The Clinical Utility of Typhidot in the Diagnosis of Typhoid Fever* Ferdin A. Membrebe. M.D. and Jennifer A. Chua. M.D.** (*2nd Prize Winner PSMID-UAP Junior Research Contest 20th Annual Convention, Westin Philippine Plaza Hotel,

November 26, 1998. **Department of Medicine, Cardinal Santos Medical Center) ABSTRACT

Typhoid fever is a severe systemic infection endemic in many developing countries, including the Philippines. Because of this, there is a clear need for a sensitive and specific test that will permit its rapid laboratory diagnosis.

One such test, the typhidot, was assessed among 185 febrile patients with blood culture as gold standard. Using IgM (+) and Igm/IgG (+) as indicators of a positive typhidot test, results revealed a sensitivity of 72% and specificity of 52%. Likelihood ratio for a positive test was 1.50 and likelihood ratio for a negative test was 0.54. With these values, given a positive typhidot test there is only 31% probability that patient has typhoid fever and given a negative typhidot test, there is only 36% probability that the patient has no typhoid fever. Analysis of typhidot with blood culture and "clinical typhoid" as gold standard was also assessed which did not significantly alter the outcome of the study. Cross-reactions with other members of the Salmonella family and other febrile illnesses were noted. (Phil J Microbiol Infect Dis 1999; 28(1):1-4)

Key words: sensitivity, specificity, IgG, IgM, typhoid fever, typhidot INTRODUCTION

Typhoid Fever is a severe systematic infection caused by Salmonella typhi. It is endemic in many developing countries, particularly in South East Asia, the Indian sub-continent, South and Central America and Africa where provision of pure water supplies and sewage control are not always adequate.1,2 In the Philippines, it has an incidence of 27/100,000 population.3 Clinical diagnosis is often unreliable and isolation of S. typhi through blood culture is still used as the gold standard, although, at its best, the blood culture yield is only 70-75% in cases of typhoid fever. The other drawback in the use of blood culture for the diagnosis of typhoid fever is the delay in the result of the test, at its best, 2-3 days.4

The dot enzyme immunoassay, which detects serum antibody to antigen dotted on a nitrocellulose membrane, has been used in the diagnosis of several microbial diseases. Its results can be interpreted visually without special equipment.5,6 Asma Ismail, et al. developed a dot enzyme immunoassay for the diagnosis of typhoid fever due to Salmonella typhi.7-10 Clearly, there is a need for a more rapid test in the diagnosis of typhoid fever.

The general objective of the study is to determine if typhidot is useful in the diagnosis of typhoid fever. The specific objectives of the study are: 1) to determine specificity, sensitivity, positive, and negative predictive values and likelihood ratios of typhidot with blood culture as gold standard. 2) To determine rate of false positive typhidot. 3) To determine if there will be a significant change in the sensitivity, specificity, positive and negative predictive values, and likelihood ratios of typhidot if blood culture together with clinical typhoid was used as gold standard. MATERIALS AND METHODS Study Population

Patients included in this study were between 18-80 years old. They were admitted from January 1997 to August 1997 (8 month period). All patients with fever regardless of the duration had their venous blood taken for blood culture and typhidot. Baseline CBC and urinalysis were

taken to help arrive at a probable diagnosis. If infection other than typhoid was entertained, other laboratory examinations were also done.

For the purpose of analysis, patients are divided into three groups: Group I (TYPCP): consisted of patients with typhoid fever confirmed by isolation of S. typhi from blood culture. Group II (CLNTYP): consisted of patients whose clinical course is compatible with typhoid fever but whose blood culture is negative for Salmonella typhi (clinical typhoid). This is on the basis of some combination of at least six of the following criteria: a history of fever of five days or more prior to admission plus documentation of fever (or to 38°C) in the hospital (both are essential for diagnosis), headache, constipation, diarrhea, roseaceous rash, splenomegaly, hepatomegaly, abdominal pain, nausea, signs of toxemia, leukopenia and/or leukocytosis, response to treatment which is described as lysis of fever after four or more days of antibiotic therapy with chloramphenicol and/or cotrimoxazole, quinolones, ceftriaxone, or any antibiotic that is recommended for treatment of typhoid fever. In this group are patients whose blood culture is (+) for Salmonella paratyphi because of the premise that typhidot will be positive only for patients who are infected with Salmonella typhi. In the analysis of sensitivity, specificity, and predictive values, this group was classified as having non-typhoidal fever since culture was used as the gold standard. Group III (NON-TYP): consisted of patients with fever due to illnesses other than typhoid fever. Isolation of S. typhi

