Abstract

DEVELOPMENT OF A SIMPLE AND RELIABLE COMMERCIAL

DIAGNOSTIC KIT FOR SALMONELLA SEROVAR SPECIFIC ANTIGENS

Francis Kariuki (1, *), Peter Kinyanjui (1), Joseph Kamau (1), Gerald Juma (1),

Atunga Nyachieo (1).

Department of Biochemistry, University of Nairobi. Nairobi, Kenya (1)

CAUSES OF TYPHOID FEVER:

1.Caused by the bacterium Salmonella Typhi .

2. Ingestion of contaminated food or water.

3. Water is contaminated where inadequate sewerage systems and poor sanitation.

4.Contact with a chronic asymptomatic carrier.

5.Eating food or drinking beverages that handled by a person carrying the bacteria.

Typhoid fever is still a significant health problem in many developing countries.

Worldwide, an estimated 17 million cases occur annually with most of the

disease burden occurring among citizens of low-income countries, particular

those in South East Asia, Africa, and Latin America. Certain serovars of

Salmonella enterica subsp. enterica cause invasive diseases (e.g., enteric fever,

bacteremia, septicemia, meningitis, etc.) in humans and constitute a global

public health problem [2, 6]. Despite the presence of several serological [4], and

molecular tests [5] for the diagnosis of Salmonella Typhi (S. Typhi) infection in

patients with typhoid fever, the Widal test is commonly used in low income

endemic areas for the detection of the pathogen. There are however a number of

controversies challenging the diagnostic utility of this test and hence continues

to be a subject of differing opinions with regards to its performance as a reliable

diagnostic test for enteric fever. The S. Typhi antibodies are shared by a large

number of organisms from the Salmonella genus and other related organisms

and this often leads to higher percentage of false positive results in several

conditions other than the actual specific infection [1]. In addition, similar cross

reacting antibodies have also been reported for some febrile illnesses such as

malaria, tuberculosis, and schistosomiasis thus indicating that Salmonella

agglutinins may occur either by specific or by nonspecific stimuli [3, 5].

For, the last three years the department of biochemistry (University of Nairobi.

Kenya) has been producing and marketing an innovative serologic Widal test kit

(salDTECT) for S. Typhi detection. However, owing to the unreliability and low

sensitivity associated with the test, as well as existing market demands, there is

a great need to develop and validate inexpensive and rapid screening and

detection methods for S. Typhi. A rapid, sensitive diagnostic and low cost test

would allow prompt initiation of treatment in individual patients and also aid in

measuring the disease burden at the population level.

Future Directions

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Microbiol. 37(9):2882-2886.

4. Parry CM. (2004). Typhoid Fever. Curr Infect Dis Rep. 6(1):27-33.

5. Wain J, Hosoglu S. (2008). The laboratory diagnosis of enteric fever. J Infect Dev Ctries. 2(6):421-425.

6. WHO: The diagnosis, treatment and prevention of typhoid fever. (2003). Communicable Disease Surveillance and Response Vaccine and Biologicals.7-18

[http://whqlibdoc.who.int/hq/2003/WHO_V&B;_03.07.pdf].

7. Tran QT, Gomez G, Khare S, Lawhon SD, Raffatellu M, Bäumler AJ, Ajithdoss D, Dhavala S, Adams LG. (2010). The Salmonella enterica serotype Typhi Vi capsular antigen is

expressed after the bacterium enters the ileal mucosa. Infect Immun. 78(1):527-535.

8. Parkhill J, Dougan G, James KD, Thomson NR, Pickard D, Wain J, Churcher C, Mungall KL, Bentley SD, Holden MT, Sebaihia M, Baker S, Basham D, Brooks K, Chillingworth T,

Connerton P, Cronin A, Davis P, Davies RM, Dowd L, White N, Farrar J, Feltwell T, Hamlin N, Haque A, Hien TT, Holroyd S, Jagels K, Krogh A, Larsen TS, Leather S, Moule S,

O'Gaora P, Parry C, Quail M, Rutherford K, Simmonds M, Skelton J, Stevens K, Whitehead S, Barrell BG. (2001). Complete genome sequence of a multiple drug resistant Salmonella

enterica serovar Typhi CT18. Nature. 413(6858):848-852.

