Volume 3, Issue 9, September– 2018 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165

Performance and Feasibility of using both Stool Culture

and Nested PCR for Improved Detection of Typhoid
Fever in Buea Health District, South West Cameroon
Rita Ayuk Ndip,1Richard Fopa Fomekong,1Manfo Tsague Faustin Pascal,
1
Boris Kingue Gabin Azantsa, 1,3 Moses Njutain Ngemenya1,2*
1
Department of Biochemistry and Molecular Biology,
2
Biotechnology Unit, Faculty of Science, University of Buea, P.O. Box 63, Buea, Cameroon
3
Department of Biochemistry, University of Yaounde 1, P. O. Box 63, Yaounde, Cameroon
Corresponding author: Moses Njutain Ngemenya; Department of Biochemistry and Molecular Biology,
Faculty of Science, University of Buea, P.O. Box 63, Buea, Cameroon.

Abstract:-
 Introduction
Presently diagnostic tests for typhoid fever include
serology and culture which both have relatively low
sensitivity and specificity. Polymerase chain reaction
(PCR) has exhibited mixed performance for blood
specimens in the detection of Salmonella. This study
compared the performance of serology test, stool culture
and nested PCR and their feasibility in the Buea Health
District in South West Cameroon.
 Methods
Three hundred and sixty (360) patients suspected of
typhoid fever and sixty one (61) apparently healthy
controls were selected for the study. Blood specimens were
analyzed using Widal serology test. Stool was culturedand
grown cells further analyzed using biochemical tests and
nested PCR targeting the flagellin gene of Salmonella
species. Performances of tests were determined using
standard formulas.
 Results
Fifty (50) test group participants (13.9%), were stool
culture positive for Salmonella following identification
with API 20-E test kit. Nested PCR had the highest
sensitivity of 91.9%, P = 0.000,while Widal slide and
tube tests had the overall lowest performance. When
nested PCR was considered as gold standard, stool culture
had the highest specificity of 94.6%, P= 0.000. Based on
cost, turnaround time and performance, stool culture and
PCR appeared as suitable methods for reliable diagnosis of
typhoid fever.
 Conclusion
Stool culture could be used as gold standard in
conjunction with serology to improve diagnosis of typhoid
fever in the study area. Additionally an algorithm should
be explored usingPCR for suspected or severe cases
negative for both serology and stool culture.
Keywords:- Typhoid fever,Salmonella, diagnosis, stool
culture, nested polymerase chain reaction.
I. INTRODUCTION
Typhoid fever remains a public health concern
particularly in developing countries. In 2010, 26.9 million
cases of typhoid were reported globally, with a mortality rate
of about 1%. Africa features among the regions with the
highest incidence estimated at 724.6 cases per 100,000
persons [1]. Some studies in South West Cameroon have
reported a prevalence of 5.1 to 7.9% [2, 3].Typhoid fever is a
potentially severe and life threatening disease caused by the
bacterium Salmonella enterica serovar typhi
(Enterobacteriaceae). Survivors may be left with long-term or
permanent neuropsychiatric complications [4]. Salmonella
serotypes, particularly Salmonella enterica serotype paratyphi
A, B or C can cause a similar syndrome [5].The causative
agentwhich thrives in conditions of poor sanitation and
hygiene is transmitted by faecal - oral and urine – oral routes
through contaminated water, milk, food or by flies[6].
Clinical diagnosis is challenging due to similarity of
symptoms to other febrile illnesses hence laboratory
investigation is necessary.Several techniques are available for
typhoid diagnosis. A variety of immunochemistry-based kits
are used for sero-diagnosis of typhoid fever;these include the
Felix-Widal, haemagglutination, countercurrent immuno-
electrophoresis and rapid diagnostic kits such as Typhidot and
Tubex. The Felix-Widal test measures agglutinating antibody
levels against O and H antigens [7]. Though still widely
used serological tests havemoderate sensitivity and
specificity, are unreliable in prior antibiotic therapy, show
cross-reactivity of S. typhi O and H antigens with other
Salmonella serotypes and other Enterobacteriaceae yielding
false-positives [8]. In endemic areas there is often a low
background level of antibodies in the healthy population [9],
necessitating establishment of the antibody threshold level of
active disease in this population. The gold standard for
diagnosis of typhoid is isolation of S. typhi by culture (of
blood, bone marrow, stool and urine), widely considered
100% specific [10]. Culture of bone marrow aspirate is
limited by invasiveness and cost [11]. Blood, intestinal
secretions and stool culture results are positive for S. typhi in