Blood specimens were incubated in liquid broth. They were sub-cultured on blood agar

and Maconkey agar. If turbidity was noted, Salmonella organisms were identified. Serotyping was then used to identify the specific Salmonella organism. Typhidot

One umL containing 0.3 ug of the 50 kD protein of S. typhi was dotted onto nitrocellulose strips. After probing with a 1:100 dilution of serum, the strips were washed and antigen-antibody complexes were visualized 1 hour after the addition of horseradish peroxide-conjugated antiserum to human IgG or IgM. A substrate, 4-chloro-l-naphthol, was added for color development. Serum containing either IgM or IgG antibodies to specific antigen gives a blue color as intense or more intense than the color of the positive control (i.e. serum from a patient with culture-positive typhoid). The EIA was considered positive if the IgM and IgG + IgM titer was ≥ 1:100.

The manufacturer recommends the following clinical interpretation of Typhidot results: 1) IgM (+) and IgM/IgG (+/-), definite typhoid fever. 2) IgG (+), correlate with clinical symptoms, possibilities include; a) previously successfully treated case of typhoid fever b) re-infection with typhoid fever c) typhoid carrier 3) IgM (-) and IgG (-), not typhoid fever.

Data Analysis

Sensitivity, specificity, positive, and negative predictive values were computed with positive blood culture for S. typhi as the gold standard. Likelihood ratios, pretest and post-test probabilities were also computed. In this study, IgM (+) and IgM/IgG (+) were used as indicator of a positive test because of the ambiguity in the interpretation of the typhidot test with regards to the IgG component. Validity of typhidot test was also measured by using blood culture and "clinical typhoid" as gold standard. RESULTS

There were 185 patients whose sera were tested both for blood culture and typhidot. There were 94 females and 91 males with a mean age of 30 years old. Results of these tests are summarized in Table 1. Table 1. Summary of blood culture and typhidot results IgM (+) and IgM/IgG (+) IgG (+) IgM/IgG (-) No. of subjects No. (%) No. (%) No. (%) Group I Blood culture (+) S. typhi 43 31 (72) 12 (28) 0 Group II Clinical typhoid 39 22 (56) 12 (31) 5 (13) Group IIA Blood culture negative 11 9 (82) 2 (18) 0 Group IIB Blood culture S. paratyphi A 28 13 (46) 10 (36) 5 (18) Group III Non-typhoidal fever 103 46 (45) 33 (32) 24 (23) Total 185 99 (53) 57 (31) 29 (16)

Out of 185 patients whose sera were tested, 43 were culture positive for S. typhi (Group I) with 31 of the 43 testing positive for typhidot (72%). Thirty-nine patients were classified under the clinical typhoid group (Group II). Eleven of these were culture negative while the remaining 28 patients had blood culture positive for Salmonella paratyphi A. Of the 28 patients with typhoid fever due to Salmonella paratyphi A, 13 had positive typhidot results.

The remaining 103 patients had febrile illnesses due to diseases other than typhoid fever, forty six (45%) of which showed positive typhidot test. Table 2 shows the diseases other than due to Salmonella typhi that resulted in (+) typhidot test. Final diagnoses for these patients were arrived at after careful review of history, physical examination, laboratory results, ancillary procedures and course in the ward of these patients. All in all, 45% of non-typhoidal fever were positive for typhidot. (46 out of 103 cases.) Table 2. Summary of non-typhoidal fever (+) for typhidot IgM and IgM/IgG Non-typhoidal fevers No. (%) UTI 3 (4.3) Hepatitis A 2 (4.3) ATP 1 (2.2) DFS/DHF 3 (4.3) URTI 4 (8.7) Pneumonia 1 (2.2) SVI 31 (67.4) Hepatic granuloma (probable TB) 1 (2.2)

Based on the blood culture results, typhidot was found to have a sensitivity of 72%, specificity of 52%, false positive rate of 68.6% and false negative rate of 14%. The likelihood ratio of a positive test is 1.5 with a predictive value of a positive test at 31 %. The likelihood ratio of a negative test is 1.8 with a predictive value of a negative test at 86%.

