A PROJECT REPORT

ON
“MEDICAL LABORATORY TECHNOLOGY”

Bachelor of Vocation in Medical Laboratory Technology

Rajiv Gandhi South Campus-Barkachha, Mirzapur
DDU KAUSHAL KENDRA
Banaras Hindu University- Varanasi

AT
N F RAILWAY CENTRAL HOSPITAL, MALIGAON (GUWAHATI)

Name: Rohit Kumar ,VI Semester (2022)

Enrolment No.: 415432
Roll No.: 19230 MLT 043

Submitted to:
Dr. Raghvendra Raman Mishra, Assistant Professor
Medical Lab Technology, RGSC
DDU KAUSHAL KENDRA
Banaras Hindu University- Varanasi
 CERTIFICATE

This is to certify that the a project report entitled Serology (Typhii) has
been carried out by the candidate MS./Mr. ROHIT KUMAR (Student of
Bachelor of Vocation in Medical Laboratory Technology at DDU
KAUSHAL KENDRA, Banaras Hindu University, Roll No.: 19230MLT043
during academic session 2019-2022.

This project work had been completed in between 1 st January 2022 to

30 April 2022. A part of this experimental work of this project carried
out at department of Pathology.

(Dr. Raghvendra Raman Mishra)

Assistant Professor-MLT
 DECLARATION

I hereby declare that my project work entitled “typhii” Submitted by

me to Coordinator Medical Laboratory Technology courses DDU
KAUSHAL KENDRA, Banaras Hindu University is a record of original
work done by me under department of Serology at N F RAILWAY
CENTRAL HOSPITAL.

I Performed this work during 01 January 2022-30 April 2022. I also

declare that no part of this work has previously been submitted to any
University of any examining body for acquiring any diploma or
degree.

ROHIT KUMAR
[Link], MLT, VI Semester, 2022
Roll No.:19230MLT043
 ACKNOWLEDGEMENT
I owe a debt of deepest gratitude to Dr. M.P Ahirwar
Coordinator-DDU Kaushal Kendra RGSC,Banaras
Hindu University, for providing all study support.

I am indebted To my subject teacher Dr. Raghvendra

Raman Mishra Assistant Professor for their guidance,
support, motivation and encouragement throughout
the project work as well as during Academic Training.

I am thankful other subjects teacher Dr. Manoj Kumar

Singh ,Dr. BMN Kumar ,[Link] Kumar Yadav, Dr.
Manish, Mohammed Irfan Sir for their support during
class as well as in practical.

I also thankful to Lab assistant [Link] for his

kind supports.

ROHIT KUMAR
 [Link], MLT, VI Semester, 2022
Roll No.:19230MLT043

INDEX
[Link]. TITLE PAGE

1 ABSTRACT I

2 LIST OF FIGURES AND TABLES II

3 ABBREVIATIONS III

4 CHAPTER 1: INTRODUCTION 9-12

5 CHAPTER 2: REVIEW OF LITERATURE 13

6 CHAPTER 3: METHODS AND MATERIALS 13-18

7 CHAPTER 4: RESULTS AND DISCUSSIONS 19-29

8 CONCLUSIONS 30

9 PHOTOS WHILE WORKING IN (N F RAILWAY CENTRAL 31

HOSPITAL)

10 REFERENCES 32
 ABSTRACT
Typhoid fever is most prevalent in the Asian part of the world especially in
the developing countries of Asia like Pakistan and India, caused by a gram-
negative bacterium Salmonella enteric serval Typhi. It is an orally
transmitted communicable disease caused by consuming contaminated food
and impure water. The incubation period of the disease is 7 to 14 days.
Symptoms include high fever, rash, weakness, abdominal pain constipation,
headache, and poor appetite. Antibiotic resistance is a major problem to
treat it effectively. Firstline drugs are mostly not used to treat typhoid and
the resistance is emerging in fluoroquinolones. The only choice of drug
remaining is ceftriaxone and azithromycin. A counteractive action of typhoid
fever is chiefly by individual and household cleanliness. The provision of
clean water and safe disposal of faeces should be implemented to
eradicate S. Typhi . Good surveillance, better diagnostics, more sensible use
of antibiotics and efficient vaccine will be significant to reduce the burden of
disease caused by S. Typhi.
 LIST OF FIGURES & TABLE
[Link]. Figures and Tables PAGE
1 Figure 1: Symptoms of typhoid 12

