Characterization of a phase 1-d epitope on Salmonella typhi flagellin and its role in the serodiagnosis of typhoid fever.

A monoclonal antibody (MAb) directed against Salmonella typhi 52 kDa flagellin protein has been previously produced by our group. In this study, we have demonstrated that the epitope specific to the MAb is unique to phase 1-d. To map the epitope, plasmids encoding different regions of S. typhi flagellin gene were constructed. Analysis of protein produced from each recombinant plasmid indicated that the epitope specific to the MAb resided within amino acids 171-303 (region IV) of S. typhi flagellin protein. The recombinant region IV flagellin was used to develop an ELISA for the detection of IgM antibody to S. typhi in serum. In the hemoculture-positive typhoid group, the developed ELISA was positive in 77 of 92 cases. In patients with non-typhoidal Salmonella, gram-positive and gram-negative bacteria or dengue virus, the ELISA was negative in all 78 cases. Two from 116 healthy control subjects had positive reactions with the assay. The calculated sensitivity, specificity, positive and negative predictive values of the test were 83.7%, 99.0%, 97.5% and 92.8%, respectively. With such high validity together with the requirement of only a single serum specimen and one day for performing the test, the developed ELISA should become a valuable diagnostic test for typhoid fever.

Detection of IgM antibody against region IV flagellin of Salmonella paratyphi A.

Salmonella paratyphi A is a pathogenic bacterium that causes paratyphoid fever. The current laboratory diagnostic techniques are unsatisfactory. To improve diagnosis, a plasmid (pSK-8E) encoding phase 1 flagellin gene nucleotide position 452-890 from S. paratyphi A has been constructed. The recombinant protein expressed from the plasmid has been used to develop an indirect ELISA for IgM antibody detection. Sera from patients with hemoculture positive for S. paratyphi A, S. typhi, other gram-positive and gram-negative bacteria, and dengue hemorrhagic fever as well as from healthy control subjects were tested. Sensitivity, specificity, positive and negative predictive values of the test were 56.9%, 98.8%, 90.6% and 92.1%, respectively. Since the sensitivity was low, the explanation for this result was investigated. It was found that the sensitivity of the test could be increased to 83.3% if the sera were obtained 9-12 days after onset of fever. The sera obtained earlier or later gave only 33.3% and 66.6% sensitivity, respectively. This result suggests that the IgM antibody detection assay which we have developed is a valuable tool for diagnosis of S. paratyphi A infection when the serum samples are taken at the appropriate time.

Metabolic and immune effects of dietary arginine, glutamine and omega-3 fatty acids supplementation in immunocompromised patients.

To evaluate the nutritional, metabolic and immune effects of dietary arginine, glutamine and omega-3 fatty acids (fish oil) supplementation in immunocompromised patients, we performed a prospective study on the effect of immune formula administered to 11 severe trauma patients (average ISS = 24), 10 burn patients (average % TBSA = 48) and 5 cancer patients. Daily calorie and protein administration were based on the patient's severity (Stress factor with the range of 35-50 kcal/kg/day and 1.5-2.5 g/kg/day, respectively) Starting with half concentration liquid immune formula through nasogastric tube by continuous drip at 30 ml/h and increasing to maximum level within 4 days. The additional energy and protein requirement will be given either by parenteral or oral nutritional support. Various nutritional, metabolic, immunologic and clinical parameters were observed on day 0 (baseline), day 3, 7, and 14. Analysis was performed by paired student-t test. Initial mean serum albumin and transferrin showed mild (trauma) to moderate (burn and cancer) degree of malnutrition. Significant improvement of nutritional parameters was seen at day 7 and 14 in trauma and burn patients. Significant increase of total lymphocyte count (day 7, P < 0.01), CD4 + count (day 7, p < 0.01), CD8 + count (day 7, p < 0.0005 & day 14, p < 0.05), complement C3 (day 7, p < 0.005 day 14, p < 0.01), IgG (day 7, and 14, p < 0.0005), IgA (day 7, p < 0.0005 & day 14, p < 0.05), in all patients. C-reactive protein decreased significantly on day 7 (p < 0.0005) and day 14 (p < 0.005). 3 cases of burn wound infection, one case of UTI and one case of sepsis were observed. Two cases of hyperglycemia in burn, 3 cases of hyperbilirubinemia in trauma, 10 cases of elevated LFT (5 trauma/5 burn), and one case of hyponatremia in cancer patients were observed. Two cases of nausea, 4 cases of vomiting, 5 cases of diarrhea (< 3 times/day), 2 cases of abdominal cramp, 1 case of distension were observed. The feeding of IMMUNE FORMULA was well tolerated and significant improvement was observed in nutritional and immunologic parameters as in other immunoenhancing diets. Further clinical trials of prospective double-blind randomized design are necessary to address the so that the necessity of using immunonutrition in critically ill patients will be clarified.

