, which has a span of over 70 miles (110 km). It is
located between the Indus River and the Jhelum River.

There are three lakes in the district.

 Ochali,
 Khabbaki
 Jahlar

Kanhatti Garden is the largest forest in Khushab district, near Khabbaki village in the Soon
Valley. Khabikki Lake is a salt-water lake in the southern Salt Range. The lake is one kilometer
wide and two kilometers long. Khabikki is also the name of a nearby village. Sakesar is the
highest mountain in the Salt Range and is the site of the ancient Amb Temples. Sakesar’s summit
is 1522 meters / 4946 feet high. It is also situated in Khushab District.
Study Area
Typhoid rapid screening test kits were performed and samples were collected from the
students who were positive for typhoid in the district Khushab. Most of the sampling has been
done form the district headquarter Jauharabad as it has the major schools and colleges which
holds the students from lower class to upper class.

Population Analysis
All the subjects included in the study were students either from Jauharabad or the vicinity
areas with urban and rural residence and varied socioeconomic status. Identification of the
typhoid positive subjects was done by The Typhoid Rapid Test Kits. After the samples have been
taken, they are further confirmed by positive culturing of the samples. Both male and female
students were included in control and experimental groups.
Inclusion and Exclusion Criteria
At first the subjects with fever are checked by typhoid rapid test kits and only those patients
are included in the experimental group:

 Which are diagnosed with typhoid by typhoid rapid test kit and later on their blood
culturing confirmed it.
 Only the students are included in this study.
 The subjects with other disease history such as Hepatitis, diabetes, cardiac diseases are
excluded from the study.
 In control group, all the subjects are normal healthy students with no disease history.

Sampling Technique and Duration

Random sampling technique in which every member of the population has an equal
chance of selection was used and samples were collected within the duration of January 2020 to
March 2020. Sampling was done after the approval of ethical committee.

Questionnaire Survey
Self-designed questionnaires were given to every individual to know about their
demographic data, contact with family or relatives or friends that have typhoid fever ,
socioeconomic status, hygienic conditions, food and water resources conditions. And a
special case questionnaire was designed to note appeared symptoms in the positive
subjects, their travel history and hygienic conditions along with food and water resources.
Similar information of control group was obtained through questionnaire. Questionnaire
was in English but also translated into Urdu and Punjabi to the subjects in order to extract
best information during interviews.

Sampling Questionnaire

Questionnaire for Typhoid

 Age: …………….. Years Date:
 Gender: Male Female Area:
 Typhidot Test: Positive Negative
 Knowledge of Typhoid Fever:
 Years of Education:
 Socioeconomic status:

Risk Factors
 Water Sources:
 Tap water: Filtered water:
Hand Pump: Tube well:
Motor Pump: Mineral water:
Boiled water:
 Food:
Food from outside: Raw fruit or vegetables:
Following how many times in a week
 Junk Food:
 Milk:
 Any type of meat:
 Vegetables:
 Pulses:
 Personal Hygiene:
Wash hands before eating: Wash hands after eating:
Wash hands after using washroom: Wash hands with soap:
Take bath frequency: Change clothes frequency:
 Contact with Typhoid Patient:
 Typhoid Immunization History:

 Case Questionnaire
 Travel  Yes No  Places Visited:
………………………
………………………
………………………
………………………
 Type of
Accommodation:
………………………
………………………
………………
 Departure: ___ /
___ / ___ Return: ___
/ ___ / ___
 Illness while away:
Yes No

 Household / Close  Yes No  Relationship:

 contact of person ………………………
known to have ………………………
typhoid infection ……………….
 or similar illness
  Had previous  Yes No  Approx. date: ___ /
typhoid/paratyphoid infection? ___ / ___

 Class fellow known to  Yes No

have current or near  Approx. date: ___ /
past ___ / ___
typhoid/paratyphoid
infection?