2 Figure 2: Test of typhoid 13

3 Figure 3: Result of typhoid 19

4 Figure 4: widal slide agglutination Result 20

5 Table 1: Compression between blood culture and widal slide 21

test

6 Table 2: frequency distribution of semi quantitative tube 22

7 Table 3: The sensitivity specificity 24

8 Table 4: antimicrobial resistance patterns 25

9 Table 5: Multiple drugs resistance 25

10 Figure 5: Some pic of mine in hospital 31

 ABBREVIATIONS
% Percentage
C Degree Celsius
ul Microliter
um Micro Metre
DNA Deoxyribonucleic acid
DNase Deoxyribonuclease
EDTA Ethylene diamine tetra acetate
PBS Phosphate buffer saline
LB Luria Bertini
M Molar
MDR Multi Drug Resistant
Mg Miligram
MHA Muller Hinton Agar
MI Mililitre
OD Optical density
PEG Polyethylene glycol
SDS Sodium do-decylculphate
NS Normal Saline
RPM Rotation per minute
APS Ammonium per Sulphate
 INTRODUCTION
Typhoid fever is the clinical syndrome caused by the enteric
bacterial pathogen Salmonella enteric serotype Typhi (S. Typhi). An
English physician Thomas Willis (1621–1675) made perhaps the first
clinical descriptions of the syndrome in 1659. A clinically similar
syndrome called paratyphoid is caused by antigenically and
phylogenetically distinct serotypes of S. enteric, namely S. Paratyphi
A, S. Paratyphi B, and S. Paratyphi C. Initially, discrimination
between typhoid and typhus fever was not routinely made and this
continued to be the case until 1837 when William Wood Gerhard
defined these as distinct syndromes. Identification of the typhoid
bacillus in 1880 by Carl Joseph Eberth and development of the
Widal agglutination test by Georges Widal in 1896 resulted in
considerable progress in the diagnosis of typhoid fever. Typhoid
was endemic in most countries of Europe and North America in the
late 19th and early 20th centuries at least in part due to
urbanization of populations during the industrial revolution and
poorly planned public water systems. At that time the disease
burden was estimated to be 200–800 cases per 100,000 population
and an associated 10–20% mortality (JAMA, 1920), rates that are
similar to the current estimates for disease burden in some regions
of Asia where typhoid remains endemic. Introduction of sanitation
in the form of water chlorination and sand filtration drastically
reduced the incidence of typhoid fever in the first half of the 20th
century in the Western world.
Typhoid fever is still endemic in many areas of Asia, Africa, and
South America, where sanitation of water supply and waste
treatment is inadequate. In developed countries the risk of
contracting typhoid fever is limited to travel in areas with endemic
typhoid and for laboratory workers, where vaccination and regular
boosting or revaccination is recommended. The severity of typhoid
fever is underlined by the fact that up to 32% mortality is reported
in some regions of the world (Hoffman et al., 1984).
The potential use of S. Typhi in bioterrorist attacks is generally
considered to be minimal. Since S. Typhi is normally transmitted
through water supplies and sewerage, large-scale attacks in the
Western world would likely be limited due to water treatment and
sanitation procedures that are in place in these countries. Smaller
scale attacks on the other hand are possible and this has been
underlined by events that took place in The Dalles, OR, USA in 1984.
In this case a local commune, called the Rajneeshee, attacked diners
at “Shakey's Pizza” by sprinkling a nontyphoidal S. enterica serotype
into the salad bar. Although no deaths were recorded, the resulting
outbreak was the largest in Oregon history with 751 confirmed
cases of Salmonella gastroenteritis. The attack could have been
considerably worse if S. Typhi had been used as the bioterrorist
agent in which case several deaths would have been expected.
Disturbingly, Federal investigators found an invoice at the
commune's ranch headquarters indicating that the Rajneeshee had
ordered and received several pathogenic bacterial strains from the
American-type culture collection (ATCC) including S. Typhi. Similar
attacks in the future cannot be discounted.
Objective
To determine diagnostic value of the Widal test, treatment
pattern of febrile patients and antimicrobial drug
susceptibility pattern of blood isolates.

Methods
Using cross sectional methods, blood samples were collected
for culture and Widal test from 502 febrile outpatients
attending Mekelle hospital and Mekelle health center with
similar symptoms to typhoid. Sensitivity, specificity for anti-
TH and anti-TO titers using culture confirmed typhoid fever
cases, and Kappa agreement between Titer and slide Widal
tests were calculated. Treatment pattern of patients and

antimicrobial susceptibility pattern of the blood isolates was

assessed.

Results
From the 502 febrile patients, 8(1.6%) of them had culture-
proven typhoid fever. However, patients who have results
indicative of recent infection by O and H antigens of the
Widal slide agglutination test were 343 (68.5%), with
specificity and sensitivity of 33% and 100%, respectively.
Over prescription of antibiotics was seen by Widal slide test
for Ciprofloxacin 268 (76.1%), Amoxicillin- Clavulanic acid
9(2.6%), Amoxicillin 8(2.4%) and Chloranphenicol 8(2.4%).
Tube titer positivity was seen in 23(5.3%) patients with 75%
sensitivity and 95.8% specificity. Widal slide and Tube titer
tests showed poor agreement for both antigens (kappa=0.02
for O) and (Kappa=0.09 for H). A single anti-TH titer of ≥
1:160 and anti-TO titer ≥ 1:80 higher in our study showed an
indication for typhoid fever infection. Drug resistance pattern
of blood isolates ranges from 0-89.7% for gram positive and
0-100% for Gram negative, with an overall multi-drug
resistance rate of 61.7%.