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approximately 85%-90% of patients with typhoid fever within
the first week of onset.Later in the illness, stool culture results
are positive because of bacteria shed through the gall bladder.
Multiple blood cultures (>3) yield a sensitivity of 73%-97%.
Stool culture alone may yield a sensitivity of less than 50%.
Overall, culture is expensive, time-consuming and killing of
bacteria by prior antibiotic use may lead to false
negatives[11].
Another option to overcome the above limitations is
molecular diagnosis based on nucleic acid amplification. The
S. typhi flagellin gene, fliC, has been targeted in numerous
polymerase chain reaction (PCR) based studies conducted on
blood samples [8, 12]. PCR can detect low numbers of live
and dead bacterial cells hence potentially highly sensitive. A
study employed semi nested PCR primers targeting the fliC
gene of S. typhi in blood and reported a 62% positivity rate in
culture negative specimens and 100% positivity in culture
positive specimens[13]. However, some PCR diagnostic
studies for typhoidin blood have not yielded reliable results.
Using real time PCR,the fliC gene of S. typhi was amplified
in all culture positive and negative blood samples, with a
much higher gene copy number in culture positive specimens
(1,000-45,000) [14]; this differed with microbiological data,
which reports a generally low number of bacteria, the
majority of patients having <1 organism/mL of blood [15].
Also, a three color real-time PCR assay gave a less than 50%
sensitivity for DNA extracted from 2mL of blood from culture
confirmed typhoid patients; though it recorded 100%
sensitivity on culture positive bone marrow samples known to
harbour significantly more bacteria [11]. This thus confirms
that PCR results are related to the actual colony forming units
found in the blood.
Some PCR studies have targeted the fliC gene, utilizing
nested PCR to improve sensitivity. Nested PCR employs two
rounds of PCR using two different sequences within the H1-d
flagellin gene of S. typhi thus offering enhanced sensitivity
and specificity [8].
Though various PCR-based studies on typhoid suggest
good performance, in the case of typhoid there is as yet no
PCR test in common use, except for in-house assayswhich
vary in protocol, quality control and interpretation[16]. Hence
the need to pursue the development of a PCR based assay of
acceptable performance.
Considering the limitations of the serology and culture
methods and the potential advantage of PCR, this
studycompared the sensitivity and specificity of Widal
serology and stool culture to nested PCR, in the diagnosis of
typhoid fever in Buea, South West Region of Cameroon, with
stool culture as the reference method. We also attempted an
assessment of the feasibility of these tests in the study area in
order to propose a possible algorithm for diagnosis of the
disease.
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International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
II. METHODS
A. Study area and design
The study was conducted at the Buea Regional Hospital
Annex and the University of Buea in the South West Region
of Cameroon.Ethical clearance (Ref 2015/318/UB/FHS/IRB)
was obtained from the Faculty of Health Sciences Institutional
Review Board, University of Buea. Administrative
authorizationRef:R11/MINSANTE/SWR/RDPH/PS/282/284)
was obtained from the Regional Delegation of Public Health,
South West Region and theBuea Regional Hospital to access
their laboratory archives and facilities for sample collection
and analysis. This study lasted 7 months (April to October,
2015). This was a hospital based cross-sectional study
involving 360 clinically diagnosed persons and 61 control
participants reporting to the medical laboratory of the Buea
Regional Hospital for Widal test and stool analysis.The study
subjects were of both sexes aged ≥ 7 years.The sample size
was estimated to be about 255 according to Daniel et al,
(2015) [17] using a prevalence of 21% for
Buea[18].Participantswho had not taken antibiotics in the
previous one month wereincluded in the study only after they