 Drank untreated  Yes No  Specify type:

water? ………………………
………………………
 Date :
_____/_____/_____
 Location:
………………………
………………………
………………………

 Participated in  Yes No  Activity:

swimming / water …………………………
sports? …………..…………….
 Date :
_____/_____/_____
 Type of water (e.g.
pool, river, etc.):
…………………………
………………………..
 Address:
…………………………
…………………………
………………
 Exposure to  Yes No  Date:
raw/untreated _____/_____/_____
sewage?  Exposure/activity:
…………………………
…………………………
……………………….
 Any other disease:
 Sewerage system of
area:
 Type of Toilet system:

Symptoms
  Malaise Yes No  Body aches Yes No
 Anorexia Yes No  Diarrhea Yes No
 Fever Yes No  Constipation Yes No
 Headache Yes No  Vomiting Yes No
 Cough Yes No
 Rash / skin spots Yes No

Response Rate
Overall response rate was 97%. Almost all the subjects did not try to hide the personal
information.

Quantitative Approach

Typhoid Rapid Test Screening

Introduction
It is a rapid test kit which is used for immediate detection. Its detection is due to anti
Salmonella Typhi IgG and IgM. These antibodies are present in human serum or plasma.

Proposed use
It is a screening test named as The Typhoid IgG/IgM Rapid Test Device. It helps
diagnose the infection with Salmonella typhi. The specimens which have IgG/IgM must be
confirmed with alternative test methods.

Summary
Typhoid is a fever. It is caused by a gram negative bacterium. This bacterium is
Salmonella typhi. The patients having aids are significantly at risk. They have increased chances
of clinical infection. H. pylori infection may increase the chance of acquiring typhoid. The
diagnosis depends upon the detection of bacterium in the blood. The typhoid IgG/IgM rapid
device test is a simple test which is used for simultaneous detection of bacteria and differentiates
IgG and the IgM antibodies to Salmonella typhi specific antigen in serum or plasma.

Principle
It is a serum/plasma device. It is based upon lateral flow chromatographic immunoassay.
The test cassettes comprises of following:
 1. A burgundy colored conjugate pad containing recombinant S. typhoid H antigen
and O antigen conjugated with colloid gold (Typhoid conjugates) and rabbit IgG
Gold conjugates
2. A nitrocellulose membrane strip containing two test bands (IgM and IgG bands)
and a control band (C band)

A definite volume of sample is put on the well present is cassette. The sample moves in
cassette by capillary action. Anti S typhi IgM is attached to the conjugate if present in the
sample. The immunocomplex is then captured on the membrane by the precoated anti human
IgM antibody. It forms a burgundy colored IgM band. It indicates a positive test.

Precautions
 Manual should be read carefully before using the devices. Invalid test results due to
failure to follow the instructions.
 Do not use expired devices.
 All reagents should be used at room temperature.
 Hemolized blood shouldn’t be used for testing.
 Wear protective gear.
 Wash hands after carrying out tests.
 Don’t perform test in room with heavy air flow.
 Don’t smoke, eat or drink while performing tests.
 Dispose the devices after use as it is biohazardous.
 Testing results should be read within 15 minutes after the specimen is put on the device.

Storage and Stability

The package should be stored at room temperature. The test device should not be expired.
The device should be held in sealed pouch until use. Do not freeze the device.

Specimen collection and Preparation

Any material of human origin considered infectious. They were handled using standard
safety procedures.

Plasma
 Collect blood specimen in EDTA tube.
 Separate the plasma by centrifugation.
 Carefully withdraw the plasma by micropipette into ne pre labeled tube.
Serum
 Collect blood specimen into a red top collection tube by venipuncture. This tube has no
anti coagulants.
 Allow blood to clot.
 Separate the serum by centrifugation.
 Carefully withdraw the serum by micropipette into ne pre labeled tube.

After the collection of specimen, the testing should be as soon as possible. Specimen should
be stored at 2 to 8 C, if the testing is not possible immediately. Avoid multiple freeze thaw
cycles. Before testing, froze specimens should brought at room temperature and gently mixed. If
the visible particles are present in the sample then the centrifugation should be repeated.

Materials Provided
 Test Devices
 Buffer
 Droppers
 Package Insert

Directions Used
 All the materials used should be allowed to settle at room temperature before use.
 Mix the specimen well.
 Device seal should be open only when the sample is ready to be tested.
 Fill the pipette dropper with specimen.
 Vertically, hold the pipette. Transfer 1 drop of specimen and 1 drop of buffer on
cassette.
 Set up timer.
 Results can be read in 5 to 10 minutes.
 Don’t read results after 15 minutes.
Interpretation of results
Refer to images.