Conclusion
Patients were wrongly diagnosed and treated for typhoid
fever by Widal test. The tube titration method was relatively
good but still had poor sensitivity. Blood isolates showed
multi drug resistance, which may be due to the indiscriminate
prescription as seen in this study. Based on our results, the
slide Widal test is not helpful in the diagnosis of typhoid,
hence other tests with rapid, feasible, better sensitivity and
specificity are urgently needed in Ethiopia.

1. Introduction
Typhoid (enteric) fever is an important health problem.
Reports by the World Health Organization revealed that about
21 million cases and >600,000 annual deaths from typhoid
fever occur throughout the world. Developing nations share
the highest burden due to rapid population growth, increased
urbanization, and limited safe water and health systems.
Serotype Typhi isolation from blood, bone morrow, urine or
stool is the most reliable way of confirming typhoid infection.
Yet, this requires laboratory equipment and technical training
that are not feasible for most primary health care facilities in
developing world. Thus, most typhoid infections are
diagnosed on clinical grounds and treated presumptively. But
as its clinical symptoms are similar with many other bacteria,
it may lead patients to receive unnecessary and
inappropriate antimicrobial treatment.
In many developing countries, Widal test, which was first
introduced by F. Widal in 1896, is widely used in the
diagnosis of typhoid fever. This is because it is relatively
cheaper, easy to perform and requires minimal training and
low sophisticated equipment. This test depends
on agglutination reaction between S.
typhi somatic Lipopolysaccharides O antigen (TO) and
flagellar H antigen (TH), where these antigens are shared by
many other Enterobacteriaceae; for this reason, its test
valueshas been debated for many years.
In addition, interpretation of results has also been a problem
as different cut-offs points have been reported from different
places. moreover; patient treatment cannot wait for results
obtained with convalescent phase samples. Hence, the
treatment decision is made on the basis of the results
obtained with a single acute-phase sample.
Due to the low prevalence of typhoid, access to safe drinking
water, better laboratory facilities to isolate the bacteria, and
the low sensitivity and specificity of the Widal test, the test is
no longer used as a diagnostic assay in developed nations
but it is the commonest test in developing countries. Widal
test recommends a slide test to be used for screening only
and positive results to be confirmed by tube titration method;
however, since titer results take 18-24 hours, diagnosis is
practically done on the basis of the slide agglutination
results, which are available within minutes. However, this
may lead to false diagnosis of typhoid, unnecessary antibiotic
therapy, and emergence of drug resistant strains.
In Assam diagnosis and treatment of typhoid fever is by Widal
test (slide agglutination); however, except for a single study
done in Addis Ababa which compared the Widal test with
blood culture we could not find published data that evaluate
test validity of the Widal test. Again the study did not address
the treatment pattern of typhoid-suspected patients and
the antimicrobial drug susceptibility pattern of the isolates; it
was also done in a small sample size (230 patients) in a
different study area in Maligaon, 7 km from our study area.
The present study was therefore designed to address the
diagnosis and treatment of Typhoid fever and the associated
prevailing drug resistance pattern in Norther Assam using a
standard blood culture method

2. Materials and Methods

2.1. Study design and specimen collection

Cross sectional study was conducted in NF Railway hospital
and Mekelle health center (MHC) from January to april 2022
consecutively upon receiving informed consent among febrile
patients suspected for typhoid fever. Study areas are located
7 km North of Maligaon the capital city of Assam. Five
hundred and two venous blood samples, 8-10 ml from adults
and 3-5 ml from children were collected aseptically using
70% alcohol and 2% tincture of iodine. Then, 5-7 ml from
adults and 2-3 ml of blood from children was dispensed into a
sterile bottle containing 45 ml of Tryptic soy broth culture
medium (BBLTM USA), mixed with the broth, and incubated at
37 °C, and the remainder was used for the Widal test.
Participants already on antibiotic treatment and those who
were diagnosed for other known febrile illness were excluded
from the study.

2.2. Isolation and identification of bacteria

Incubated blood samples were checked for signs of bacterial
growth (haemolysis, turbidity, and clot formation) daily up to
7 days. Bottles that showed signs of growth were further
processed by Gram stain and subculturing onto Blood
agar, MacConkey agar, and Manitol salt agar (all Oxoid, UK)
and incubated at 37 °C for 24 hours. Blood culture broth with
no bacterial growth after 7 days was sub-cultured before
being reported as a negative result. Identification of isolates
was done by colony morphology, Gram
staining, Catalase test, Coagulase test, and biochemical tests
using Triple Sugar Iron agar (TSI) (OXOID, UK), Citrate
utilization test (BBLTM USA), Urease test (BBLTM USA) and
Lysine motility indole test (LDC) [BBLTM USA] using the
standard bacteriological methods.

