• 31 Oct 2016

    Comparative Evaluation of Tubex Tf (Inhibition Magnetic Binding Immunoassay) for Typhoid Fever in Endemic Area

    Typhoid fever remains an important cause of disease in developing countries like India. Isolation of Salmonella typhi is the current gold standard method for confirming a diagnosis of typhoid fever. For the isolation of Salmonella typhi now-a-days either costly automated blood culture machines are required or trained staff is needed for manual methods. These are not found in primary health care settings in a developing country like India. As the symptoms of typhoid fever are diverse and non specific, sometimes the patients having similar symptoms due to other diseases are put on unnecessary antimicrobial treatment or the treatment is delayed in some cases [1,2]


    However, Salmonella typhi can be isolated from blood, urine, stool and bone marrow but these tests takes 2-3 days so the diagnosis is delayed [3]. Serological tests based on antibody detection have been used as an alternative for blood culture in the diagnosis of typhoid fever. Most widely used serological test is widal test that detects agglutinating antibodies to TO and TH antigen of salmonella typhi. Some researchers however found false positive and false negative results with this test and processing time of upto 18 hours limits its usefulness [4,5]. Many commercial tests that detect the presence of Salmonella enterica serovar typhi antigen/antibody have been developed for early diagnosis. These commercial tests are rapid and user friendly and do not require specially trained staff. IMBI (Inhibition Magnetic Binding Immunoassay) is a semi quantitative test based on visual interpretation of the test results. It detects infection specific Salmonella typhi anti O9 IgM antibodies in patient's serum. These antibodies inhibit the reaction between the antigen coated on magnetic particles and antibody coated on coloured latex particles. The colour is proportional to the concentration of inspection specific antibodies in the patient's serum. Results are calculated by visual interpretation of the colour developed. The scores are 0-10 on the colour scale (zero corresponds to absence of infection specific antibodies in the patients serum). Another new generation of rapid test is Typhidot (dot enzyme immunosorbent assay) which detects both IgM and IgG antibodies against typhoid antigen [6,7]. While tubex test was the most sensitive and specific in the Phillipines whereas neither tubex nor typhidot was both sensitive and specific in an evaluation in Vietnam [8,9]. Performance was also poor in trails in Bangladesh and Egypt [10,11]. We undertook this study, firstly to determine a area specific cut off point for the widal test and secondly to compare the performance of widal test, typhidot and tubex test in the diagnosis of typhoid fever.


    MATERIALs AND METHODS
    This cross-sectional study was done in the Department of Microbiology, Sri Guru Ram Das Institute of Medical Sciences and Research, Amritsar, during the six month period from March 2014 to August 2014. Blood samples were collected from three groups of individuals observing strict aseptic precautions after taking the informed consent.

    Group I comprised of 100 normal healthy individuals residing in Amritsar (Punjab).

    Group II comprised of 50 patients with a definite diagnosis of typhoid fever as indicated by isolation of Salmonella typhi from blood. The blood was collected in the acute phase preferably in the second week of illness.

    Group III comprised of 50 patients with non enteric fever who had not been previously immunized with TAB vaccine. These included patients with urinary tract infection, respiratory tract infection, other infections like wound infection, burns and malaria. The diagnosis was made on the basis of laboratory tests such as demonstration of malarial parasite in peripheral blood film, sputum smear, positive urine culture, pus and blood culture.

    Blood samples from patients in all the three groups were collected under aseptic conditions. A 5ml of the blood collected was put in the bile broth for blood culture and 2 ml was put in a vial for separation of serum to be used for widal test.

    A co-incidental salmonella infection was excluded by blood cultures in group I and III also.

    The blood was dispensed in blood culture bottles containing 50 ml bile broth. After incubation of 48 hours at 37°C sub culturing was done on blood and McConkeys agar (In case there was no growth then blood culture bottles were reincubated further for seven days. These bottles were observed for signs of growth daily and were subcultured before being discarded). Suspecting nonlactose fermenting colonies on the above media were screened by biochemical tests and agglutination with specific antisera (CRI Kasauli) for final confirmation. The serum was separated with sterile precautions from 2 ml of the clotted blood taken from the patient earlier along with blood collected for culture. Widal test was carriedout by tube dilution technique with the antigens obtained from CRI Kasauli [12].

    The tubex test (IDL Sweden) was conducted on all the three group of samples. To the reaction well strip provided with the kit added 45 microlitre of Tubex TF brown reagent. Then added 45 microlitre of the patient's serum sample and incubated for two minutes. After incubation 90 microlitre of blue reagent was added and the well strip was sealed with a tape. After shaking the well for two minutes, the well was placed on tubex colour scale. The reading was taken after five minutes by comparing the colour with tubex colour scale (colour ranging from clear pink (negative) to intense blue (Positive) are given scores of 0 and 10 respectively. Scores of ? 4 were taken as positive [7].

    Typhidot was carried out as per the manufacturer's instructions. (Typhidot: Malaysian Biodiagnostic Research, Kuala Lampur, Malaysia). A 250 micro liters of diluent was added to the test strip and then 2.5 micro liters of the patients serum was added to it. This mixture was incubated at 37° C for 20 minutes. After washing the strip three times added 250 micro litres of Antihuman IgM conjugate and incubated for 15 minutes. Again after washing the strip thrice added 250 micro litres of colour development solution and incubated for 15 minutes. After that the strip was washed and result read. If the dots were dark or darker than the positive control, the result was reported as positive. Absence or faint dots than the control were reported as negative [6]. Taking the blood culture results as gold standard, the sensitivity and specificity of the kits were assessed. Sensitivity of the test kit was the percentage of culture positive patients which were correctly identified with these kits. Whereas specificity of the test kit was the percentage of the culture negative patients which were correctly identified with these kits

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