Positive
 Alongside with the presence of C band, if only the IgM band is present, the result is
positive. As the IgM presence shows the anti S IgM.
 In addition to the presence of C band, if only the IgG band is present, the result is
positive.
 If both IgG and IgM bands are present and the C band is present, the result is also
positive.

Negative
 Only C band is present. No IgM or IgG bands present. The result is negative.

Invalid
 Control line fails to appear.

Limitations
It has following limitations:
  The sample should be tested with another testing method if the test is positive with
Typhoid Rapid Test Device.

Blood Collection
Sample collection was done by venipuncture from both control and case groups by sterile
injections. Blood then collected into EDTA tubes. Total ml blood drawn from the subjects.
Further, Complete blood count (CBC) test, Liver function Test (LFT) and analysis for oxidative
stress was done.

Sample Management and Storage

Blood samples were immediately placed into ice box. It is to maintain the temperature.
After collection of samples, CBC and LFT tests were performed and reports were collected from
laboratory. Mein hematological and liver function test parameters were analyzed.

Plasma Extraction
Blood samples were centrifuged for 1 minute at 4100rpm in Humax 4K centrifuge at
DHQ laboratory. Micropipette was used to separate the plasma present on the upper layer. Then
plasma was placed into labelled Eppendorf. Plasma samples then immediately stored at 20 C in
ultra-freezer.
Analytical Instruments

Automated Hematological Analyzer

Complete Blood Count (CBS) was done through SYSMEX Kx-21 Haematological
Analyzer.it is capable of multiple parameter analysis. It has the processing speed of 0 samples
per hour. It displays on LCD screen. These are the parameters that are analyzed by this analyzer.

 Hemoglobin concentration (Hb)

 Red blood cells (RBCs)
 White blood cells (WBCs)
 Hematocrit percentage (HCT)
 Mean corpuscular hemoglobin (MHC)
 Mean corpuscular hemoglobin concentration (MCHC)
 Platelets % (PLT)
 Lymphocytes % (LYM)
 Neutrophils % (NEUT)
 Mixed cell count % (MXD)
Light Spectrophotometer
UV Visible spectrophotometer is a reliable instrument for the absorbance measurement of
different substances in biological samples. This spectrophotometer was used for measuring the
absorbance readings of antioxidants in plasma of the typhoid fever subjects. A liquid crystal
display shows the values of absorbance.
 Biochemical Markers

1. OSI Protocol
Oxidative stress index (OSI) is a measurement of Total Antioxidant Capacity (TAC) and
Total Peroxides (TP). For determination of OSI, plasma was divided into 2 parts. One was
subjected for Total Anti-oxidant Capacity (TAC) measurement and other was used for Total
Peroxides (TP) measurement.

Total Anti-oxidant Capacity Measurement

Principle

Total antioxidant capacity was measured through FRAP assay. FRAP assay is based on
principle that at low pH, Ferric Tripyridyl Triazine (Fe III TPTZ) get reduced to ferrous
Tripyridyl Triazine and develop intense blue colour (Benzie & Strain, 1996).

Table 3.2 Reagents for FRAP Assay

Sr. # Case Number Reagent Company

1 7782-63-0 Ferrous Sulphate Merk K GaA
2 10025-77-1 Ferric Chloride Sigma Aldrich
3 64-19-7 Glacial Acetic Acid Merk K GaA
4 7647-01-0 Hydrochloric Acid BDH-Chem
5 127-09-3 Sodium Acetate Sigma Aldrich
6 3682-35-7 2,4,6-tri-pyridyl-s-triazine Glentham Life Sciences

Procedure

Preparation of 300mM Acetate Buffer

16ml of glacial acetic acid (C₂H₄O₂) and 3.1 gram of sodium acetate (CH₃COONa) were
mixed in 1000ml of distilled water. Solution was stored at 4 centigrade after adjusting pH to 3.6.
Preparation of 40mM dilute HCl

1.46ml of conc. HCl was dissolved in 1000ml of distil water. Solution was stored at room
temperature.