A study of cell-mediated immune response to pancreatic antigens in patients with fibrocalculous pancreatic diabetes.

In order to investigate whether there was any association between autoimmunity to pancreatic antigens with FCPD as well as IDDM, cell-mediated immune response to pancreatic antigens was studied by lymphoproliferation assay in 7 FCPD, 17 IDDM, 33 NIDDM patients and 102 normal controls. Optimal pancreatic antigen concentrations used were 100, 150 and 200 micrograms/ml. Positive results were considered for each concentration of antigens tested, at stimulation index (SI) > (mean +/- 2 SD) SI obtained from normal age-matched controls with the use of the corresponding concentration of antigen. The one who gave positive result with any of these optimal antigen concentrations was considered to be the responder to pancreatic antigens. With this criterion, the responders were found to be 3/7 (42.9%) FCPD, 6/17 (35.3%) IDDM and 6/33 (18.2%) NIDDM patients; while there were 11 of all 102 (10.8%) normal controls.

Opsonophagocytosis and intracellular killing activity of neutrophils in patients with human immunodeficiency virus infection.

In this study, neutrophils isolated from asymptomatic HIV positive individuals, patients with AIDS-related complex (ARC), ARC patients receiving zidovudine (AZT) and full-blown AIDS patients were assayed for their opsonophagocytic and intracellular killing activities. Progressively decreasing opsonophagocytosis of C. albicans by neutrophils correlated with increasing severity of the disease in all groups of HIV infected individuals, as compared to neutrophils isolated from healthy controls. The intracellular killing of C. albicans by neutrophils of asymptomatic and ARC patients did not differ significantly from controls. Neutrophils of ARC patients receiving AZT and AIDS patients showed a slightly decreased killing activity in comparison to that of neutrophils from healthy controls.

Monoclonal antibodies against protein antigens of salmonellae causing paratyphoid fever and their diagnostic application.

Hybrid clones producing monoclonal antibodies (MAbs) specific for Salmonella paratyphi A (72 clones), S. paratyphi B (9 clones) and S. paratyphi C (8 clones) were produced by using the affinity purified Salmonella protein (Bp) as immunogens. MAbs to S. paratyphi A and S. paratyphi B reacted specifically with the 52 kDa homologous flagellin protein components while those to S. paratyphi C reacted with a 61 kDa flagellin protein component. The MAbs against S. paratyphi A and S. paratyphi B were used to establish a double antibody sandwich ELISA for detection of the 52 kDa flagellin antigens in serum samples from patients with acute paratyphoid A and paratyphoid B fever. With this assay system, 6.25 ng per ml of flagellin antigens of S. paratyphi A and S. paratyphi B could be detected. However, the assay system could not detect the flagellin antigens in patients' sera. The presence of IgM antibodies to the 52 kDa antigens of S. paratyphi A and S. paratyphi B in the acute sera from paratyphoid A or paratyphoid B patients suggested that the 52 kDa protein components of both salmonellae are good immunogens for human and might be used as antigens for early diagnosis of paratyphoid A and paratyphoid B fever.