2.3. Antimicrobial Susceptibility tests

The disk diffusion assay method was used to determine the
antibiotic resistance/susceptibility pattern of blood isolates on
Muller-Hinton agar (Oxoid, England) against Amoxicillin-
Clavulanic acid (30 μg) (Oxoid, UK), Ceftriazone (30 μg)
BBLTM,USA), Vancomycin (30 μg) (BBLTM,USA), Ciprofloxacin (5 μg)
(BBLTM,USA), Gentamicin (120 μg)
(BBLTM,USA), Norfloxacin (10 μg)
(OXOID,UK), Doxycycline (30 μg) (OXOID
TM USA
UK), Erythromycin (15 μg) (BBL ), Nitrofurantonin and
Trimethoprim-Sulphamethoxazole (25 μg) [BBLTM,USA]. The
criteria used to select the antimicrobial agents tested were
based on the availability and frequency of prescription for the
management of bacterial infections in Assam. To standardize
the inoculum density for a susceptibility test, a BaSO4
turbidity standard, equivalent to a 0.5 McFarland standard
was used by strictly following the SOP for the preparation and
standardization. Multidrug resistance was defined as
resistance of an isolate to three or more of the antimicrobial
agents tested.
2.4. Widal test
Qualitative slide and semi quantitative
tube agglutination methods were done using febrile antigen
kits of Salmonella typhi (Chromatest Febrile Antigens kits,
linear chemicals, Spain). Slide agglutination Widal tests were
done by laboratory professionals who were blind to the study
and were based on the manufacturers guidelines, and results
were given to doctors who requested the tests for patient
management. Slide test reactive serums were transported to
Ayder referral and teaching hospital microbiology laboratory
and further tested by standard tube agglutination test
(titration) method. According to the manufacturer's manual,
serum samples were serially diluted using a fresh 0.95%
saline preparation from 1:20 to 1:640 for anti TO and anti TH
separately in 12 test tubes. An equal amount of O and H
antigens were then added to all test tubes. Based on the
manufacturer's manual, an antibody titer of ≥ 1:80 for anti
TO and ≥ 1:160 for anti TH antibodies were taken as a cutoff
value to indicate recent typhoid infection.

2.5. Antibiotics given by doctors for positive slide agglutination

results
We reviewed the treatment patterns of patients based on
their Widal slide test results and clinical grounds by doctors
who did not know about the ongoing study. We used patients’
charts to review the treatment profiles using the chart
number of the patients and date visited in both health
institutions from each patient's questioner.
A senior clinician who is member of the research team
handled this task in each card room of the health institutions.
2.6. Data quality control and management
A standard bacteriological procedure was followed to
maintain the quality of all laboratory tests. American Type
Culture Collection (ATCC) strains S. aureus (ATCC 25923); S.
Typhi (ATCC 13311) and [Link] (ATCC 25922) were used as
positive controls for culture and sensitivity testing. Negative
control was performed by randomly taking prepared culture
media and incubating overnight to check for any growth.
Standard operational procedures were followed during
processing of each sample, and all the instruments used for
sample processing were checked every morning for proper
functioning. SPSS software Version 20 was used for the
analysis of the data. Sensitivity, specificity, positive
predictive value (PPV) and negative predictive value (NPV)
were calculated for Widal test using culture confirmed
typhoid fever. We also calculated the Kappa to determine the
agreement between the slide and tube titration method.

2.7. Ethical issues

Ethical clearance was obtained from Research Ethical
committee/IRB of the College of Health Sciences. Permission
was also obtained from the General Hospital and health
center administration. Data and samples were collected after
written informed consent was obtained from each volunteer
and guardian.
3. Results
From the total 502 febrile patients involved in the study, 269
(52.3%) were female and 245 (47.7%) male. Their age
ranged from 1-81 years (mean 27.97 ± 1.59 [SD]). Two
hundred seventy two (54.2%) of the participants were in the

age range of 15-32 years.

3.1. Qualitative Slide agglutination Widal test
Three hundred forty three (68.3%) patients were reactive for
both O and H antigens, while 34 (6.8%) were reactive only for
O antigen. One hundred twenty three (24.5%) patients
showed no reaction result for both antigens. Overall 376
(74.9%) patients have a reactive slide agglutination Widal
test by either or both O and H antigens (Table 1).
Table 1. Qualitative Slide agglutination results of Widal test among febrile patients suspected
of typhoid fever in N F RAILWAY CENTRAL HOSPITAL, January 2022.
Widal slide agglutination results Frequency Percent

Both O and H antigens reactive 343 68.3

Only O antigens reactive 34 6.8

Only H antigens reactive 8 1.6

Non reactive for O and H antigens 126 25.1

Total 502 100

From the total 343(68.3%) patients with reactive Widal slide agglutination test, only 8 (1.6%) patients
had culture proven S. typhi in their blood, while the remaining 66.7% were treated wrongly as typhoid
fever (Fig. 1).