had responded to the study questionnaire and given their
consent by signing the informed consent and or assent forms
for subjects below 21years. Healthy individuals with no recent
history of fever upon screening for Widal test and malaria
parasite and no Gram negative bacilli infection in the recent
past were selected same as above to serve as control[18].
Persons who did not fulfil the above selection criteria or
presented difficulties in providing blood and stool sample
were excluded. Widal serology and stool culture were
performed on all specimens and nested PCR was performed on
selected samples. Each participant’s information was tracked
using a code and was kept confidential.
B. Serological test using Widal kit
Venous blood (3mL) was collected from participants and
allowed to clot and then centrifuged at 3000 rotations per
minute (rpm) for 2minutes. After centrifugation,the
semiquantitative Widal slide agglutination test and the Widal
quantitative tube agglutination test were performed using a
commercial kit (Omega Diagnostics,United Kingdom)
containing somatic (O) and flagellar (H) antigens of
Salmonella typhi, Salmonella paratyphi A, B, and C, following
manufacturers’instructions. Saline negative and a serum
positive controls were included in each run. Samples positive
for slide agglutination test were subjected to Widal tube
agglutination test by serial dilutions from 1/20 to 1/1280 in
0.9% normal saline and the result read immediately to
determine antibody titers. Titers of 1/80 and above were
considered diagnostic for typhoid fever in this study based on
the manufacturer’s instructions.
C. Culture and biochemical identification of Salmonella
Stool specimens were collected using a sterile spatula
into a clean plastic wide mouthed container and analyzed
within one hour of collection. Pre-enrichment of samples was
performed by homogenizing 1g of stool samplesin 5mL of
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Selenite F broth and incubating for 24 hours at 37ºC (DHP-
9052, England).This culture was divided into two aliquots. One
aliquot and the bacterial isolates from nutrient agar in 10%
glycerol were stored at -20ºC for DNA extraction [10].The
other aliquot was subcultured on Eosin Methylene Blue
(EMB) agar and Salmonella- Shigella (SS)/Hektoen Enteric
agar (HE) by streaking and incubated for 24 hours at 37ºC
(Liofilchem®S.r.l). Non lactose fermenters producing
hydrogen sulphide(H2S) gas on SS/HE agar were further
subcultured on nutrient agar and incubated at 37ºC for 24
hours. With a sterile Pasteur pipette, characteristic colonies
were picked and stabbed t hrough the center of KIA to the
bottom of the tube. Then, the sample was streaked in the
surface of the KIA slant.The tubes were incubated at 37ºC for
18-24hours. Isolates which produced H2S with a characteristic
black buttorring without gas bubbles and an alkaline (red) slant
were considered Salmonella positive and were again grown
onto SS-Nutrient agar t o obtain pure colonies for biochemical
identification.
For identification, the strips of API 20E test kit
(BioMerieux®,Inc., France) was used following
manufacturers’ instructions. A single pur e colony was picked
from an isolation plate and emulsified in 5mL of 0.85%
sodium chloride giving a homogeneous suspension.
Anaerobiosis where required was maintained by overlaying
the cupule with mineral oil. The incubation box was closed and
k e p t at 36±2°C for 18 to 24hours. The reactions were read
and interpreted according to the reading table and the
identification software (BioMerieux, apiweb™).
D. Polymerase chain reaction for S. typhi flagellin gene
Nested PCR was done on 171 specimens (110 randomly
selected cases and 61 controls) comprising both serology and
culture positive specimens and also specimens which tested
negative for these two methods in order to detect S. typhi
infections which were not detected bythese two
tests.TheflagellingenesequencewasdetectedbyPCRaccordinga
publi shed protocol [8].
E. DNA extraction
DNA was extracted from afresh culture of the stored
bacterial aliquot by the boiling method [19]. Briefly, 1 mL of
