The Distribution of Anti-salmonella Antibodies in the Sera of Healthy Blood Donors in

Baquba city
Abdul-Razak Shafiq Hasan*
Abbas Abood Al-Duliami**
Adawia Fadil Abbas***
Abstract:
Background: Misuse and interpretation of the Widal test for the laboratory diagnosis of typhoid fever usually result in over
diagnosis of the disease particularly in endemic areas which lack standard baseline anti-salmonella antibody titer for
healthy individuals.
Aim of study: to determine the baseline anti-salmonella antibody titer in healthy individual in Baquba city, and to evaluate
the widal tube agglutination test in the diagnosis of clinically suspected typhoid fever.
Subjects and methods: one hundred and twenty three apparently healthy blood donors and 127 clinically suspected patients
as having typhoid fever were enrolled in this study. The Widal test was done using tube agglutination on serially
diluted sera. Data were statistically analyzed.
Results: The baseline anti-(O) antibody titer of S. typhi, S. paratyphi A,B, and C in healthy blood donors were 1:160, 1:160,
1:160 and 1:80 respectively. While the baseline anti-(H) antibody titer of S. typhi, S. paratyphi A,B, and C were
1:640, 1:640, 1:320 and 1:80 respectively. The mean anti-(O) and anti-(H) of S. typhi and S.paratyphi A, B was
significantly higher in patients compared to healthy controls. According to baseline anti-salmonella antibody titer in
healthy blood donors, a total of 89 (70%) of typhoid fever patients were positive by Widal test. 34(26.8%), 15
(11.8%), 26(20.5%) and 14(11%) had S. typhi, S. paratyphi A, B, and C respectively.
Conclusion: pre-knowledge of baseline anti-salmonella antibody titer of healthy individuals in a community may improve
the efficacy of Widal tube agglutination test in laboratory diagnosis of clinically suspected typhoid fever in an
endemic area.
Key words: typhoid fever, Widal test, Salmonellosis.

Introduction:
yphoid ever continue to be a global health
problem, especially in tropics and subtropics
with an annual global burden of about21,600,000
illness and 220,000 death during 2000 and that
paratyphoid cause 5,410,000 illness [1]. The
diagnosis of typhoid fever in developing countries is
based mainly on clinical ground and is difficult, as
the presenting symptoms are diverse and similar to
those observed in other common febrile illness [2,3].
The traditional salmonella laboratory confirmation
of clinical suspicion of typhoid fever is based on
culture on selective media and identification of
suspected colonies by biochemical and serological
tests [4]. These methods are generally timeconsuming, laborious and may give false negative
results [5]. An alternative is typhoid serology using
the Widal test that is widely used in many
developing countries including Iraq, because of its
feasible, fast and easy. Over 100 years since its
introduction as a serological means of detecting the
presence of typhoid fever, the Widal test continues
to be plagued with controversies involving the
quality of the antigens used and interpretation of the
result, particularly in endemic areas [6-9].However,
several reports affirmed that when performed
reliably and interpreted with care it can be of value
in the diagnosis of typhoid fever [10-12]. In order to
interpret the Widal test properly, the baseline antisalmonella antibody titer in a community must be
standardized [13-15].

Subjects and Methods:

One hundred and twenty three apparently healthy
blood donors were enrolled. Those were neither

241

infected with typhoid or paratyphoid fever, nor

received anti-typhoid vaccine for previous 3 years
from the date of enrollment. They were 65 (52.8%)
female and 58 (47.2%) males. Additionally, 127
patients who were clinically diagnosed as having
typhoid or paratyphoid fever. They were referred
from outpatient clinic for laboratory confirmation.
85 (66.9%) were females & 42(33.1%) were males.
Venous blood sample was collected from each
participant, left to clot for 15 minutes in the room
temperature. Sera were separated using automatic
pipette and disposable plan tubes. The Widal test
was first done by slide agglutination technique, and
then positive sera were further subjected to tube
agglutination for quantitative titration by making
serial serum dilutions: 1:20, 1:40, 1:80, 1:160,
1:320, 1:640, and 1:1280. The anti-(O) and anti-(H)
antibodies of S. typhi and S. paratyphi A,B and C
were determined according to the procedure
described by (Nustebu et al., 2002) [11]. For patients,
Widal test was done first on undiluted serum,
thereafter; positive sera were processed with serial
dilutions as described for blood donors.
Results:
The results in table (1) revealed the baseline anti(O) and (H) of S. typhi and S. paratyphi A, B, and
C. Based on the 95% percentile, the baseline anti(O) antibody titer of S. typhi was 1:160, since 117
(95.1%) of the blood donors had an antibody titer
1:160. Similarly, the baseline anti-(H) antibody titer
of S. typhi was 1:640, because 118(95.9%) of the
blood donors had a titer of 1:640.
Likewise, the baseline anti-(O) antibody titer of S.
paratyphi A, B, and C were 1:160[120(97.6%],