Preparation of 10mM TPTZ

0.031 grams of TPTZ (2, 4, 6-tri-pyridyl-s-triazine) was mixed in 10ml of HCl in

corning tube. Suspension was placed in water bath at 50 centigrade.

Preparation of 20mM Ferric chloride

0.054 grams of ferric chloride (FeCl₃.6H₂O) was dissolved in 10ml of distilled water.
Solution was made on the day of assay.

Standard solution of 1mM Ferrous Sulphate

For preparation of 1mM of Ferrous sulphate (FeSO₄.7H₂O), 0.278 grams were mixed in
1 litre of distilled water and used as standard solution.

By mixing 1 ml of 10mM TPTZ in 4omM HCl, 10 ml of 300mM acetate buffer and 1ml
of 20mM ferric chloride, FRAP reagent was prepared. Before starting reaction, 0.1ml of plasma
and 0.9ml of FRAP reagent were incubated at 30 centigrade for 5 minutes. Reaction mixture
(1ml) contain;

0.1mlof plasma

0.9ml of FRAP reagent

Standard solution of ferrous sulphate was used as blank and absorbance was recorded at 630 nm
wavelength through spectrophotometer.

Calculation

Total Antioxidant Enzyme (µmol/L) = Test Absorbance /Standard Absorbance×STD conc.

(1000)
 Total Peroxides Measurement

Principle

Total peroxides were measured by FOX 2 method. Principle depends upon the ability of
ferrous ion get oxidized to ferric ion due to presence of peroxides in plasma and form ferric-
xylenol complex and produce characteristic orange colour (Nourooz-Zadeh, 1999).

Table 3.3 Reagents for Total peroxides

Sr. # Case Number Reagent Company

1 7783-85-9 Ammonium Ferrous Sulphate Sigma Aldrich
2 7664-93-9 Sulphuric Acid Riedel-de-haen
3 128-37-0 Butylated Hydroxyl Toulene Sigma Aldrich
4 3618-43-7 Xylenol Orange Sigma Aldrich

Preparation of 25mM H₂SO₄

25mM H₂SO₄ was prepared by dissolving 12.5ml H₂SO₄, making volume of 100ml by
adding distilled water.

Preparation of 250mM Ammonium ferrous sulphate and

9.8mg of ammonium ferrous sulphate [(NH₄)₂Fe(SO₄).6H₂O] was added in 10ml of

25mM H₂SO₄ making a final concentration of 250mM ferrous iron in acid.

Preparation of 4nM BHT in 90% methanol

For making solution of ion of 4nM BHT [(CH₃)₃C] ₂C₆H₂ (CH₃) OH, 79.2 mg of BHT
in 90ml HPLC grade methanol.
 FOX reagent was prepared by 250µM ammonium ferrous sulphate in to 250 mM H2SO4.
4nM BHT solution this solution and stirred well. Then 100µM xylene orange (7.3mg) was added
making a final volume of 100ml.

1800µL FOX 2 reagent and 200μL of plasma were incubated in cuvette for 30 minutes
and centrifuged at the speed of 12000 rpm for 10 minutes. FOX2 reagent excluding ferrous
sulphate was used as blank. Absorbance of supernatant was measured at 560 nm wavelength.

Calculation

Absorbance of standard solution of H₂O₂ having concentration of 5, 10, 15, 20, 25 and 30
µmol/L was measured through spectrophotometer at wavelength of 560nm. Slop equation was
calculated by plotting graph against y-intercept.

Total peroxides = sample Absorbance – Y intercept/Slope

Where Y-intercept is 0.0153 and slope is 0.023

Calculation of OSI

Oxidative stress index is calculated as follow;

OSI= [Total Peroxides (TP) × 100]/Total Antioxidant Capacity (TAC)

 2. Superoxide Dismutase
Principal

Principle is based on inhibition of Nitro blue tetrazole (NBT) by superoxide dismutase

(Beauchamp & Fridovich, 1971).