sample was transferred to a 1.5 mL micro-centrifuge tube and
centrifuged for 10 minutes at 14,000 x g. The supernatant was
discarded carefully. The bacterial pellet was re-suspended in
300µL of autoclaved distilled water by vortexing andprevious
step repeated with centrifuging for 5 minutes. The pellet was
vortexed in 200µL of distilled water and the tube incubated for
15 minutes at 100°C and chilled on ice. The tube was
centrifuged as above for 5 minutes at4°C, the supernatant
carefully transferred to a fresh tube and 5µL of it was used as
template DNA in the PCR reaction. Another 5µL was
electrophoresed in1% agarose gelat 100V for 15 minutes and
visualized under UV trans-illuminator (BioMetra).
IJISRT18SP178 www.ijisrt.com
International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
F. Nested polymerase chain reaction
DNA was amplified using the nested PCR protocol as
first described[20] and used by others [8].Two sets of primers
were used in two successive PCRs. The ST1-5′-TAT GCC
GCT ACA TAT GAT GAG-3′ andST2-5′-TTA ACG CAG
TAA AGA GAG-3′(first pair) were used to amplfy a 497bp
fragment corresponding to nucleotides 1024-1044, and 1504-
1521, respectively in the flagellin gene; and the second pair
ST3-5′-ACT GCT AAA ACC ACT ACT-3′ and ST4-5′-TGG
AGA CTT CGG TTG CGT AG-3′ toamplify a 366bp
fragment corresponding to nucleotide 1060-1077 and 1407-
1426 respectively of the gene. The reaction was performed in
a 25μL volume using REDTaq Master Mix (Sigma). Thermo-
cycling conditions were as follows: for the first round, the
cycling program started with an initial dissociation step (pre-
denaturation at 94°C for 5 minutes and denaturation at 94°C
for 2 minutes) to ensure complete separation of DNA stands.
This was followed by 40 cycles of primer annealing at 57°C
for 1 minute 30 seconds and primer extension at 72ºC for
1minute. Next was the final extension step of 5 minutes at
72°C which ensured that all amplicons are fully extended.
Amplification conditions for second round of PCR (nested
PCR) were similar to the first round PCR with the exception
that annealing was done at a higher temperature of 68ºC for 1
minute 30seconds [8].
G. Electrophoresisofamplicons
A1.5% agarose gel was prepared, then 5µL of amplified
product was slowly loaded into the wells using disposable
micropipette tips. A wide range bp ladder (Direct Load™
Wide Range DNA Marker 50 bp - 10,000 bp) was loaded in an
adjacent well to determine size of amplified PCR products; a
well was loaded with sample containing no DNA to serve as
negative control and simultaneously electrophoresed.
Electrophoresis was carried out at 100V for 35 minutes with
2µL ethidium bromide added to the gel. Then the bands(366bp
in second round of nested PCR) were visualized using a trans-
illuminator, the gel photographed with a digital camerawas
transferred to computer for further analysis. A graph of the
distance versus log MW of the molecular weight marker was
plotted using Microsoft Excel2010, an equation obtained and
the amplicon size was estimated from the plot.
H. Assessment of feasibilityof test methods in study area
The study assessed the feasibility of each of Widal, stool
culture and nested PCR in the study area. The assessment was
based on laboratory performance, cost of a single test based on
laboratory price list, duration of test, staffing, cost of
equipment, maintenance of technology. Information on PCR
was obtained from Centre Pasteur Yaounde, under
Cameroon’s Ministry of Public Health in the Centre region of
Cameroon whereit is performed routinely and the rest of the
information from the Buea Regional Hospital.
I. Statistical Analysis
The statistical package for social science (SPSS) version
20.0 (SPSS Inc. Chicago, USA) was used to analyze the
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ISSN No:-2456-2165
results obtained by nested PCR, Widal and stool culture tests.
Differences between tests were calculated using Chi Square
test and P < 0.05 defined significance for 95% of the
population. Positivity, sensitivity and specificity values of the
different tests used for diagnosis of typhoid fever were
calculated using standard formulas.

