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TUBEX Test Versus Widal Test In The Diagnosis Of Typhoid Fever In Kafr El -Shekh, Egypt
Wafaa M.K. Bakr* , Laila A. El Attar , Medhat S. Ashour , Ayman M. El Tokhy Microbiology Department, High Institute of Public Health, Alexandria University ABSTRACT Background: The value of the Widal test for the diagnosis of typhoid fever has been debated for as many years as it has been available. TUBEX test is a serological test which was stated to have the advantages of the Widal test without its controversies. The aim of this study was to evaluate TUBEX test versus the Widal test regarding sensitivity and specificity for the diagnosis of typhoid fever in an endemic area like Kafr El Shekh, Egypt. Materials and Methods: Serum samples were collected from typhoid (n=91) and febrile non-typhoid patients (n=25) and used to evaluate the performance of both Widal and TUBEX tests in diagnosis of typhoid fever using IgM anti-LPS ELISA as a reference test. Results: TUBEX test had sensitivity, specificity, accuracy, positive predictive value and negative predictive value of 74.6%, 75%, 74.7%, 89.2% and 58% respectively. Widal test had higher results. Conclusion and recommendation: TUBEX test results are not superior to Widal test. TUBEX has a very serious shortcoming regarding its color scoring system. We do not recommend the use of TUBEX test for diagnosis of typhoid fever in Egypt as Widal test which is the test commonly used in diagnosis gave better performance.
Keywords: Serologic tests, TUBEX, typhoid fever, Widal. Corresponding Author: Dr. Wafaa Bakr Microbiology Department, High Institute of Public Health, Alexandria University E-mail: wafaabakr@hotmail.com Phone: (02)0124583627 Fax:(02)034288436

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INTRODUCTION Today most of the burden of typhoid fever occurs in the developing world, where sanitary conditions remain poor.(1,2) Reliable data to estimate the burden of the disease in these areas are difficult to obtain, since many hospitals lack facilities for blood culture, and up to 90 % of patients with typhoid are treated as outpatients. Community based studies have consistently shown higher levels of typhoid fever than public health figures suggest.(3) The definitive diagnosis of typhoid fever requires the isolation of Salmonella enterica subspecies enterica serovar Typhi (S. Typhi) from the patient. Cultures of blood, stool, urine, rose spots, blood mononuclear cellplatelet fraction and bone marrow can all be useful for diagnosis.(4) The above mentioned cultural techniques for the isolation and identification of S. Typhi from clinical samples require laboratory equipments and technical training that are beyond the means of most primary health care facilities in the developing world. In addition cultural techniques are long and cumbersome, and are not always successful.(5) Therefore, developing an inexpensive and rapid diagnostic test for typhoid fever that is both sensitive and specific has become a public health priority. Several new serologic tests for typhoid fever have been introduced which detect IgM or IgG antibodies to various purified antigens of S. Typhi as TUBEX test.(6) Studies evaluating TUBEX test revealed marked variation in its results.(7,8) This study aimed to evaluate the performance of TUBEX test in a typhoid fever endemic area like Kafr El- Shekh, Egypt.

MATERIAL AND METHODS This comparative cross sectional study was carried out at Desouk fever hospital, kafr El-Shekh governorate, Egypt after obtaining the approval of

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the ethical committee of the Egyptian Ministry of Health. Patients were recruited after obtaining their (or their parents) consents .

Typhoid fever patients group

This group included 91 patients clinically suspected of typhoid fever (suffering from continuous fever greater than 38C in addition to headache, constipation or diarrhea).

Febrile non typhoid patients group

This group included 25 patients with febrile diseases other than typhoid fever that have been diagnosed after both clinical examination and laboratory investigations.

Samples collection
Blood samples were collected from typhoid and non typhoid patients. They were centrifuged and sera were separated and stored at -20 oC.

Tests carried out 1- Widal test

Widal test, both slide and tube agglutination tests were performed according to the manufacturers instructions (Remel stained Salmonella O and H suspensions, Remel Europe ltd, UK).