9. Pratap CB, Kumar G, Patel SK, Verma AK, Shukla VK, Kumar K, Nath G. (2013). Targeting of putative fimbrial gene for detection of S. Typhi in typhoid fever and chronic typhoid

carriers by nested PCR. J Infect Dev Ctries. 7(7):520-527.

10. Al-Dahhan AH, Muhammed Ali AC, Al-Ammer M. (2015). Phenotypic and Genotypic Characterization of Salmonella Typhi Virulence Factors Isolated From Patients with Typhoid

Fever in Najaf Province /Iraq. Int J Res Stud Biosci. 3(6):77-84.

11. Trkov M, Avgustin G. (2003). An improved 16S rRNA based PCR method for the specific detection of Salmonella enterica. Int J Food Microbiol. 80(1):67-75

Approaches (Methods)

Detection of various Salmonella serovars (local isolates) using the target genes of interest

(staA, viaB and sopE).

Results

Introduction To avoid severe complications or even the loss of life as a result of Salmonellae

infections, definite and accurate diagnosis and treatment need to be initiated as

soon as the onset symptoms of the infection begin to manifest. However, the

lack of adequate diagnostic capabilities in poor resource settings common in

most public health facilities in Kenya and Africa in general, hinder prompt

diagnosis of Salmonellae infections particularly typhoid fever. Currently, the

available diagnostic tests are often complicated, time consuming, expensive,

unreliable and more so not readily available in most resource poor endemic

areas. This has often led to misdiagnosis of the disease, thereby delaying

appropriate treatment and making typhoid fever endemic in most resource poor

areas. Therefore, there is a great need for a rapid, reliable and inexpensive

laboratory test for early and accurate diagnosis of patients with typhoid fever

and other Salmonella infections.

ELISA is a serological method that has been used in the diagnosis of typhoid

Fever. This method is mainly based on the ability of antibody or antigen-coated

polymers to bind specific antigen or antibodies. Majority of these assays have

been based on the detection of S. Typhi anti-lipopolysaccharide (O) antibodies

though recent ELISA detection of antiflagellum (H) and anti-polysaccharide

(Vi) antibodies have also been developed. However, S. Typhi is a member of the

Enterobacteriaceae family and shares most of the surface antigens (O and H)

used in serology with other salmonella serotypes and also cross-reacting

epitopes with other Enterobacteriaceae. This makes it difficult to develop a

specific diagnostic kit for typhoid because any such kit would likely yield

significant false positives. An alternative would be to use an antigen detection

method that detects only an active infection and is species/strain specific.

Thus, to improve diagnostic tests for Salmonella infections, this study aims to

develop a serovar-specific antigen based ELISA kit for sensitive, rapid, and

inexpensive detection of Salmonella serovar enteritidis Typhi in Kenyan strains.

This will provide an efficient, less expensive and timely test that can be

effectively used in resource poor pathogen prone endemic areas.

Salmonella Typhi :

A major cause of foodborne illness throughout the world

Enterobacteriaceae family

Member of the genus Salmonella

rod shaped bacilli

gram-negative

facultative anaerobic,

Research question Would the alternative recombinant proteins: Capsular antigen Vi (viaB), Salmonella outer

protein E (SopE) and putative fimbrial protein (staA) provide a better diagnostic tool for

early S. Typhi detection?

Capsular antigen Vi (viaB): A virulence factor that protects the bacilli against

Phagocytosis and is absent in non-typhoidal Salmonella that is induced when S. Typhi

transits from the intestinal lumen into the illeal mucosa [7].