III. RESULTS

A. Characteristics of study participants

Of the421 participants included in this study, 152
(36.0%) were males and 289 (64.0%) females. The ages of the
test group of 360 ranged from 7-76 years (mean age of
29.64±14.6); 125 (34.7%) were males and 235(65.3%)
females.In the control group of 61 participants ages ranged
from 6 -64 years (mean age of 31.08 ±12.65;27(44.5%) were
males and females 34 (55.5%). All suspected cases presented
with fever (100%), followed in decreasing frequency by
headache, abdominal discomfort, fatigue, and joint pains in
about half of them among other less frequent signs and
symptoms.

B. Antigens, serotypes and antibody titers by Widaltest

Agglutination sero-positivity of somatic O antigens
was138 (38.3%) among the febrile patients with 104
(28.8%)positive for flagella H antigens. Salmonella typhi
somatic T-O/H antigens, were58 (16%) / 59
(16.3%)respectivelyand were the most frequentspecies in the
patients while Salmonella paratyphi C-O/H,19 (5%) / 6 (1.7%)
respectively were the least frequent (Table 1). No control
group participants reacted for Somatic O antigens while only 6
(9.8%)tested positive for flagella H antigen. For the antibody
titers in the tube test, the majority of the cases reacted at a titer
of 1/80 (n = 27; 7.5%) for the O antigen. For the H antigen the
titers were spread over 1/80 to 1/320 with the highest number
of cases, 26 (44%) reacting at 1/320. The total number of S.
paratyphi cases detected was 80 (22.2%).

C. Observations from Stool culture

On Salmonella-Shigella (SS) agar, discrete colourless
colonies with black centers and or colonies completely
covered with black deposit due to the H2S produced was
observed after 24 hours incubation. Preliminary identification
of Salmonella species using KIA was based on observation of
a red slant and yellow butt, no cracks in the medium with
black deposit of H2S in the medium. The species confirmed by
the API 20E reactions are shown in Table 1 above. Fifty (50)
i.e. 13.9% of the 360 suspected cases were positive for the
stool culture and all the 61 controls were negative.

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ISSN No:-2456-2165
Test Group (n= 360)
Antigens Serotypes Positives Titers

1/40 1/80 1/160 1/320 1/640 1/1280

Somatic O S.typhi O 58 (16) 0 (0) 27(7.5) 20(5.6) 7(1.7) 3(0.8) 1(0.3)
antigens S. paratyphi 30 (8) 10(2.8) 15(4.1) 2(0.6) 2(.6) 1(0.3) 0(0)
A-O
S. paratyphi 31 (9) 12(3.3) 8(2.2) 3(0.8) 5(1.4) 2(0.6) 1(0.3)
B-O
S. paratyphi 19(5) 5(1.4) 4(1.1) 3(0.8) 2(0.6) 0(0)
C-O 5(1.4)
Subtotal 138 (38.3) 27 (7.5) 55 (15.2) 29 (8.1) 17 (4.7) 8 (2.2) 2 (0.6)

Flagella H S. typhi H 59 (16.3) 0(0) 15(4.1) 6(1.6) 26(7.2) 11(3.0) 1(0.3)

antigens S. paratyphi 27 (7.5) 1(0.3) 19(5.3) 1(0.03) 5(1.4) 1(0.3) 0(0)
A-H
S. paratyphi 12 (3.3) 0(0) 5(1.4) 2(.6) 4(0.6) 0(0) 1(0.3)
B-H
S. paratyphi 6(1.7) 0(0) 1(0.3) 0(0) 4(1.1) 1(0.3) 0(0)
C-H
Subtotal 104 (28.8) 1 (0.03) 40 (11.1) 9 (2.5) 39 (10.8) 13(3.6) 2 (0.6)
Table 1. Frequency of somatic (O) and flagella (H) antigens and antibody titers for the Widal tube test
culture had the highest positive predictive value (91.9%) while
D. Nested polymerase chain reaction products nested PCR had the highest negative predictive value of 94.6
Of the 110 cases tested, 55 (50%) were positive. Nested %. With respect to positivity nested PCR showed a higher
PCR amplification of flagellin gene showed a single band of positivity than stool culture at all durations of the fever
expected size (366bp) shown in Figure 1. A total of 39 investigated (Figure 2).
(35.4%) of the 110 samples which were positive in the stool
culture tested positive in the nested PCR while no control was
positive.