Iraqi J. Comm. Med., July. 2011 24 (3)

The distribution of anti-salmonella in the sera of blood donors in Baquba

1:160 [118 (95.9%)], and 1:80 [117 (95.1%)]

respectively. While the baseline anti-(H) antibody
titer of S. paratyphi A,B, and C were 1:640
[119(96.7%)], 1:320 [117 (95.1%)], and 1:80 [118
(95.5%)] respectively.
Table (2) showed the distribution of anti-salmonella
antibody titers. According to the base line antisalmonella antibody titers in the healthy control
(table 1), the number and percentage of positive
patients for different salmonella serotypes were also
included. For instance, 34 (26.6%) patients were
considered positive for S. typhi, since they have

Abdul-Razak Shafiq Hasan et.al.

anti-(O) titer > 1:160. Similarly, 15(11.8%), 26

(20.5%) and 14 (11%) were positive for S. paratyphi
A,B, and C respectively. So the total positivity rate
was 89 (70%).
The results also revealed a significant increase in the
median of anti-(O) antibody titer of S. typhi, S.
paratyphi A, B, but not S.paratyphi C. Similarly, a
significant increase in the median of anti-(H)
antibody of S. typhi, S. paratyphi A and B, but not S.
paratyphi C, table (3).

Table (3): the median anti-Salmonella (O)and (H) antibody titers in healthy and patients.
Median antibody titer
Type of antibodies
P value
Healthy
Patients
Anti- S. typhi
1:20
1:160
<0.001
Anti-S. paratyphi AO
1:20
1:80
<0.001
Anti- S. paratyphi BO
1:20
1:80
<0.001
Anti-S .paratyphi CO
1:20
1:20
0.42 [NS]
Anti-S. typhi H
1:40
1:80
<0.002
Anti-S. paratyphi AH
1:40
1:20
<0.001
Anti-S. paratyphi BH
1:20
1:40
<0.001
Anti-S. paratyphi CH
1:20
0
0.94 [NS]
Discussion:
The 95% percentile were used in this study to
determine the anti-salmonella (O) and (H) antibody
titer, because it represent more reasonable balance
between the sensitivity and specificity.
Unfortunately, there was no previous study on the
distribution of baseline anti-salmonella antibody titer
among healthy subjects in Baquba city. However, in
comparison with studies conducted elsewhere [6,8,1214]
. The elevated baseline of anti-salmonella
antibody titers obtained in this study may be
attributed to the habitual exposure of individuals in
the community to salmonella antigens through
contaminated water and food. Although these
subclinical doses of salmonellae are unable to induce
an overt clinical illness; however, they are
continuously boosting the host's immune system
resulting in cumulative accumulation of these
antibodies in the circulation.
Salmonella infections may induce a broad
polyclonal stimulation of the immune response. This
result in the production not only of specific
antibodies to the causative agent but also of
antibodies to cross reacting group or other groups of
(O) and (H) antigens. This may explain the
multifaceted picture of serological results which
may often be encountered during serodiagnostic
evaluation of salmonella infections [5,7,10,16]. The high
prevalence of baseline anti-(H) antibody may result
from cross reactions, anamenestic reactions or the
persistence of (H) antibody in the sera of subjects for
years after clinical recovery. On the other hand,

242

these H agglutinins often precede salmonella

gastroenteritis that may cause by non-typhoidal
salmonellae e.g. S. typhimurium [17,20]. However, the
prevalence of H agglutinin within the population of
different geographical regions depend o the
corresponding prevalence rate of salmonella
infections [1,3]. Therefore, it has been well
documented that O agglutinin titer is more valuable
than the H agglutinin in helping to establish the
diagnosis of acute salmonella infections [7,12,19].
According to the resultant baseline anti-salmonella
antibody titer in healthy control, 70% of patients
who were clinically suspected as having typhoid or
paratyphoid fever were positive by Widal tube
agglutination test. These include S. typhi, S.
paratyphi A,B and C infections. As far as the studies
conducted in this field, different positivity rates were
[6,7,10,12-15,18,19]
obtained
.
Nevertheless,
the
composition of the antigen suspension used in Widal
tube agglutination test should reflect the prevalence
of the typhoid salmonella serotypes encountered in
each particular geographical region. In Europe, for
example, typhoid fever is mainly caused by S. typhi
and S. Paratyphi B, whereas, in Africa and Asia, S.
paratyphi A is important and in the Middle East and
Asia, S. paratyphi C is another possible serotype [1214]
. It can be concluded that pre-knowledge of
baseline anti-salmonella antibody titer of healthy
individuals in a community may improve the
efficacy of Widal tube agglutination test in
laboratory diagnosis of clinically suspected typhoid
fever in an endemic area.