Fig. 1. Coparsion between blood culture and Widal slide test for typhoide diagnosis.
The Widal slide agglutination test in our study showed that both O and H antigens had 100% sensitivity
and 100% negative predictive value, but showed low positive predictive value of 2.7% and 4.6% for O
and H antigens respectively. Poor specificity was seen in O antigen and H antigen, 33% and 35.4%
respectively.
3.2. Semi quantitative tube agglutination test (titration)
Serum samples with reactive slide agglutination test results
were further analysed by standard tube Titration method.
Two hundred (52. %) and 111(34.1%) of the slide reactive
patients showed reaction for anti TO and anti TH antibody
respectively (Table 2).
Table 2. Frequency of distribution of semi quantitative tube agglutination test in febrile patients
suspected of typhoid fever, January-April 2022..
Titer O –Antigen H-Antigen

No % % % %
Frequency Frequency
agglutination (n=377) (n=502) (n=351) (n=502)

137 36.3 27.3 99 23.7 19.3

1:20 144 38.2 28.7 129 36.8 25.7

1:40 63 16.7 12.6 91 25.9 18.2

1:80 12 3.2 2.4 17 3.5 3.4

1:160 15 4 3 8 2.3 1.7

1:320 0 0 0 5 1.4 1.2

1:640 0 0 0 2 0.7 0.4

Total 377 100 74 351 100 69.9

Taking antibody Titer of ≥1:80 for O and ≥1:160 for H

antigens as cutoff values to indicate recent typhoid infection
(positive titer), 25 (4.9%) and 15 (2.9%) patients had results
indicative of recent typhoid infection by O and H antigens,
respectively. The total number of patients who had results
indicative of recent infection by either or both O and H
antigens was 23 (5.3%).
In our study we have calculated a statistical method to show
agreement between slide agglutination and standard tube
titer tests and we found very poor agreement for both
antigens (kappa=0.02 for O) and (Kappa=0.06 for H).

3.3. Blood culture results

Out of 502 blood cultures, there were
8(1.6%) [Link] isolates, and 107(21.3%) other non-
Salmonella pathogenic bacteria. No bacteria growth was seen
from blood cultures of 387 (77.1%). Non-Salmonella species
included: Staphylococcus aureus (n=41), Coagulase
negative Staphylococcus (n=39), Escherichia
coli (n=12), Citrobacter species (n=9),
[Link] (n=6), Pseudomonas spp (n=2) and Klebsiella spp
(n=3).
Anti TO agglutination titer of 1:80 and higher were detected
among 7 (87.5%) of the culture confirmed typhoid cases
by [Link] as compared with 12 (9%) by other non salmonella
bacterial species. But positive titer for TH was seen in 6
(75%) and other non salmonella bacteraemia 8 (6%). The
specificity and NPV of both antigens was high but the PPV
which is important measurement in the diagnosis of the
disease was very low (28% and 33.3% for TO and TH,
respectively) (Table 3).

Table 3. The sensitivity, specificity, PPV, and NPV of titers of anti TO

(≥1:80) and anti TH (≥1:160) Widal tests for diagnosis of typhoid among
febrile patients in N F Railway Central hospital 2022.
Measuremen O-Antigen H -Antigen Both antigens
t (%) (%) (%)

Sensitivity 87.5 62.5 75

Specificity 96.4 98 95.9

PPV 28 33.3 22.2

NPV 97.8 99.4 99.6

In this current study, the overall Widal Titer positive among
culture confirmed patients of [Link] was 6(1.2%), which all
have positive titer of anti TH and one has also a positive titer
for anti TO. Thus the sensitivity, specificity, PPV and NPV of
the overall positive titer was 75%, 95.9%, 22.2% and 99.6%,
respectively (Fig. 2).

Fig. 2. Comparsion of blood culture and Widal titer tests.

Patients’ charts of Widal slide test positive results showed
that the following antibiotics were over
prescribed: Ciprofloxacin 268 (76.1%), Amoxicillin- Clavulanic
acid 9(2.6%), Amoxicillin 8(2.4%), Chloranphenicol 8(2.4%)
and 24(4.7%) other antibiotics. Twenty four patients had no
chart available during collection time; hence no information
was obtained about their treatment.

3.4. Antimicrobial susceptibility pattern of blood culture isolates

The In vitro antibiotic susceptibility pattern (Table 4) showed
that gram positive bacteria resistance ranges from 0 to
89.7%. Thirty six (87.7%) S. aureus isolates were resistant to
Trimethoprim-sulphamethoxazole, 34(82.9%) to Ceftriazone
and 31(75.6%) to Doxycycline. In Thirty five (89.7%) and
25(64%) resistance was seen by CoNS to Trimethoprim-
sulphamethoxazole and Doxycycline,
respectively. Vancomycin resistant was seen in
22% [Link] and 23% CoNS.
Table 4. Antimicrobial resistance pattern of bacterial isolates of febrile
patients attending N F Railway Central Hospital January-April 2022,
No (%).
Antibio
Empty
tics Bacteria isolated
Cell
Tested

[Link]
[Link] CoNS [Link] S. Total
Empty i Citrobacter spp( Klebsiella spp(
us (n=39 en typhi (n=11
Cell (n=1 n=6) n=3)
(n=41) ) (n=6) (n=8) 5)
2)

7(17.1 27(23.
AMC 9(23) 2(33.3) 3(25) 2(33.3) 2(25) 2(75)
) 5)

34(82. 21(53. 73(63.