Figure 1: Detection of flagellin gene of S. typhi on a 2%

agarose gel. * Data shows size of flagellin gene amplicon with
366pb. Lane MM: size marker; lanes 1-7 and lane 9 are
samples from test group under study while lane 8 is negative
control (water as template DNA). Lanes 1-7 showed visible Figure 2: Positivity of Widal serology, stool culture and
bands hence positive while lane 9 showed no band, hence nested polymerase chain reaction in the course of typhoid
negative for nested polymerase chain reaction (PCR). fever.

E. Performance of tests
Cross tabulations of number of positives and negatives
based on 110 selected cases for the three test methods revealed
that with culture as gold standard, nested PCR had the highest
performance (sensitivity of 91.9% and specificity of 71.2 %,
P =0.000) while Widal serology had the lowest performance
for both the slide test (P = 0.024) and the tube test (P =
0.011). With nested PCR as gold standard, stool culture had
the highest specificity (94.6 %, P = 0.000), Table 2. Stool

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Volume 3, Issue 9, September– 2018 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
Method Sensitivity Specificity Disease prevalence Positive Negative predictive P value
(%) (%) (%) predictive value value (%)
(%)
Gold standard: stool culture*

Widal slide 75.7 46.6 33.6 41.8 79.1 0.024

(58.8 - 88.2) (34.8 - 58.6) (24.9 - 43.3) (35.1 - 48.8) (67.0 - 87.5)
Widal tube 56.8 68.5 33.6 47.7 75.8 0.011
(39.5 - 72.9) (56.6 - 78.9) (24.9 - 43.3) (37.0 - 58.6) (67.7 - 82.4)
Nested PCR 91.9 71.2 33.6 61.8 94.6 0.000
(78.1- 98.3) (78.1 - 98.3) (24.9 - 43.3) (52.7 - 70.2) (85.3 - 98.1)
Gold standard: nested PCR*
Stool 61.8 94.6 50.0 91.9 71.2 0.000
culture (47.7- 74.6) (84.9 - 98.9) (40.3 - 59.7) (78.7- 97.2) (63.8 - 77.7)
Widal 45.5 65.5 50.0 50.0 54.6 0.243
tube test (32.0 - 59.5) (51.4 - 77.8) (40.3 - 59.7) (45.3 - 67.7) (46.9 - 62.0)
Widal slide 76.0 45.5 57.7 65.5 58.1 0.171
test (64.8 - 85.1) (32.0 - 59.5) (48.7 - 66.3) (59.1 - 71.4) (45.8 - 69.5)
*Stool culture as gold standard for Widal and nested PCR; Nested PCR as gold standard for stool culture and Widal. P <
0.05 is significant. 95% confidence interval in parentheses.
Table 2. Performance of Widal, stool culture and nested polymerase chain reaction in diagnosis of typhoid fever

F. Feasibility of typhoid diagnostic methods in study area

Following an assessment of the feasibility of providing Stool culture and nested PCR afforded good performance but
typhoid diagnosis test by the three methods in the study area, are expensive though nested PCR has a relatively shorter
cost, turnaround time and performance of test method were turnaround time of 2 days duration compared to culture with 3
most determinant factors of which test method could be used. to 7 days (Table 3).

