Typhoid Fever Detection by Dot-eia-omp Test

Typhoid fever is still an important public health problem in many developing countries especially in tropical parts of the world, as in Indonesia. This problem opens the way for a further study with the aim of finding an alternative serological test with a high degree of reliability for the detection of typhoid fever. Given the above mentioned purpose, a study on the reliability of a laboratory test, the dot-enzyme-immunoassay outer membrane protein (DOT-EIA-OMP) was conducted comprising sera from 207 subjects (44 adult typhoid patients, 43 adult nontyphoid patients and sera from 120 adult healthy individuals serving as controls. The result of the study revealed that the diagnostic sensitivity of the DOT-EIA-OMP test for the detection of typhoid fever can be classified as high (93.16%), the specificity as moderate (76.74%), the efficiency (accuracy), positive predictive value and negative predictive value as high (85.06%, 80.39% and 91.66% respectively). The within run and between days reproducibility of this test was very high (CV=0%). Analysis of data obtained indicated that the DOT-EIA-OMP test was a reliable screening test for the establishment of the diagnosis of typhoid fever in health centers with simple laboratory facilities. The application of this test has to be more contemplated in countries where the cost of laboratory test is a problem. SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH Vol 32 No. 3 September 2001 508 sitive, specific and applicable even in primary health centers. MATERIALS AND METHOD This laboratory study was performed on sera obtained from 207 subjects divided into 3 groups: 1. A group of 44 adults patients (24 males and 20 females aged 15-60 years) with positive blood culture for S. typhi and / or with positive polymerase chain reaction (PCR) for S. typhi, and / or with a four fold increase of the titer of Widal test, and with clinical signs and symptoms suggestive of typhoid fever. 2. A group of 43 adult patients (22 males and 21 females aged 15-42 years) with negative blood, urine and stool culture for S. typhi, with negative PCR test for S. typhi and with negative Widal test, but with fever caused by other diseases than typhoid fever. 3. A group of 120 healthy adults as control. In the list of nontyphoid diseases with fever were dengue, malaria, hepatitis, pneumonia, urinary-tract infections, paratyphoid fever A and B. Sera obtained were tested by dot-enzymeimmunoassay to detect OMP antigen (DOTEIA-OMP). The primary antibody used to detect the OMP antigen in sera was anti-OMP polyclonal antibody (rabbit IgG against OMP) obtained from a rabbit (body weight 2.5 kg) which had received an intramuscularly injection of 0.25 μg/μl of a mixture of equal quantities of OMPs derived from 5 different strains of S. typhi that are locally prevalent. The OMPs were prepared according to the procedure as described previously (Gam, 1992; Verdugo-Rodrigues et al, 1993 a,b). In order to obtain anti-OMP polyclonal antibody, rabbit hyperimmune sera were purified by affinity chromatography technique using Hi Trap column (protein-A-sepharose column) (Hebert, 1996; Pang et al, 1991). The conjugate used in this test was alkaline phosphatase conjugate goat antirabbit IgG (Sigma) and the chromogenic substrate used was 5-bromo-4-chloro-3-indoylphosphate and nitroblue tetrazolium (Promega) in Tris buffer solution containing 0.1 M NaCl and 50 mM MgCl 2 . DOT-EIA-OMP test procedure As results of chequerboard titration (Leung et al, 1991; Portsmann and Kiessig, 1992; Kemeny, 1992), the optimal serum dilution was 1:8, the optimal anti-OMP polyclonal antibody dilution was 1:150, the optimal conjugate dilution was 1:1,500 and the optimal reading time was 5 minutes. The DOT-EIA-OMP test is a modification of the method used by Sadallah et al (1990) and is in short performed as follows. One μl appropriately diluted human sera (1:8) was added to 5x10 mm nitrocellulose paper with a pore size of 0.45 μm (Optitran) and left to dry at room temperature. The nitrocellulose paper was afterwards blocked with 5% skimmed milk in buffer blocking solution. After one hour incubation at room temperature the nitrocellulose paper was washed 3 times with phosphate buffer saline tween 20 (PBST) solution with a pH of 7.4 during a 10 minutes washing period each time. During the next step, the nitrocellulose paper was soaked in the same chamber containing a 1:150 dilution of anti-OMP polyclonal antibody in PBS solution with pH of 7.2 and incubated for one hour at room temperature on an oscillator. After 3 washes with PBS-T solution as mentioned above, the nitrocellulose paper was soaked in the same chamber containing a 1:1,500 dilution of alkaline phosphate conjugate goat antirabbit IgG in PBS solution (pH 7.4) containing 0.2% gelatin and 0.2% bovine serum albumin (BSA) and incubated for one hour at room temperature. The nitrocellulose paper was washed again with PBS-T solution, three times as mentioned above followed by the addition of a chromogenic substrate (10 μl NBT and 5 μl BCIP) in 10 ml substrate buffer solution containing 0.1M NaCl and 50 mM TYPHOID FEVER DETECTION BY DOT-EIA-OMP TEST Vol 32 No. 3 September 2001 509 MgCl 2 with pH of 9.5. Incubation was made afterwards in a dark room for 5 minutes and then washed with distilled water to stop the reaction and the result of the test was made discernible with naked eyes. The result was positive if the intensity of the blue dot was the same or sharper when compared to the positive control obtained by making a 1:32 dilution of 0.25 μg/μl OMP in PBS solution (pH 7.2) (Chaicumpa et al, 1992; Handojo, 1996; Leung, 1991). Blood, urine and stool cultures and the PCR test for S. typhi (Prihatini, 1996) were done according to the standard procedures of the Microbiology Division, Department of Clinical Pathology, Dr Sutomo Hospital in Surabaya (Baron, 1990; Cheesbrough, 1985). The Widal tube test was performed by using antigen obtained from locally prevalent strains (5 strains) of S. typhi. The cut-off value of the Widal test was based on the titer of 1:200 (Prihatini et al, 1982).