CRO 1(16.7) 9(75) 4(66.6) 2(25) 3(100)
9) 8) 5)

3
CN 16(39) 7(18) 0 3(25) 2(33.3) (37.5 0 31(27)
)

31(75. 5(62. 78(67.

Do 25(64) 4(66.7) 6(50) 4(50) 3(100)
6) 5) 8)

10(25. 1(8.3 1(12. 35(30.

CIP 18(43) 1(16.7) 3(50) 1(33.3)
6) ) 5) 4)

36(87. 35(89. 10(8 94(81.

SXT 5(89.3) 1(16.7) 4(50) 3(100)
7) 7) 3) 7)

8(66. 22(19.
F NA NA NA 5(83.3) 6(75) 3(100)
7) 1)

19(48. 41(35.
E 21(51) 1(16.7) NA NA NA NA
7) 7)

5(41. 3(37. 45(39.

NOR 18(43) 14(36) 1(16.7) 1(16.7) 3(100)
7) 5) 1)

V 14(34) 9(23) 0 NA NA NA NA 23(20)

CoNS=Coagulase negative Staphylococci, AMC=Amoxicillin-
clavunilic acid, CRO=Ceftriazone, CN=Gentamicin,
E=Erythromycin DO=Doxycycline, CIP=Ciprofloxacin,
SXT=Trimethoprim-sulphamethoxazole, NOR=Norfloxacin,
F=Nitrofurantonin, V=vancomycin, NA=Not Applicable.
Over all, high resistance was seen by gram positive bacteria
to Trimethoprim-sulphamethoxazole 89.7%, Doxycycline
75.6% and Ceftriazone 82.9%. Relatively, Amoxicillin-
clavunilic acid and Vancomycin were effective against Gram
positive isolates in our study.
Resistance levels of gram-negative organisms ranged from 0
to100%. E. coli showed high resistance to Ceftriazone 75%
and Nitrofurantonin 66.7%. Isolated S. typhi were resistant to
Nitrofurantonin 75%, Doxycycline 62.5% and Trimethoprim-
sulphamethoxazole 50%. Overall gram genitive isolates
showed high resistance to Nitrofurantonin75.9%,
Trimethoprim-sulphamethoxazole 69%, Ceftriazone 62% and
Doxycycline 55.6%. On the other hand, low level of resistance
was seen by gram negative bacteria to Ciprofloxacin
14%, Gentamicin 28% and Amoxicillin-clavunilic acid 31% in
this study.
Antibiogram drug resistance pattern showed that 65.9%,
68.9% and 50% of [Link], CoNS
and [Link] showed multi drug resistance, respectively with
an overall gram positive MDR rate of 66.3%. On the other
hand, 50% MDR was seen for [Link], Citrobacter spp
and [Link] with overall gram negative MRD rate of 44.8%. In
general the multi drug resistance rate of in this study was
seen in 71(61.7%) of the isolates (Table 5).
Table 5. Multiple drug resistance patterns of bacterial isolates from blood of
febrile patients in N F Railway Central Hospital January April 2022.
 Empty
Bacterial isolates and their Antibiogram (Resistance pattern) No (%)
Cell

S. [Link]
Emp CoNS Citrobac [Link]
[Link] (n= pyog i Klebsiella Total
ty (n=3 ter spp hi
41) en (n=1 spp (n=3) (115)
Cell 9) (n=6) (n=8)
(n=6) 2)

5(9.1 3(37. 12(10.

R0 3(7.3) ____ ____ ____ 1(33.3)
) 8) 3)

4(9.1 1(16. 1(12. 18(15.

R1 7(17.1) 4(.3) 1(16.7) ____
) 7) 5) 7)

8(20. 2(33. 2(13. 21(18.

R2 4(9.8) 1(16.7) 2(25) 2(75)
5) 3) 3) 3)

8(20. 1(16. 1(12. 18(15.

R3 5 (12.2) 2(20) 1(16.7) ____
5) 7) 5) 7)

5(13. 1(16. 3(26. 18(15.

R4 5(12.2) 2(33.3) ____ 2(75)
6) 7) 7) 7)

3(6.8 1(16. 1(12. 10(8.7

R5 4(9.8) ____ ____ 1(33.3)
) 7) 5) )

5(9.1 1(6.7 12(10.