No Factor WidalSlide/Tube Stool culture Nested PCR

1 Cost of single test 4,200FCFA 10,000FCFA (16.13 15,000FCFA
(6.8 USD) USD) (24.2 USD)
2 Turnaround time 2 Hours 3-7 Days 2 Days

3 Sensitivity 75.7/56.8 61.8 91.9

4 Specificity 46.6/68.5 94.6 71.2

5 Staff Readily available Available Available

6 Cost of Equipment Minimal, very Moderate, affordable Expensive, affordable
affordable
7 Maintenance of Very affordable Affordable Expensive, affordable
technology
8 Overall Feasible in setting Feasible in setting
Feasible in central or
reference lab
USD: United States of America dollars at 620 FCFA per dollar December, 2016
Table 3. Evaluation of feasibility of Widal serology, stool Culture and nested PCR in Buea Health District

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IV. DISCUSSION
Each diagnostic method for typhoid fever has major
limitations as earlier mentioned. No single method therefore is
adequate to give a reliable result except where clinical
presentation and experience may be important factors in
medical decision making. Findings of this study show that
stool culture and nested PCR had the highest specificity and
sensitivity respectively whereas the serological test had a poor
performance based on stool culture as gold standard. These
findings suggest that more than one method may be required
for a more reliable diagnosis of typhoid fever depending on
the clinical presentation.
All the suspected cases presented with fever hence this
symptom is crucial when interpreting laboratory results among
others; fever was also reported in all suspected cases in
another study [18]. Based on the reactions of the majority of
cases in the serology test, the diagnostic titers are ≥1/80 and
≥1/320 for both the O and H antigens respectively. The titer of
1/80 agrees with that of the kit used for the serology test but
differs from that of a similar study which using a different
commercial test kit recorded ≥1/160 and ≥1/320 respectively
[18]. The serology test shows that most of the cases of typhoid
were caused by S. paratyphi with S. paratyphi A being the
most common.
Of the 360 cases more were positive for S. typhi in the
serology test (16.3%) than the stool culture (13.9%) probably
reflecting the low specificity of the Widal test, considering
stool culture as gold standard and exclusion of prior antibiotic
use cases in this study. The overall low detection rate of
positives (maximum 16.3% using the Widal test) among the
suspected cases may be due to the non-exclusion of malaria
cases which has a high incidence in the study area and present
with fever and similar clinical features. A study recorded a
similar finding in Cameroon where all cases of fever were
included [21]. More cases (50%) were positive in the nested
PCR than for stool culture (35.4%) suggesting low sensitivity
in the stool culture.
In comparison of the three methods using stool culture as
gold standard, that nested PCR had an overall higher
performance than Widal serology in terms of sensitivity,
specificity, positive and negative predictive values (Table 2).
Nested PCR showed a higher positivity when compared to
stool culture showing it has higher reliability in detecting
positive cases (Figure 2). These data demonstrate the likely
benefit of including PCR as one of the methods of diagnosis of
typhoid fever. Some studies have reported a low positivity for
nested PCR for stool specimens. For example one reported
higher nested PCR positives than stool culture [22]; with high
sensitivities and specificities when nested PCR was performed
on DNA extracted directly from urine and blood compared to
DNA extracted from stool. Hatta and Smits [23] had similar
results for nested PCR with high sensitivities for nested PCR
on blood and urine while PCR on faeces and blood culture had
lower sensitivities. Apart from earlier mentioned limitations,
the poor performance of blood and stool cultures may be also
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International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
be due to poor adherence to culture procedure, materials,
conditions, type and quality of media [10, 24], implying that
the PCR method is less subject to variation or more efficiently
performed by laboratory staff.
To better assess the performance of the three methods,
nested PCR was considered the gold standard showing that
stool culture had the highest specificity (P = 0.000). Given the
highest specificity recorded for stool culture and the highest
sensitivity for nested PCR, these two methods may be used in
a complementary approach alongside serological tests taking
into consideration the clinical picture.
Widal had the overall lowest performance. Few studies
have used stool culture as gold standard to assess the
performance of serological tests though it recorded more
positives than the serological tests. One study recorded higher
