Antibody response in typhoid fever in endemic Indonesia and the relevance of serology and culture to diagnosis


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Slide 1 : Antibody response in typhoid fever in endemic Indonesia and the relevance of serology and culture to diagnosis Prof. Dr. Mochammad Hatta, Ph.D. Clin MicroDepartment of Medical Microbiology, Molecular Biology and Immunology Laboratory, Faculty of Medicine, Hasanuddin University, Makassar, Indonesia.
Slide 2 : Abstract. Culture and serology were performed on blood and serum samples collected at or shortly after admission from 473 patients presented with suspected clinical typhoid. Culture confirmed the diagnosis in 65.39% of the patients with typhoid fever as the final diagnosis. The sensitivity (58%) and specificity (98.1 %) of a rapid dipstick assay for the detection of S. typhi-specific immunoglobulin M were somewhat lower than those of culture but higher than those of the Widal test. The dipstick assay thus may well be used in the serodiagnosis of typhoid in situation where culture facilities are not available. Combination of test results of dipstick and culture improved sensitivity to 82% Sensitivity of the dipstick assay strongly increased with duration of illness and was higher for culture positive than for culture negative patients. Duration of illness and presence of S. tvphi in the blood are major factors that determine severity of disease.
Slide 3 : MATERIALS AND METHODS
Slide 4 : Patients and sample collection Blood and serum samples were collected from all patients presented with suspected clinical typhoid fever at the Hasanuddin University Hospital of South Sulawesi, three primary health care centers in Makassar, and a district hospital in Gowa district, a suburban area of Makassar, Indonesia. Laboratory diagnosis Blood culture was performed by inoculation of 15 ml of ox bile broth (Merck) with 5ml of freshly collected blood. Cultures were incubated for 24 hours at 37°C. The Widal test using the 0 antigen was performed by incubation of two-fold serial serum dilutions (1/20 -1/1.280) with an equal volume of the antigen (0) suspension at 50°C. Tubes were checked for agglutination after 4 hours. According to routine diagnostic criterion. a titer > 1:320 was considered to be positive.
Slide 5 : Dipstick assay. The dipstick assay was performed by incubation of a wetted dipstick in a mixture of 5 ul serum and 250 ul detection reagent for 3 hours at room temperature. At the end of the incubation. the dipsticks were thoroughly rinsed with water and dried. The staining intensity of the antigen band was then graded by comparison with a colored reference strip. The test was scored negative when no staining was observed. 1+ when a weak staining was observed and 2+. 3+ or 4+ when a moderate to strong staining was observed (ref. Hatta M, et al. Am. J. Trop. Med. Hyg., 66(4), 2002, pp. 416–421) Calculations The sensitivity. specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated using the following formulae: sensitivity is a/(a + c)100%. Specificity is d/(d + b) 100%. PPV is a/(a + b)100% and NPV is d/(d + c)100% in these formulae. a is test positive and true positive. b is test positive and true negative. c is test negative and true positive. and d is test negative and true negative. 95% confidence intervals (CI) were determined by using the Epi-info computer package. Chi-square analysis was used to determine statistical significance between groups. Mann-Whitney U test was used to determine significance for continuous variables. A p-value <0.05 was considered to indicate statistical significance
Slide 6 : RESULTS
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Slide 12 : Negatif (1+) (2+) (3+) (4+)
Slide 13 : Conclusions The value of the Widal test in the serodiagnosis of typhoid fever thus is limited, as results are difficult to interpret. In this study, the sensitivity and specificity of the dipstick assay were significantly higher than those of the Widal test. In about 10% of the culture-proven and culture-negative typhoid fever no specific IgM antibodies could be detected in the dipstick assay during a 2 week follow-up. Any of the suspected factors including strain variation, poor antigenicity, prior antibiotic treatment and host factors could play a role in the absence or weakness of the immune response in the patients. The dipstick assay is easy to use and does not require specialised training or equipment, and the components are stable without a requirement for refrigeration. The dipstick assay is highly specific and has an acceptable sensitivity. All these factors make the test ideal for developing countries and rural and suburban settings in endemic areas where culture facilities are not readily available and the need for a rapid tests that can be applied in district hospitals and primary health care is high.
Slide 14 : ACKNOWLEDGEMENTS This study was supported by EC grant no IC18CT9980381 Correspondence: Prof. Dr. Mochammad Hatta, Ph.D., Clin Micro Head Department of Medical Microbiology, Molecular Biology and Immunology Laboratory, Faculty of Medicine, Hasanuddin University, Kampus Tamalanrea, Km 10, Makassar, Indonesia. Tel / Fax: 062-411-586971; E-mail: hattaram@indosat.net.id

 


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