Iraqi J. Comm. Med., July. 2011 24 (3)

The distribution of anti-salmonella in the sera of blood donors in Baquba

Abdul-Razak Shafiq Hasan et.al.

Table (1): Anti-salmonella antibody titers among healthy control.

Salmonella serotype

Anti-salmonella antibody titers

Total

1:20

1:40

1:80

1:160

1:320

1:640

1:1280

(95%)

S. typhi O

30(24.3%)

31(25.2%)

25(20.3%)

23(18.6%)

8(6.5%)

5(4%)

1(0.8%)

117(95.1%)

S. typhi H

20(16.2%)

15(12.1%)

25(20.3%)

18(14.6%)

21(17%)

14(11.3%)

5(4%)

5(4%)

118(95.5%)

S. paratyphi AO

49(39.8%)

11(8.9%)

26(21.1%)

22(17.8%)

12(9.7%)

3(2.4%)

120(97.5%)

S. paratyphi AH

27(21.9%)

13(10.5%)

24(19.5%)

17(13.8%)

12(9.7%)

15(12.2%)

11(8.9%)

4(3.4%)0

119(96.7%)

S. paratyphi BO

33(26.8%)

14(11.3%)

33(26.8%)

29(23.5%)

9(7.3%)

3(2.4%)

2(1.6%)

118(95.5%)

S. paratyphi BH

53(43%)

16(13%)

23(18.6%)

18(14.6%)

5(4%)

2(1.6%)

1(0.8%)

5(4%)

117(95.1%)

S. paratyphi CO

54(43.9%)

21(17%)

26(21.1%)

16(13%)

6(4.8%)

117(95.1%)

S .paratyphi CH

51(41.4%)

19(15.4%)

25(20.3%)

23(18.6%)

3(2.4%)

1(0.8%)

1(0.8%)

118(95.9%)

Table (2): Anti-salmonella antibody titers among patient group.

Salmonella serotype

Anti-salmonella antibody titers

Total

1:20

1:40

1:80

1:160

1:320

1:640

1:1280

S. typhi O

17(13.3%)

9(7%)

16(12.5%)

26(20.4%)

25(19.6%)

28(22%)

5(3.9%)

1(0.8%)

S. typhi H

33(25.9%)

13(10.2%)

11(8.6%)

21(16.5%)

13(10.2%)

11(8.6%)

19(14.9%)

6(4.7%)

S. paratyphi AO

49(38.5%)

4(3.1%)

12(9.4%)

29(22.8%)

18(14.1%)

11(8.6%)

3(2.3%)

1(0.8%)

S. paratyphi AH

67(52.7%)

11(8.6%)

9(7%)

15(11.8%)

10(7.8%)

10(7.8%)

2(1.5%)

3(2.3%)

S. paratyphi BO

36(28.3%)

6(4.7%)

10(7.8%)

29(22.8%)

20(15.7%)

21(16.5%)

4(3.1%)

1(0.8%)

S. paratyphi BH

59(46.4%)

8(6.3%)

15(11.8%)

22(17.3%)

11(8.6%)

5(3.9%)

4(3.1%)

3(2.3%)

S. paratyphi CO

67(52.7%)

7(5.5%)

16(12.5%)

23(18.1%)

9(7%)

5(3.9%)

S. paratyphi CH

8(6.9%)

8(6.3%)

15(11.8%)

13(10.2%)

5(3.9%)

6(4.7%)

6(4.7%)

243

Iraqi J. Comm. Med., July. 2011 24 (3)

(95%)
34(26.8%)

15(11.8%)

26(20.5%)

14(11%)