A two by two table was also done correlating blood culture and clinical typhoid with typhidot which gave the following results; sensitivity of 65%, specificity of 55%, false positive rate of 55% and false negative rate of 31 %. The likelihood ratio of a positive test is 1.30 with a predictive value of a positive test at 44%. The likelihood ratio of a negative test is 1.4 with a predictive value of a negative test at 69%. DISCUSSION

Based on the results, typhidot was found to be lacking in sensitivity (72%) and specificity (52%). False positivity was due to number of factors including cross-reactions with Salmonella paratyphi A. (46%) and other non-typhoidal fever who tested positive (45%). Predictive value for

a positive test was computed at 31 %, which means given a positive typhidot, there is only 31% probability that the patient has typhoid fever. Predictive value for a negative test was computed at 86%, which means given a negative typhidot, there is an 86% probability that the patient has no typhoid fever.

There is no significant difference in the results of typhidot when clinical typhoid was added as a gold standard to blood culture. Blood culture was found out to be positive in 75% of cases based on our criteria of clinical typhoid which was comparable with the report of Hoffman which claimed that during the first seven days of fever, blood culture is positive in 72.4% of cases and its sensitivity decreases thereafter.4 Fifty-six percent of clinical typhoid fever is positive for typhidot. CONCLUSIONS

Locally, typhidot lacks the sensitivity and specificity for it to replace blood culture in the diagnosis of typhoid fever as claimed in studies done abroad. REFERENCES

1. Keusch GT. Salmonellosis, Harrison's Principles of Internal Medicine (14th) ed). vol. 1, 1998, 950-956. 2. EdelmanR, Levine MM. Summary of an International Workshop on Typhoid Fever. Rev Infect Dis 1986;

8:329-349. 3. Paje-Villar E, Carlos JC, Carandang E (eds). Salmonellosis. Handbook of Infectious Diseases (1992 ed).

Philippine Pediatric Society Inc. 1992, 302-309. 4. Hoffman SL, Punabi NH, Rockhill RC, Sutomo A, Rivai AR, Pulungsih SP. Duodenal string capsule compared

with bone marrow blood and rectal swab cultures for diagnosing typhoid and paratyphoid fever. J Infect Dis 1984; 149:157-161.

5. Schroeder SA. Interpretation of serologic tests for typhoid fever. JAMA 1968; 206:839-840. 6. Sivadasan K, Kurien B, John TJ. Rapid diagnosis of typhoid fever by antigen detection. Lancet 1984; 1:34-35. 7. Choo KE, Oppenheimer SJ, Ismail AB, Ong KH. Rapid serodiagnosis of typhoid fever by dot enzyme

immunoassay in an endemic area. CID 1994; 19 (July):172-176. 8. Ismail AB, Kader ZSA, Ong KH. Dot enzyme immunosorbent assay for the serodiagnosis of typhoid fever.

Southeast Asian J Trop Med Public Health 1991; 22:563- 566. 9. lsmail AB, Hai OK, Kader ZA. Demonstration of an antigenic protein specific for Salmonella typhi. Biochem

Biophys Res Commun 1991; 181:301-305. 10. lsmail AB, Hai OK, Choo KE, Kader ZA, lbrahim TA. Recent developments in the diagnosis of typhoid fever.

Supplement to JAMA Southeast Asia 1994; 321-323. Typhidot TM Protocol. Dot EIA for the Rapid Detection of Specific IgM and IgG Antibodies to Salmonella typhi.

Malaysian Biodiagnostic Research SDN BHD, May 1996.