positives for stool culture than in the Widal test with high
sensitivity (85%) and specificity (95%) for the Widal test [18],
another had similar findings but with relatively lower
sensitivity in the stool culture [25]. On the contrary a
serological test, Tubex, showed higher performance against
stool as a gold standard [26].
This study also assessed the feasibility of these three
tests being offered to clients by the medical laboratories in the
Buea Health district in South West Cameroon. Seven factors
(Table 3) were considered; all factors had about the same
influence on the choice of each test in the study area except
cost, turnaround time and performance. Some of these factors
among others have been documented in developing countries
[27]. Skilled staff are available in Cameroon following
increased training in molecular biology techniques in higher
education institutions. Widal being rapid is the first line test
most commonly used and no further testing is done where the
clinical picture strongly suggests presence of the disease.
Culture being more expensive and time consuming is likely
therefore to be done for doubtful cases with mild symptoms
which are Widal negative. If the advantages of relatively
shorter duration and good performance of PCR are considered
then PCR constitutes a good option to avoid potential life
threatening complications of the typhoid disease due to a false
negative from serology and culture tests. PCR therefore is
likely to be very useful in cases of possible false negative for
both serology and culture where there are signs and symptoms
of the disease. However given the high cost of PCR, it could
be performed in a central point (referral laboratory) in the
health district as proposed some authors, to attenuate the cost
[27]. Also, malaria should be excluded in endemic areas to
avoid unnecessary costly testing for typhoid [21]. Hence the
following algorithm (Figure 3) is proposed for the study area
and others with the same socioeconomic environment whereby
at least two tests are used to make a diagnosis with culture
serving as gold standard and offering the additional advantage
of susceptibility testing for effective treatment. In this
algorithm, one of the two tests offers higher sensitivity and the
other higher specificity.
315
Volume 3, Issue 9, September– 2018
Figure 3: Proposed algorithm for improved diagnosis of
typhoid fever in resource limited areas using serology, culture
and polymerase chain reaction. Diagnosis is made using at
least two tests; + positive, − negative. * Assessed based on
nature of signs and symptoms including body temperature of
35.5 -37.5°C for mild and > 37.5 °C for severe cases. PCR is
done in a referral laboratory.
V. CONCLUSION
Based on the high sensitivity recorded for nested PCR
and the high specificity of stool culture, we propose that in a
resource limited environment such as in developing countries,
stool culture be used as gold standard in conjunction with
serology to improve diagnosis of typhoid fever. Additionally
PCR should be employed for severe suspected cases negative
for serology and stool culture. This proposed algorithm should
be further tested in such environments.
A. What is known about this topic
 Diagnosis of typhoid fever using serology and culture is
limited by relatively low sensitivity and specificity.
 Polymerase chain reaction (PCR) has been used to detect
Salmonella targeting the flagellin gene flic.
 Polymerase chain reaction (PCR) has exhibited mixed
performance for blood specimens in the detection of
Salmonella.
B. What this study adds
 Nested PCR showed the highest sensitivity compared to
serology and stool culture.
 Stool culture showed the highest specificity compared to
serology and nested PCR.
 Considering cost, turnaround time and performance, stool
culture and PCR appear as suitable methods for reliable
diagnosis of typhoid fever and PCR could compliment
stool culture.
C. Competing interests
The authors declare that they have no competing
interests.
IJISRT18SP178
International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
D. Authors’ contributions
RAN and RFF performed the field and bench
experiments and data analysis. MTFM and BKGA contributed
to the study design and supervised the bench work. MNN
conceived and designed the study, performed data
analysis,general supervision of the work and wrote the draft
manuscript. All authors corrected the draft and approved the
final manuscript.
ACKNOWLEDGEMENTS
The authors acknowledge the University of Buea (UB)
for funding of this project through the budget of professional
Master of Science programmes in the Department of
Biochemistry and Molecular Biology, Faculty of Science. The
Buea Regional Hospital Annex, South West Cameroon and the
Biotechnology Unit of UB provided some materials and
access to essential equipment.
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