Salmonella outer protein E (SopE): invasion-associated secreted protein [8].

Putative fimbrial protein (staA): a member of the fimbrial gene family specific

to Salmonella Typhi only [9] and has also been linked to S. Typhi pathogenicity [10].

Approaches (Methods)

Gene and Primer Length Tm

(oC)

GC

content

(%)

Amplified fragment

size (bp)

Source (GenBank

Accession

numbers)

viaB

vi- Forward (5’- ATG AGG TTT CAT CAT TTC TGG CC-3’) vi- Reverse (5’- TTA CAG TAA AGT AAC TGA ATC CGG C-3’)

23

25

55.6

54.9

43.5

40.0

540

(4524679- 4524140)

NC_003198.1

(AL513382.1)

sopE

SopE- Forward (5’-ATG CTT CAA ACG CTC AAT GAT ATA G-3’) SopE- Reverse (5’-TCA GGG AGT GTA TTG TAT ATA TTT ATT AGC -3’)

25

30

53.7

52.8

36.0

30.0

465

(4482059-4482523)

NC_003198.1

(AL513382.1)

staA

StaA-Forward (5’- ATG AAA AAA GCG ATT TTA GCT GC -3’) StaA-Reverse (5’- TTA CTG GTA AGT AAA GGT ATA CAT TGC -3’)

23

27

52.9

52.9

34.8

33.3

585

(217411-216827)

NC_003198.1

(AL513382.1)

Primer design targeting full length (staA) or partial (viaB or sopE) genes of interest.

Collect local isolates of Salmonella

Extract genomic DNA

Confirm the Salmonella Spp. Using PCR.

Results

Figure 1. 1% Agarose gel of the PCR products using 16S rRNA target primers and 5

DNA templates extracted from local Salmonella isolates. (Expected product size is 402 bp)

The PCR products using 16S rRNA target primers (Minf 5’-ACGGTAACAGGAAGCAG-3’ and Minr 5’-TATTAACCACAACACCT-3’)[11] and various DNA templates were ran and analyzed by using a 1% agarose gel

containing ethidium bromide (0.5ug/ml). M represents 1kb DNA ladder, Lane 1 represents negative control. Lane 2

contains Positive control (S. Typhi ). Lane 3 contains isolate 7a (S. Typhimurium). Lane 4 contains isolate 16a (S.

Enteritidis). Lane 5 contains isolate 30a (S. Gallinarum). Lane 6 contains isolate 40a (S. Dublin).

M 1 2 3 4 5 6 bp

500

400

100

Cloning, Expression and Purification of the target antigens i.e. proteins / peptides (viaB, SopE, staA).

Immunize source organism (Rabbit or Rat) with multiple injections of the antigen to boost antibody levels.

Affinity purify antibodies from the source animal serum.

Test the specificity of the purified antibodies using Enzyme-Linked Immunosorbent Assay (ELISA) techniques.

M 1 2 3 4 5 6 7 M 1 2 3 4 5 6 7 M 1 2 3 4 5 6 7

viaB PCR products sopE PCR products staA PCR products

bp

1000

500

200

Figure 2. 1% Agarose gel of the PCR products using staA, viaB and sopE target primers and 5 DNA templates extracted from

local Salmonella isolates and 1 Escherichia coli (E. coli) DNA template. (Expected product sizes are 540 bp, 465 bp and 585 bp respectively)

The PCR products using staA, viaB and sopE target primers and various DNA templates were ran and analyzed by using a 1% agarose gel containing ethidium

bromide (0.5ug/ml). M represents 1kb DNA ladder, Lane 1 represents negative control. Lane 2 contains S. Typhi PCR product. Lane 3 contains S.

Typhimurium PCR product. Lane 4 contains S. Enteritidis PCR product. Lane 5 contains S. Gallinarum PCR product. Lane 6 contains S. Dublin PCR

products. Lane 7 contains isolate E. Coli PCR product.