The distribution of anti-salmonella in the sera of blood donors in Baquba

References:
1-Crunp, J.A.; Luby, S.P. and Mintz, E.D. The
global burden of typhoid fever. Bull. WHO
2004; 82(5): 346-53.
2-Ohanu, M.; Mbah, A.; Okonkwo, P and Nwagbo,
F. Interference by malaria in the diagnosis of
typhoid fever using Widal test alone. West Afr.
J.Med. 2003; 22(3): 250-2.
3-Heymann, D.L. Control of Communicable Disease
Manual. 8th. Ed. American Public Health
Association. 2004, 577-83.
4-Ngwu, B.A. and Abo, J.A. Typhoid fever: Clinical
diagnosis versus laboratory confirmation. Niger.
J.Med. 2003; 12(4): 187-92.
5. Hosoglu, S.; Bosnak, V.; Akalin, S.; Geyik, F.M.
and Ayaz, C. Evaluation of false negativity of the
Widal test among culture proven typhoid fever
cases. J. Infect. Developing Countries, 2008;
2(6): 475-8.
6-Pang, T. and Puthucheary, S. Significance and
value of the Widal test in the diagnosis of
typhoid fever in an endemic area. J. Clin. Pathol.
1983; 36:471-5.
7-Parry, C.; Hoa, N.: Diep, T.; Wain, J.; Chinh, N.;
Vinh, H.; Value of a single- tube Widal test in
the diagnosis of typhoid fever in Vietnam. J.
Clin. Microbiol. 1999; 37: 2882-6.
8-House, D.; Eain, J.; Ho, V.; Diep, T.; Chinh, N.;
Serology of typhoid fever in an area of
endemicity and its relevance to diagnosis.
J.Clin.Microbiol.2001; 39(2): 1002-7.
9. Olopoenia, L.A. and King, A.L. Widal
agglutination test- 100 years later: still plagued
by controversies. Postgrad. Med. J. 2000; 76:8084.
10-Saha, S.; Ruhulamin, M.; Hanif, M.; Islam, M.
and Khan, W. Interpretation of the Widal test in
the diagnosis of typhoid fever in Bangladesh
children. Trop. Pediat. 1996; 16: 75-8.
11-Nustebu, E.; Ndumbbe, P. and Koulla, S. The
increase in occurrence of typhoid fever in

244

Abdul-Razak Shafiq Hasan et.al.

Cameron: Over diagnosis due to misuse of the

Widal test. Trans. Roy. Soc. Trop. Med. Hyg.
2002; 96: 64-7.
12. Willke, A.; Ergonul, O. and Bayar, B. Widal test
in diagnosis of typhoid fever in Turkey. Clin.
Diag. Lab. Immunol. 2002; 9(4): 938-41.
13. Pokhrel, B.M.; Karmacharya, R.; Mishra, S.K.
and Koirala, J. Distribution of antibody titer
against salmonella enteric among healthy
individuals in Nepal. Ann. Clin. Microbiol.
Antimicrob. 2009; 8(1): 1-4.
14. Zailani, S.B.; Oyelese, A.O. and Aboderin, A.O.
Determination of baseline antibody titer to S.
typhi/paratyphi in Ile-Ife, Nigeria. Afr. J. Med.
Med. Sci. 2003; 32(3):307-10.
15. Malhotra, V.L. and Khandpur, N. Significance of
Knowing baseline Widal test titers in pediatric
population in Delhi. J. Commun. Dis. 2004;
36(1):70-1.
16-Nustebu, E.; Ndumbbe, P. and Adiogo, D. The
distribution of anti-salmonella antibodies in the
sera of blood donors in Yaound, Cameron.
Trans. Roy. Soc. Trop. Med. Hyg. 2002; 96: 689.
17-Mastroeni, P. and Menager, N. Development of
acquired immunity to salmonella. J. Med.
Microbiol. 2003; 52: 453-9.
18-Kist, M. Salmonellosis .In: Clinical Laboratory
Diagnosis. Thomas, L. 1st. Ed. TH Books, 1998,
1194-8.
19-Levine, M.M. and Grados, O. Diagnostic value
of Widal test in areas endemic for typhoid fever.
Am. J. Trop. Med. Hyg. 1978; 27:795-800.
20. Chart, H.; Cheasty, T.; de Pinna, E.; Siovanes,
L.; Wain, J.; et al. Serodiagnosis of salmonella
enteric serovar typhi and S. enteric serovars
paratyphi A, B and C human infections. J. Med.
Microbiol.2007; 56(9):1161-66.
*College of Medicine- Diyala University
**College of Education- Diyala University
***College of Medicine- Diyala University

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