2- TUBEX test
In TUBEX, antibodies are detected in a 5-min procedure by their ability to inhibit the binding between 2 reagent particles: An indicator (colored) particle coated with a monoclonal antibody specific for the O9 antigen found in S, Typhi lipopolysaccharide (LPS) and a magnetic particle coated with S. Typhi LPS.(7) Reaction of the indicator particle is revealed by magnetic separation of the particles and consequent color development. Interestingly, only IgM antibodies and not IgG antibodies appeared to be

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detectable by this test from whole serum.(8)

Kit content: ( IDL Biotech AB, Sweden)

BROWN reagent : 1 vial, LPS O-9 antigen coated latex particles with embedded ferric oxide in protein stabilized buffer. BLUE reagent: 1 vial, mouse monoclonal anti LPS O-9 antibody coated blue latex particles in protein stabilized buffer.

Assay procedures:
The reaction wells strip was placed on a flat surface. All reagents and samples were allowed to adjust to room temperature. All reagents were shaken thoroughly prior to use until all sediment had dispensed into solution. One drop (45 micro-liters) of the BROWEN reagent was dispensed in each well. To each of these wells, 1 drop of control or serum sample was added. The BROWN reagent and the control / sample were mixed carefully using the pipette for a minimum of 10 times. The reaction mixture was left on a flat surface for 2 minutes. Two drops (90 micro-liters) of the BLUE reagent were added to each well in use. The reaction wells were sealed using the sealing tape provided and the tape was pressed hard against the plastic to prevent leakage. The assay was left at room temperature for 2 minutes with gently and continuously tilting the strip. To read the result, the reaction well strip was placed on the magnetic rack, with 2 wells above the magnet (center). A minimum of 45 seconds were allowed for all brown matter to sediment and to obtain a clear separation. The separation procedure was repeated for the next 2 wells. Before final reading the strip was slided once across the magnet to obtain optimal separation. The result was obtained within 15 minutes after separation. - The result of the samples was graded by comparing its color with the color code (0 10). Samples with score 2 and below were

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considered negative, those with score 4 or above were considered positive. - The test run was considered valid providing the following criteria were met: negative control scored 2 and positive control scored 8.

3- ELISA test (Salmonella Typhi IgM anti-LPS ELISA BIOQUANT INC, USA)
In the present study detection of S. Typhi by culture method was not done, instead ELISA test was performed as a comparative reference test to both Widal and TUBEX tests, on the collected 91 serum samples .

Statistical analysis
The statistical tests used were mainly sensitivity, specificity, positive predictive value, negative predictive value and accuracy. These were calculated by using the following formulas: sensitivity is a/(a + c), specificity is d/(d + b), positive predictive value a/(a+b), negative predictive value d/(c+ d), accuracy a+d/(a+b+c + d), where a is positive ELISA and Widal test, b is negative ELISA, but positive Widal test, c is positive ELISA, but negative Widal test, and d is negative ELISA, and negative Widal test. In addition, chi-square and Pearson correlation were performed. Differences were considered significant at P value <0.05.

RESULTS TUBEX and Widal tests were both evaluated on serum samples collected from 91 clinically diagnosed typhoid fever cases and 25 febrile non typhoid patients (12 patients with urinary tract infection, 5 patients with chest infection, 4 patients with brucellosis and 4 patients with measles). Sera from the 25 febrile non typhoid patients were TUBEX negative, while one serum sample gave positive Widal test result at a titer of 1/80,

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(the Egyptian Ministry of Health and Population consider a diagnostic titer for typhoid fever to be of 1/160).(9) For evaluation of TUBEX sensitivity, specificity, accuracy, positive and negative predictive values, comparison with ELISA test results was performed (Table 1). Sensitivity, specificity, accuracy, positive predictive value and negative predictive values were 74.62 %, 74.62 %, 74.72 %, 89.28 % and 58.06% respectively. The difference between TUBEX and ELISA results among positive cases was very highly significant (chi-square= 22.396 and P = 0.001).

Table (1):

Distribution of the TUBEX Test Results of the Serum Samples of 91 Typhoid Cases In Relation to Their Corresponding Igm Anti-LPS ELISA Results ELISA Total

TUBEX test results

Positive No. % 19.40 74.62 5.97 100.00

Negative No. 18 6 0 24 % 75.00 25.00 0.00 100.00 No. 31 56 4 91 % 34.06 61.53 4.39 100.00

Negative Positive Non-interpretable Total

Chi-square = 22.396 ,

13 50 4 67
P = 0.001

Considering Widal test results compared to ELISA (Table 2), it was calculated from this table that Widal anti-O sensitivity, specificity, accuracy, positive and negative predictive values were 89.50%, 83.33%, 87.91%, 93.75% and 74.07% respectively, while Widal anti-H test was associated with sensitivity, specificity, accuracy, positive and negative predictive values of 83.58%, 20.83%, 67.03%, 74.66% and 31.25% respectively.