Molecular cloning and expression of Salmonella paratyphi A 52 kDa specific protein gene.

Monoclonal antibodies (MAbs) specific to Salmonella paratyphi A have been established by our group in 1989. These MAbs were proven to be species-specific for 52 kDa protein of S. paratyphi A but the nature of this protein is unknown. However, our group have proved that the 52 kDa protein which is specific to S. typhi was flagellin. This present study has characterized the 52 kDa protein of S. paratyphi A and identified its encoded gene. The plasmid containing the specific 52 kDa antigen gene was cloned from the S. paratyphi A genome, herein designated pSKA-4. Partial nucleotide sequences from this clone was analysed by computer program and found to be phase 1-a flagellin gene of S. paratyphi A. In addition, the nucleotide sequence analysis from such clone also showed that the structural gene for phase 1 flagellin has amino acid sequences conserved at the terminal whereas the central region is variable among Salmonella spp. Therefore, the central portion of flagellin which highly polymorphic in amino acid sequences would be the most specific to S. paratyphi A, thus, should be used as specific antigen for developing specific diagnosis of S. paratyphi A infection. Using the PCR technique, an expression plasmid containing the antigen gene producing only the variable region in the central portion of flagellin from S. paratyphi A, namely pSKA-7, has been established. The recombinant protein produced by the established plasmid has a MW 33.5 kDa as detected by immunoblotting using specific MAbs. Further study by using this specific flagellin protein for immunodiagnosis of S. paratyphi A infection is being carried out in our laboratory.

Fusion protein of Salmonella typhi flagellin as antigen for diagnosis of typhoid fever.

We previously established the specific 52 kDa antigen of Salmonella typhi, detected by our monoclonal antibodies, which was a flagellin protein. Comparison of the nucleotide sequences of phase-1 flagellin of Salmonella species available through GenBank database showed high homology at both ends of the genes with lower degree of homology in the middle portion which contained the antigenically variable regions. Thus, proteins from the central regions of flagellin genes should be species specific and could be used as specific antigens for the immunodiagnostic tests. In this report, recombinant protein derived from the central region of S. typhi flagellin was produced as a fusion protein with glutathione-S-transferase. This fusion protein was used as specific S. typhi antigen for the immunodiagnostic test to detect IgM antibodies in sera using enzyme-linked immunosorbent assay. The sensitivity, specificity, accuracy, positive predictive value and negative predictive value of this test were 53.5, 98.0, 91.5, 82.1 and 92.4%, respectively.

Production of monoclonal antibodies to Vi polysaccharide antigen of Salmonella typhi.

Twenty-four Vi antigen-specific monoclonal antibodies were produced in this study. The MAbs were found to be highly specific to Vi possessing bacteria. Selected MAbs were used in a direct agglutination assay for rapid identification of S. typhi in primary bacterial culture and also used to develop an assay to detect Vi antigen in clinical specimens. The result showed that they could not detect the antigen in urine and serum from acute patients even they could detect as low as 0.02 micrograms/ml of Vi antigen added in normal urine. The study has shown that these MAbs are very useful for rapid identification of S. typhi in primary bacterial culture and they can replace polyclonal anti-Vi antibodies which have been used routinely in bacteriological laboratories.

Molecular cloning and expression of Salmonella typhi flagellin: characterization of 52 kDa specific antigen of S. typhi.

We previously reported monoclonal antibodies (MAbs) specific to S. typhi 52 kDa antigen which do not cross react with related protein antigens from 11 bacteria causing enteric fever and enteric fever-like illness. Using the combination of these specific MAbs and recombinant DNA technology, expression plasmids containing the antigen gene producing substantial amount of the S. typhi protein antigen have been established. Plasmid pSKM-T7 containing the specific 52 kDa antigen gene was cloned and the antigen expressed was detectable by immunoblotting using specific mAbs. The complete nucleotide sequence of this gene was compared with other bacterial sequences and found to be highly homologous with the flagellin gene H1-d of S. muenchen except in the hypervariable region in the central portion. The specific 52 kDa antigen of S. typhi detected by our MAbs is thus a flagellin.