R6 6(14.6) ____ ____ ____ ____
) ) 3)

5(9.1
R7 4(9.8) ____ ____ ____ ____ ____ 9(7.8)
)

1(2.3
R8 3(7.3) ____ ____ ____ ____ ____ 4(3.5)
)

R9 ____ ____ ____ ____ ____ ____ ____ ____

CoNS - coagulase negative Staphylococci; R0 - sensitive to all antibiotics tested; R1, R2, R3,
R4, R5, R6, R7, R8, R9, resistant to one, two, three, four, five, six, seven, eight, nine
antibiotics, respectively.
4. Discussion
Though definitive diagnosis of typhoid is by isolation of the
bacteria from blood, bone morrow or other body fluids, most
developing nations like Ethiopia due to limited access to
laboratory facilities, use the old Widal test In our current
study 343 (68.3%) of the febrile patients showed positive
slide Widal test. This test was found good as a screening test
[p=0.002] with 100% sensitivity and negative predictive
values. It was however, very low specificity for both antigens
(33% for O and 35.4% for H. This result was similar with the
study report from India
Since positive predictive value (PPV) represents the
proportion of patients with positive test results that are
correctively diagnosed, it is considered as the most important
clinical diagnosis method. In our current result the PPV was
very low for both antigens [2.7% for O and 3.02% for H].
Similar results were reported from the study finding of febrile
patients from India, which proves that slide test is good in
screening out negative samples but not helpful in the
diagnosis of the disease. This is the reason why previous
studies have found it the test performing worst in the
diagnosis and recommended that it should not be used for
the diagnosis of the disease.
We found 335(66.7%) febrile patients with Widal false
positive and treated wrongly as typhoid fever while only
8(1.6%) patients were culture-proved to have typhoid fever.
This high false positive rate may be due to cross reacting
antibodies of other bacterial and non bacterial infections. We
also reviewed the treatments given for those slide positive
patients by the clinicians from each patient chart and found
that patients were give: Ciprofloxacin 268 (76.1%),
Amoxicillin- Clavulanic acid 9(2.6%), Amoxicillin 8(2.4%),
Chloranphenicol 8(2.4%) and 24(4.7%) other antibiotics. This
incorrect treatment based on Widal slide test results and
clinical round of patients may lead to unnecessary treatment
costs and pressure the normal gut flora to develop drug
resistance, and most importantly, highly fatal diseases of
febrile patients such as malaria, non typhoidal
salmonelasis, endocarditis and urinary tract infection may be
missed, ultimately leading to bad patient outcomes. Again we
found that one of the reasons that may lead clinicians to the
misdiagnosis of patients is the way Widal results are reported
i.e. results should be reported as 0(no agglutination), +1
[25% agglutination], +2[for 50% agglutination], +3[for 75%
agglutination] and +4[for 100% agglutinations rather than
reactive or non reactive. This problem in reporting was also
seen in our study areas and needs to be corrected.
The tube titration method was done for those patients whose
serum was positive for slide Widal test, and from the 343 a
positive titer (≥1:80 for TO and ≥1:160 for TH) was seen
among 23 (6.7%) with sensitivity and specificity of 75% and
95.8% respectively. Similar findings were reported by other
researchers. A study from Kenya has shown much lower
sensitivity (26%) of Titer result. This low sensitivity of Titer
test could be due to variation in the blood collection time.
Titer test showed very low PPV (22.2%) and high NPV
(99.6%). Similar reports were seen from Egypt (5.7% of PPV
and 98% of NPV) and Ethiopia (98.9% NPV and 5.7% NPV).
Though not as high as the slide Widal test, a significant
number of patients were still reported falsely as positive
(PPV=22.2%) by Tube titration methods, which could be due
to cross reacting antibodies by infections other than typhoid
fever.
In our study positive titer was found in 12 (9%) and 8 (6%)
patients for TO and TH, respectively by other non salmonella
species. This was clearly seen in a study conducted in
Cameroon where out of the total in febrile patients clinically
similar to typhoid, 45% were malaria cases and only 2.5%
were true typhoid cases proving that there are febrile
infections that induces cross reacting antibodies with the
somatic and flagellar antigens. In this study there were two
culture confirmed cases of typhoid but had a negative titer.
Possible reasons for this are early blood collection time
before disease or inadequate bacterial inoculation to
induce antibody production and more importantly previous
antibiotic treatment of patients even if no patients told us of
taking any antibiotics during our study.
Slide agglutination and standard tube titration results were
compared and results revealed that there was statistically
poor agreement between both antigens (kappa=0.02 for O)
and (Kappa=0.09 for H), similar to the study conducted in
India with poor agreements between the tests but in contrast
to this, fair agreement results were reported from other areas
The antimicrobial susceptibility pattern of blood isolates was
determined for the commonly available and prescribed
antibiotics. Overall the range of drug resistance pattern for
gram positives was from 0% - 89.7% and from 0% -100%
for gram negative bacteria, which is similar to the result from
other part of Ethiopia, which was 0- 85.7% and 0% - 100% for
gram negative and positive respectively. This increased
resistance in this study may be an indication of indiscriminate
and continuous use of antibiotics as clearly seen in our study,
where more patients (66.7%) were put on the wrong
treatments.
Thirty six (87.7%) of the S. aureus isolates were resistant to
Trimethoprim-sulphamethoxazole, 34(82.9%) to ceftriazone
31(75.6%) to doxycycline, 51% to erythromycin, 39%
to Gentamicin. Resistance of Trimethoprim-
sulphamethoxazole was comparable with reports from other
parts of Ethiopia. Our present study reveals lower resistance
to vancomycin by [Link] (22%) and CoNS (23%) than the
study done on surgical wound infection from South Ethiopia,
which was 100% and 65.2%, respectively. The consequences
of using ineffective drugs in rigorous bacterial infections
could be devastating as this can complicate the management
and increase morbidity and mortality. CoNS were mainly
recognized as a contaminant until the 1970's, nevertheless,
several studies have reported an increasing incidence of
infections due to these bacteria. This was similar to our
current study. E. coli was 75% and 66.7% resistant to
Ceftriazone and Nitrofurantonin respectively. Resistance
to ceftriaxone by E. coli may be due to production of Amp C
enzymes and BAL TEM genes (Beta-lactamse enzymes) which
inhibit the Beta-lactam rings of cephalosporin.
S. typhi isolates were resistant to Doxycycline (62.5%),
Trimethoprim-sulphamethoxazole (50%) and Nitrofurantonin
(75%). Conversely, among the antibiotics used for
susceptibility testing Amoxicillin-clavunilic acid and
Ciprofloxacin were relatively effective for gram negative
bacteria isolates. Even if Ciprofloxacin was prescribed right
and left for almost all febrile slide-test-positive patients in this
study area, it was effective for most gram negative bacteria
isolates, which could be because of its broad spectrum,
because it is new-generation and has not been used for long,
because the patients visiting the hospitals during the study
period were taking the antibiotic for the first time, and it
could also be the isolates did not develop resistance.
However, if this indiscriminate type of antibiotic prescribing
continues and no rational use of antibiotics is implemented, it
would not be long to miss these antibiotics as well.
A general overview of the anti biogram of all the bacterial
isolates indicates that multi drug resistance was observed
in [Link] 65.9%, CoNS
68.9%, [Link] 50% Citrobacter spp 50% and E .coli 50%
and [Link] 50%. The overall multi drug resistance rate in our
study was 71(61.7%). This suggests a high-resistance gene
pool perhaps due to gross misuse and inappropriate usage of
the antibacterial agents which we came across in this study
for the diagnosis of typhoid fever.
Amoxicillin clavulanic acid was found to be effective against
both gram positive and gram negative isolates in this study.
Unlike our current findings, other studies reported
Ciprofloxacin is effective for both gram positive and negative,
but Ciprofloxacin was found to be effective against gram
negative isolates here in our study, which is in line with
findings that others reported.