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Table (3) shows that there was a strong positive Pearson correlation (0.75- 0.99), between each of the following: Widal anti-O titer (Remel) and IgM anti-LPS ELISA antibody index (0.860) Widal anti- O titer (Remel) and TUBEX score (0.828) IgM anti-LPS ELISA antibody index and TUBEX score (0.804)

Table (2):

Distribution of The Widal Test Results of The Serum Samples of 91 Typhoid Fever Cases at A Titer of 1/160* in Relation to Their Corresponding Igm Anti-LPS ELISA Results ELISA (91)

Widal test 1/160* Negative Anti -O Positive Negative Anti -H Positive

Positive (67) No. 7 60 11 56 % 10.44 89.55 16.41 83.58

Negative (24) No. 20 4 5 19 % 83.33 16.66 79.16 4.16

Total

27 64 16 75

* The diagnostic titer of typhoid fever by the Egyptian Ministry of Health and Population

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Table (3):

Pearson Correlations Between Anti-O Titers of The 4 Widal Brands, Igm Anti-LPS-ELISA Antibody Index and Score Anti-O titers ELISA TUBEX Ab index Remel Biosystems Biotech Dialab score

ELISA Ab index No. of cases Remel No. of cases Biosystems Anti-O No. of cases titers Biotech No of cases Dialab No. of cases TUBEX score No. of cases

1.00 91 0.86 91 0.789 91 0.758 91 0.725 91 0.804 87

0.86 91 1.00 150 0.917 150 0.836 150 0.694 150 0.828 136

0.798 91 0.917 150 1.00 150 0.767 150 0.658 150 0.76 136

0.758 91 0.836 150 0.767 150 1.00 150 0.709 150 0.749 136

0.725 91 0.694 150 0.658 150 0.709 150 1.00 150 0.729 136

0.804 87 0.828 136 0.76 136 0.749 136 0.729 136 1.00 136

DISCUSSION Lim et al., in 1998 in Hong Kong described a test (TUBEX) to detect typhoid-specific antibodies based on particle separation in tubes and stated that it has the advantages of the Widal test and the specificity normally accorded to ELISAs that utilize purified antigens for detection.(7) In the present study, TUBEX test was evaluated for the serological diagnosis of typhoid fever. A sensitivity of 74.62 %, specificity of 75 %, accuracy of 74.72 %, positive predictive value (PPV) of 89.28 % and negative predictive value (NPV) of 58.06% were obtained. These figures are lower than those reported by Lim et al., who reported that TUBEX was found to be 100% sensitive and 100% specific.(7) However, figures of

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the present study are to some extent comparable with those obtained by Jaffery et al,(2005) in Pakistan.(10) They reported that TUBEX test had a sensitivity of 89.29% specificity of 84.62%, PPV of 86.21% and NPV of 88.00%. Also Gasem et al. (2002) in Indonesia reported that in a single blood sample collected on admission to hospital, sensitivity of TUBEX test was 69.8% as compared with bone marrow culture and 86.5% as compared with blood culture.(11) Dutta et al.(2006) in India examined the performance of TUBEX test in a community field site and compared it with Typhidot and Widal tests for diagnosis of typhoid fever.(12) The sensitivity, specificity, PPV, and NPV of Typhidot and TUBEX were not better than Widal test. They stated that there is a need for more efficient rapid diagnostic test for typhoid fever especially during the acute stage of the disease. Until then, culture remains the method of choice. This was exactly the case of the present study where the sensitivity, specificity and accuracy of Widal anti-O was higher than those of TUBEX. In a study carried out by Tam and Lim (2003) in Hong Kong, they stated that TUBEX test for the diagnosis of typhoid fever detects antibodies to the Salmonella enterica serovar Typhi lipopolysaccharide (LPS) O9 antigen(8) whereas it was previously assumed by Lim et al.(1998) that both IgM and IgG antibodies could inhibit the reaction, their study revealed that only the IgM antibodies did.(7) It was not clear why IgG anti-O9 antibodies, did not inhibit the reaction, although these can bind to the LPS-sensitized magnetic particles as efficiently as the IgM antibodies. They concluded that the finding that TUBEX detected IgM antibodies but not IgG antibodies was important, as detecting only the IgM antibodies enhances the specificity of the assay because, unlike IgG, these are found only in current infection. However, Rahman et al.(2007) in Bangladesh examined the performance of TUBEX in 234

