Serum Tcii in Patients with Scrub Typhus

Serum transcobalamin II levels were measured in scrub typhus patients. Eighteen out of fifty-two patients admitted to Maharat Nakhon Ratchasima Hospital were diagnosed with scrub typhus infection. The serum unsaturated vitamin B12 binding protein (UBBC) and total vitamin B12 binding protein (TBBC) levels in these patients were significantly higher than in normal subjects (p<0.001). The mean serum transcobalamin II level in the typhus patients was also significantly higher than in the normal subjects (p=0.004). There was a significant correlation between serum TCII levels and typhus IgM or IgG titers (p<0.05), but not to total IgM levels. These findings indicate that patients with scrub typhus had stimulation of the recticuloendothelial system as a result of a considerable increase in transcobalamin II levels. cytic systems, such as malignant histiocytosis, also had elevated serum TCII (Fehr and Vecchi, 1985). It has been suggested that monocytes and macrophages may be the sites of TCII synthesis (Laser et al, 1985; Arnalich et al, 1990). As reactive macrophage hyperplasia occurs frequently in patients with typhus, it is of interest to study TCII levels in patients with scrub typhus. MATERIALS AND METHODS Fifty-two patients with clinical signs and symptoms of scrub typhus, such as eschar, fever and headache, were investigated. Patients were admitted to Maharat Nakhon Ratchasima Hospital, Nakhon Ratchasima Province, Thailand. Clinical history, age and other pertinent information were recorded. The following tests were performed by staff of the hospital laboratory : multiple blood film examinations for malaria parasites, bacterial cultures with blood, urine and stool samples, serologic tests for melioidosis, bacterial agglutination tests for typhoid fever, and an indirect immunoperoxidase test (IIP). The diagnosis of scrub typhus was conf i rmed by a demonstrat ion of r is ing IgG (≥1:1,600) and IgM (≥1:400) titers against the rickettsial antigen. Indirect immunoperoxidase test (IIP) The IIP test for specific immunoglobulin SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH 114 Vol 36 No. 1 January 2005 against the scrub typhus antigen was modified from Suto (1980) and Yamamoto and Minamishima (1982). Serum and conjugated antibody were diluted using phosphate buffered saline (pH 7.3) and incubated for 30 minutes at 37oC. Peroxidase conjugated antihuman IgG, IgM, or whole IgM was used. Determination of transcobalamin levels Transcobalamin determination was performed by using a modified method described by Selhub et al (1976). Three transcobalamins, TCI, TCII and TCIII, were measured by filtration through stack charged cellulose (DE-81) disks. A reaction mixture containing serum was incubated with excess Co-B12 of high specific activity, diluted with 0.1 M sodium borate buffer (pH 8.5), and passed through the filter stack by applying a vacuum. Under these conditions, TCII was selectively and quantitatively adsorbed onto the cellulose-nitrate filter while both the TCI and TCIII were adsorbed onto the DE-81 filters. Then, TCIII was selectively eluted from the filters by applying a 0.05 M monopotassium phosphate solution (pH 4.6). Unsaturated vitamin B12 binding protein (UBBC) UBBC was present as the sum of TCI, TCII and TCIII in pg of Co-B12 per ml of serum. Determination of vitamin B12 Vitamin B12 level was measured using a modified method of radioisotope dilution and coated charcoal technique (Lau et al, 1965; Kidroni and Grossowicz, 1969). This technique used chicken serum as a vitamin B12 binder and PVP-coated charcoal to separate the bound vitamin B12 from the free vitamin B12. Total vitamin B12 binding protein (TBBC) TBBC was calculated from the sum of vitamin B12 and UBBC. Statistical analysis The relationships between the serum transcobalamin II and the other biochemical parameters were determined by the Pearsons correlation method. The differences between the means of the serum vitamin B12 and vitamin B12 binding protein levels in patients and controls were compared by the Student’s t-test for independent samples. A p<0.05 was considered statistically significant. RESULTS Fifty-two patients with suspected scrub typhus were investigated for scrub typhus. Only 18 patients were confirmed to be infected with scrub typhus by the Indirect Immunoperoxidase (IIP) test. These patients with scrub typhus were investigated further in this study. The mean values for serum vitamin B12, UBBC, and TBBC in the patients (1,936±946.19 pg/ml, 4,167±1,463.27 pg/ml, and 6,140± 2,587.67 pg/ml, respectively) were significantly higher than the 60 control subjects (575±157.32 pg/ml, 1,450± 364.08 pg/ml, and 1974±359.86 pg/ml, respectively) (p<0.001). The mean value of serum transcobalamin II in the patients (2,761±1,069.84 pg/ml) was also significantly higher than the controls (1,083±296.22 pg/ml) (p=0.004). Fourteen out of eighteen patients (78%) had an elevated serum TCII level, over 2,000 pg/ml. The demographic details of these 18 patients with scrub typhus are shown in Table 1. There was a direct association between serum TC II levels and IgM/IgG titres/ total IgM in this study (Fig 1). There was no correlation between serum TCII levels and total IgM levels (p=0.376),or l iver and renal function tests (p>0.117), as shown in Table 2. A correlation was found between IgM titers and total IgM levels in these infected patients (y=138.118 + 0.0602 x, r=0.767, p<0.001 (Fig 2).