Table 3.4 Reagents for SOD

Sr. # Case Number Reagent Company

1 7778-77-0 Potassium Dihydrogen Phosphate Sigma Aldrich
2 143-33-9 Sodium Cyanide Sigma Aldrich
3 215300662-2 Nitro Blue Tetrazolium (NBT) Bio PLUS
4 83-88-5 Riboflavin Sigma Aldrich
5 16788-57-1 Dipotassium Hydrogen Phosphate Sigma Aldrich
6 6381-92-6 Ethylene Diamine tetra Acetic Acid Sigma Aldrich

Procedure

Preparation of 1.5mM NBT solution

3.23mg of NBT was taken in flask and volume was raised up to 2.64ml by adding
distilled water. Solution was stored in dark cold bottle.

Preparation of 0.12Mm riboflavin solution

0.06mg of riboflavin was put in flask and volume was raised up to 1.3ml by adding
distilled water. Solution was stored in cold dark bottle.

Preparation of 0.1M NaCN/EDTA solution

 0.08 mg of NaCN and 0.16g of EDTA were taken in flask and volume was raised up to
5.4ml by adding distilled water.

Preparation of 0.067 M potassium phosphate buffer (pH 7.8)

0.74g of dibasic salt (K2HPO4) and 0.14g of monobasic salt (KH2PO4) were taken in
flask. Volume was raised up to 80ml by adding distilled water and pH was adjusted to 7.8.

3 ml of buffer was added in cuvette as a blank and its absorbance was noted after placing
it in spectrophotometer. Spectrophotometer was adjusted at zero at wavelength of 560nm after
taking blank reading. 5-6 cuvette were placed in light box with internally mounted 30 watt light
bulb.0.016ml of riboflavin and 0.05 ml enzyme extract was added in each tube. For 12 minutes,
all cuvettes were incubated in light box. After that 0.033 ml of NBT and 0.067ml of
EDTA/NaCN solution was added in illuminated reaction mixture and cuvettes were transferred
to spectrophotometer.

After 20 seconds of reaction, absorbance was noted. Superoxide Dismutase activity was
determined by measuring %age inhibition of NBT.
 Figure. 3.6 Incubation of Reaction Mixture

Calculation

%age Inhibition = Blank Absorbance – Sample Absorbamce×100 / Blank Absorbance

SOD (U) = 1/50×Percentage

SOD (U/L) = SOD (U) ×Total volume of reaction mixture/plasma volume

3. Catalase
Principal

Principle is based on ability of Catalase to lower the H₂O₂ concentration at wavelength of

240 nm (Chance & Maehly, 1955).
Table 3.5 Reagents for Catalase

Sr. # Case Number Reagent Company

1 10028-24-7 Disodium Hydrogen Phosphate Sigma Aldrich
2 13472-35-0 Sodium Dihydrongen Phosphate Sigma Aldrich

Procedure

10mM Hydrogen peroxide and 50mM sodium phosphate buffer (pH = 7.0) are required
for reaction mixture

Preparation of phosphate buffer (50mM)

0.270 gram of dibasic salt (Na2HPO4) and 0.371 gram of monobasic salt (NaH2PO4)
was taken in flask. Distilled water was added, making a total volume of 100ml and pH was 7.0.

Preparation buffer substrate solution

0.442ml of 10mM H2O2 was dissolved in 50mM phosphate buffer solution making a
buffer substrate solution. Reaction mixture (2ml) contains;

 Enzyme extract 0.05ml

 Buffered substrate solution 1.95ml
 Blank 60mM phosphate buffer used as blank
2ml of blank solution was taken in cuvette and placed in spectrophotometer, setting it to
zero at 240nm wavelength. 1.95ml of buffered substrate and 0.05ml of enzyme extract were
taken in cuvette and placed the cuvette in spectrophotometer. Reaction time was 3 minutes and
absorbance was noted after 1 minute interval.
 Figure. 3.7 Catalase determination through UV Spectrophotometer

Calculation

Activity (U/ml) = ΔA / min × Dilution × 2ml / 0.04mM1‫ ־‬cm-1 × 0.05ml

4. Lipid peroxidation
Principle

MDA, a lipid peroxidation product, forms orange to pink colour product when heated
with Thiobarbituric acid (TBA), whose absorbance is measured at 535nm (Buege & Aust, 1978).