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cases (mostly children and infants) and concluded that regression analysis of the TUBEX and ELISA results showed good concordance between them, better with the combined IgM-IgG ELISA than with IgM alone, suggesting that TUBEX detects IgM antibodies not necessarily by themselves, as previously reported, but with the help of IgG antibodies.(6) In the present study there was a strong positive correlation between TUBEX and IgM anti-LPS ELISA antibody index (Pearson correlation = 0.804). The present study revealed that TUBEX has a very serious shortcoming regarding the color scoring system. In spite that the producing company made changes in the color scoring, sharp color scores were obtained only in the two extremes (strong positive cases: score 10 and negative cases: score 0). In many cases we were not able to have a sharp score of TUBEX and even the score was subjected to variation with different operators. In some cases we were not able to have a score at all and we reported these cases as non interpretable. We think that this problem was encountered by other researchers Rahman et al,. when they considered the scores of 0-4 as negative in spite of the 0-2 recommended by the manufacturing company.(6)

CONCLUSION AND RECOMMMENDATIONS TUBEX results are not superior to Widal test results as compared to the results of IgM anti-LPS ELISA as a reference test. TUBEX has a very serious shortcoming regarding scoring of its results as sharp scores were obtained only in the two extremities (strong positive and strong negative cases). In many cases a sharp score of TUBEX was not obtained and even the score was subjected to variation with different operators. Even in some cases no score

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was obtained at all and these cases were reported as non interpretable. The following is recommended: An improvement of the scoring system of TUBEX must be done by the manufacturing company to give a more distinct scoring of the test results. Till this is achieved we do not recommend the use of TUBEX test for diagnosis of typhoid fever in Egypt as Widal test which is the test commonly used in diagnosis gave better performance.

REFERENCES
1. Kidgell C, Reichard U, Wain J, Linz B, Torpdahl M, Dougan G, et al. Salmonella typhi, the causative agent of typhoid fever, is approximately 50,000 years old. Infect Genet Evol. 2002; 2: 39 45. Merrell DS, Falkow S. Frontal and stealth attack strategies in microbial pathogenesis. Nature. 2004; 430: 250 56. Parry CM, Hien MB, Dougan MD, White NJ, Farrar MT. Typhoid fever. N Engl J Med. 2002; 347: 1770-82. Wilke A, Ergonul O, Bayar B. Widal test in diagnosis of typhoid fever in Turkey. Clin Diagn Lab Immunol. 2002; 9:938-41. Bhutta Z A. Current concepts in the diagnosis and treatment of typhoid fever. B M J. 2006; 333:78-82. Rahman M, Siddique AK, Chi-Hang F, Sabrina Sharmin S, Rashid H, Iqbal A, et al. Detection of early typhoid fever in endemic community children by the R O9-antibody test. Diagn Microbiol Infect Dis. 2007; 58(3):275-81. 7. Lim PL, Tam FC, Cheong YM, Jegathesan M. One-step 2-minute test to detect typhoid-specific antibodies based on particle separation in tubes. J Clin Microbiol. 1998; 36(8):2271-8. 8. Tam FC, Lim PL. The TUBEX typhoid test based on particle-inhibition immunoassay detects IgM but not IgG anti-O9 antibodies. J Immunol Method. 2003; 282:83-91.

2. 3. 4. 5. 6.

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Ministry of Health and Population, Egypt: Enhanced Surveillance for Communicable Diseases. Annual Summary January December 2000. Introduction. [cited 2008 Feb 16]. Available from: http:/ www.geis.fhp.osd.mil/GEIS/Training/EgyptSurv2000.htm

10. Jaffery G, Anwar MS, Hussain W, Maqbool S. Serological diagnosis of typhoid fever in children: a comparative evaluation of Salmonella Typhi O-9 antigen based rapid assay. Pak Pediatr J. 2005; 29:27-33. 11. Gasem MH, Smits HL, Goris MG, Dolmans WM. Evaluation of a simple and rapid dipstick assay for the diagnosis of typhoid fever in Indonesia. J Med Microbiol. 2002; 51(2):173-7. 12. Dutta S, Sur D, Manna B, Sen B, Deb AK, Deen JL. Evaluation of newgeneration serologic tests for the diagnosis of typhoid fever: data from a community-based surveillance in Calcutta, India. Diag Microbiol Infect Dis. 2006;56(4):359-65.

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