Studies on the 52 kDa antigen of Salmonella typhi: physicochemical stability, purification by affinity chromatography and immunochemical specificity.

The 52 kDa specific protein antigen of Salmonella typhi, as identified by monoclonal antibodies (Ekpo et al. 1990) has been studied with respect to its physicochemical stability, purification by affinity chromatography and immunochemical specificity. It was found that the 52 kDa protein was degraded into smaller antigenic fragments of MW 30-51 kDa when treated with acetone, ethanol, sodium thiocyanate, 0.3M sodium chloride and Veronal and Tris buffers. The exact chemical nature of the degradation of the protein under these conditions is not known but digestion by conventional proteases and dissociation of the non-covalent subunit type have been ruled out. It is proposed that the degradation may be the result of yet unidentified enzyme(s) which become activated by various physical or chemical treatments. Affinity chromatography using a specific monoclonal antibody has been carried out in an attempt to purify the 52 kDa protein. The binding of S. typhi protein to the column was saturable at 65.6 microgram protein/ml gel. The amount of S. typhi protein adsorbed on the column was 0.51% of the total sonicated cell protein. SDS-PAGE of the immunoadsorbent purified protein revealed bands at Mr 15-58 kDa, indicating that the protein obtained had been severely degraded. However, Western blot of the purified protein stained with a specific monoclonal antibody and with rabbit polyclonal antibody against S. typhi showed striking similarity, indicating that the protein obtained was close to immunochemical purity. The 52 kDa protein purified by affinity adsorbent was used as an antigen for the detection of specific IgM in sera of patients. It was shown that sera of patients infected with S. typhi as well as those infected with other bacteria, contained specific IgM against the 52 kDa protein. Thus, it appears that the 52 kDa protein contains species specific as well as cross-reacting epitopes. The possible development of specific diagnosis of S. typhi based on the present experimental results in discussed.

Kinetics of lymphocyte subpopulations in dengue hemorrhagic fever/dengue shock syndrome.

A kinetic study of lymphocyte subpopulations was performed in 61 dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) patients aged 8 months to 12 years and in 59 age-matched normal controls. There were 36 patients in grade 2 and 25 patients in grade 3 of the disease severity. The studies were performed on febrile stage, the day of subsidence of fever or shock stage, 3 subsequent days after subsidence of fever or shock, and once on the recovery stage (approximately 14-18 days after subsidence of fever or shock). The study revealed that the absolute total lymphocytes, CD3+, CD4+, CD8+ and HNK-1+ cells were decreased on febrile stage and their lowest values were noted on the first day of subsidence of fever or shock, while B1+ cells were in the normal range. Thereafter, all lymphocyte subpopulations were increased. The total lymphocytes, B1+ and CD8+ cells were rapidly increased and were above normal value on day 2 after subsidence of fever or shock (early convalescence), then gradually declined to the normal range. In contrast, CD3+, CD4+ and HNK-1+ cells were increased gradually and reached their normal values on day 2 after subsidence of fever or shock. The T4:T8 ratio began to reverse on the day of subsidence of fever or shock, reached its peak on day 2 after shock and returned to normal ratio rapidly thereafter. Thus, the absolute lymphopenia on the day of shock was due to the decrement or T cells (both CD4+ and CD8+ cells) and HNK-1+ cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Natural killer cells in dengue hemorrhagic fever/dengue shock syndrome.