Table 3.6 Reagents for Lipid Peroxidation

Sr. # Case Number Reagent Company

1 76-03-9 Tri Chloroacetic Acid (TCA) SRL-Chem
2 504-17-6 Thiobarbituric Acid (TBA) UMI-Chem
3 7647-01-0 Hydrochloric acid BDH
Procedure

Stock solution of 15 grams TCA, 0.375 grams TBA and 875ul HCl was prepared. 2ml of
stock solution and 0.1 ml of enzyme source were taken in eppendrops and heated at 100
centigrade for 15 minutes in water bath. After heating, solution was allowed to cool and then
centrifuged at 3600 rpm for 10 minutes. Orange to pink coloured supernatant was observed.

3 ml of stock solution was taken in cuvette, placed in spectrophotometer and set it to zero
at wavelength of 535 nm. Absorbance of orange to pink supernatant was read at 535nm.

Figure 3.8 Sample preparation and centrifugation

Calculation

Concentration of MDA was measured using following formula;

Absorbance = Extinction coefficient × 2 × Activity

Activity (mM) = Absorbance / Ɛ × 2

Ɛ= extinction coefficient (Ɛ = 156mM¹‫־‬cm¹‫)־‬

 Glutathione Peroxidase

Principle

Principle is based on the activity of glutathione peroxidase to utilize NADPH during

conversion of lipid peroxides and H₂O₂ at wavelength of 340nm (Paglia & Valentine, 1967).

Table 3.7 Reagents for Glutathione peroxidase

Sr. # Case Number Reagent Company

1 7722-84-1 Hydrogen Peroxide Merck K GaA
2 26628-22-8 Sodium Azide Daejung-Chem
3 27025-41-8 Glutathione oxidized Oakwood-Chem
4 6381-92-6 Ethylene diamine tetra acetic acid Sigma Aldrich
(EDTA)
5 16788-57-1 Dipotassium Hydrogen Phosphate Sigma Aldrich
6 2646-71-1 Nicotinamide adenine dinucleotide Chem-impex int’l
phosphate
7 7778-77-0 Potassium Dihydrogen Phosphate Sigma Aldrich
8 70-18-8 Reduced Glutathione BDH-chem VWR

Preparation of 50mM Potassium Phosphate Buffer

0.570 grams of dibasic potassium phosphate (K₂HPO₄) and 0.342 grams of monobasic
potassium phosphate (KH₂PO₄) was added in 100ml of distilled water and pH was adjusted to
7.0.

Preparation of 1mM EDTA

0.3722 grams of EDTA was added in 100ml of distilled water making a solution of 1mM.

Preparation of 1Mm Sodium Azide (NaN₃)

 0.0065 grams of sodium azide was added in 100ml of distilled water.

Preparation of 0.2mM NADPH

For making 0.2mM NADPH solution, 0.0164 grams of NADPH was added in 100ml of
distilled water.

Preparation of 1 EU/ml Glutathione Reductase (GR)

0.2mg glutathione reductase was dissolved in 1litre of distilled water.

Preparation of 1mM Glutathione oxidase (GSH)

For preparation of glutathione oxidase 0.031 grams of respective chemical was added in
100ml of distilled water.

Preparation of 0.25mM H₂O₂

0.025ml of H₂O₂ were dissolved in 100ml of distilled water.

100µl of 0.25mM H₂O₂, 100µl of GSH, 4µl of GR, 100µl of EDTA, 566µl of potassium
phosphate buffer and 10µl of NaN₃ were added in cuvette. This blank solution was placed in
spectrophotometer and its absorbance was measured at wavelength of 340nm.

100µl of plasma and 800µl of solution were added in cuvette and incubate it for 5
minutes at room temperature. After that H₂O₂ was added and absorbance was measured.

Calculation

GPx value was calculated as follow;

GPx (mU/mL) = A₃₄₀ per minute/ε

Where ε = 6.02×10ˉ³ Mˉcmˉ

Statistical Analysis
Results