5. Conclusions
Prevalence of typhoid fever in the study area was low;
however, due to the poor diagnostic value of the Widal test,
patients were wrongly diagnosed and treated for typhoid
fever. The tube titration method was relatively good but still
had poor sensitivity. The test has scarce PPV value, specificity
and correlation with serological testing tube. Therefore, the
culture must be the reference test for diagnosis of typhoid
fever. There is no doubt about the value of the presented
study and its potential local impact. However, these data
have been previously reported and the impact on other
countries is very limited because these techniques are no
longer used.
The antimicrobial drug susceptibility pattern of blood isolates
showed high multi drug resistance to commonly used
antibiotics which may be due to the indiscriminate
prescription of the antibiotics as seen in this study. Hence it
should be a call to health authorities for the establishment of
programs for the appropriate use of antimicrobials to control
the emergence of drug resistant bacterial strains. Based on
our results, the Widal test is no longer important in the
diagnosis of typhoid fever. Hence, other tests that are rapid,
feasible, and have good sensitivity and specificity are
urgently needed in Assam
Limitation of the study: We have used a single blood test due
to the problem of patient recruitment for next time.
Competing interest: All authors declare that they have no
competing interest.
Authors’ contributions: Araya Gebreyesus was the principal
researcher, conceived the study, designed and collect data,
laboratory works, conducted data analyzed and drafted the
manuscript for publication. Letemichale Negash involved in
data collection, Laboratory works and data analysis. Senay
Aregawi in collecting the antibiotics prescribed by clinicians
to patients by their slide Widal test results from the two
health institutions and reviewed the initial draft manuscript.
Tsehaye Asmelash, Tadesse Dejenie, Abadi Luel conceived
the study and contributed designing the study, Saravanan
Muthupandian contributed designing the study, analysis and
interpretation of data and reviewed the initial draft
manuscript. All authors read and approved the final
manuscript.

Acknowledgments
We are greatly thankful for the Mekelle University, NORAD III
project for grant. We are also grateful to laboratory staff of
Mekelle hospital and Ayder microbiology staff for their great
cooperation. We also thank Afework Mulugeta (PhD) for his
immense advice and guidance in how to proceed with the
research from the beginning.

Pictures of mine in Hospital during internship period