Natural killer (NK) cell activity against K-562 target cells and HNK-1+ cell levels were serially determined in peripheral blood of 62 Thai children with dengue hemorrhagic fever/dengue shock syndrome aged 4-12 years and 59 age-matched normal controls. The studies were performed on febrile stage, 1st and 2nd day of subsidence of fever (shock stage), 3rd and 4th day of subsidence of fever (early convalescent stage) and once again on the late convalescent stage (approximately 14-18 days after subsidence of fever). The study revealed that during the course of disease the NK cell activity was not changed significantly from the normal controls. In contrast, the levels of HNK-1+ cells, which exhibited almost all NK and killer cell functional activities, were significantly decreased in the febrile and the shock stages and were normal in the early and late convalescent stages. The NK cell activity, on the per-cell basis, was significantly increased in the early disease stage when compared to that of the later period of the disease and of the normal controls. The study also revealed that patients with grade III of disease severity exhibited significantly more NK cell functional activities per cell than grade II on febrile stage and the first day of shock. These results suggest that natural killer cells were active in defense against dengue viral infection and might play some role in the pathogenesis of dengue hemorrhagic fever/dengue shock syndrome. Their functions might also determine the severity of the disease.

Clinical experience with typhoid vaccine in Thailand.

A brief report of the typhoid vaccine trials in Thailand is reviewed, and discussed in relation to other clinical and field studies.

Diagnostic value of carcinoembryonic antigen in pleural fluid.

A double antibody sandwich ELISA was used for the determination of carcinoembryonic antigen levels in sera and pleural fluid samples of 25 cancer and 16 tuberculous patients. It was found that while cancer patients had pleural fluid CEA levels significantly higher than those in sera (p < 0.01), this was not true for tuberculous patients. When the CEA level at 20 ng/ml was used as the cut-off value for the diagnosis of malignancies, 8 of 25 (32%) cancer patients could be diagnosed from serum CEA level while 16 of them (64%) could be diagnosed from CEA level in the pleural fluid. In addition, none of tuberculous patients had CEA above the cut-off level. Based on the results of cytologic examination, only 11 of the 25 cancer patients (44%) could be diagnosed. However, if either positive cytologic examination or pleural fluid CEA level higher than 20 ng/ml was used as a criterion for diagnosis, then 20 of them (80%) could be diagnosed.

Detection of Salmonella typhi protein antigen in serum and urine: a value for diagnosis of typhoid fever in an endemic area.

Using haemoculture as the gold standard, a double antibody sandwich ELISA for the detection of Salmonella typhi Barber protein antigen (BP) was compared with the Widal test. Specimens used were serum and urine obtained from normal healthy individuals and from patients with typhoid fever, paratyphoid fever, pyrexia caused by other bacteria and pyrexia with negative haemoculture. The ELISA for antigenuria gave a significantly higher sensitivity, specificity, accuracy and positive predictive value than the Widal test (p less than 0.05). The ELISA for antigenaemia gave a significantly higher sensitivity and positive predictive value only. All other values were not significantly different. The timing of specimen collection was critical for sensitivity in the ELISA for antigenaemia and antigenuria, and the best results could be obtained by carrying out both assays simultaneously. The clearance of BP from serum into urine occurred around 16 days after the onset of fever in one patient. In two patients, BP could be detected in sera up to 3 weeks after the onset of fever. In two patients, serum BP could still be detected although haemoculture was negative.

Gel-filtration chromatography of Salmonella protein antigens and their implication in immunodiagnosis.

Crude Barber protein (Bp) antigens were prepared from Salmonella typhi, S. krefeld and S. derby by an original method that has been described previously. These antigens were subjected to gel-filtration chromatography using Sephadex G-200. A sharp peak that eluted together with the void volume was thus separated from a broad second peak that eluted from the column at positions equivalent to 118,000 to 12,000 daltons. The proteins eluted in the latter peak were arbitrarily divided into 5 fractions and, together with the first peak, subjected to polyacrylamide gel electrophoresis and immunoprecipitation with both homologous and heterologous rabbit antisera. The extent of immunological cross reactivities was determined by enzyme-linked immunosorbent assay. The preliminary results obtained by this technique showed species-specific protein antigens to have molecular weights ranging between 36,000 and 68,000 daltons.