Serodiagnosis and Immunotherapy in Infectious Disease最新文献

An immunodominant glycoprotein antigen (45 kD) from Aspergillus fumigatus was purified and characterized. The purified antigen exhibited strong precipitin reaction with the sera of allergic bronchopulmonary aspergillosis (ABPA) patients. Enzyme-linked immunosorbent assay (ELISA) and Western blot results revealed strong binding of gp 45 with IgG and IgE antibodies of ABPA patients. Three monoclonals of IgM isotype, raised against a glycoprotein fraction of A. fumigatus, recognized 45 and 55 kD antigens. These results suggest the presence of common epitopes in these antigens. The protein to carbohydrate ratio of purified antigen was observed to be 1.4: 1.0. Further analysis by gas liquid chromatography revealed the presence of glucose, mannose and glucosamine residues in a 4 : 3 : 1 ratio. Deglycosylation of N-linked sugars of gp 45 with N-glycosidase-F resulted in a 27 kD band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Neuraminidase treatment destroyed the IgE binding activity of the antigen but IgG binding activity was retained. The IgG binding activity on the other hand was lost on treatment with pronase. The major IgG binding epitope of this glycoprotein antigen may therefore be in the protein part of the antigen. The purified 45 kD antigen displayed protease activity at alkaline pH. It was inhibited by PMSF and ethylene diamine tetraacetic acid (EDTA). Proteolytic activity of the antigen may contribute significantly to pathogenesis by destroying the elastin present in the lung tissue.

A rapid IgM immunoblotting serological test was compared to a polymerase chain reaction (PCR) assay of respiratory specimens for the diagnosis of acute Mycoplasma pneumoniae infection. Among 112 paired specimens, the frequency of a positive diagnosis by any method was 7.1%. Both IgM serology and PCR were positive for only two out of eight infected patients. PCR positive, IgM negative patients (4) were ill for an insufficient period to allow the IgM response to be demonstrable (⩽7 days). PCR negative, IgM positive patients (2) were likely to have had negative amplification assays because of the nature of the respiratory specimens. Nasopharyngeal washings uncommonly inhibited PCR amplification. Both rapid serological and genetic amplification assays have a role, together or alone, in the diagnosis of M. pneumoniae infection and paradigms for cost-effective utilization will be required.

Monoclonal antibodies directed against group specific antigen A of Vibio cholerae 01 were obtained through hybridoma technology which involved fusions of Sp 2/0 myeloma cells with splenocytes of Balb/c mice immunized with whole cell lysates of V. cholerae 01. Specificity and crossreactivity of the monoclonal antibodies were determined against whole cell lysates prepared from 38 strains of V. cholerae 01, six strains of V. cholerae non-01, five strains of other vibrios, 45 strains of enterobacteria and Entamoeba histolytica by indirect and dot-blot enzyme-linked immunosorbent assay (ELISA). The monoclonal antibodies (MAb) reacted specifically to the whole cell lysates of the V. cholerae serogroup 01 and did not react to the antigens prepared from other organisms. The MAb were used in a dot-blot ELISA for detecting V. cholerae 01 antigen in 335 seafood specimens and in stools and rectal swabs of patients with acute watery diarrhoea. The dotblot ELISA performed on rectal swab specimens enriched in alkaline peptone water gave 96.0% sensitivity, 99.3% specificity, 94.7% positive predictive value, 99.5% negative predictive value and 98.9% efficacy, respectively, when compared to the conventional culture method. The two methods showed excellent agreement beyond chance, on the basis of a k coefficient value of 94.7%. No V. cholerae 01 was isolated from the 335 seafood samples and the dot-blot ELISA for V. cholerae 01 antigen was also negative, consistent with a high specificity of the assay. The dot-blot ELISA is easy to perform, relatively inexpensive, highly sensitive and specific. It permits multisample analysis at a single time, requires no special equipment and does not pose any disposal problem (compared with the culture method). Most of all, diagnosis of cholera cases could be made accurately within 1–3 h (the dot-blot ELISA takes 1 h, while the culture method takes 2 days). The method is recommended for rapid detection of V. cholerae 01 in contaminated foods, in environmental samples and in stools of diarrhoeic patients.

We studied the anti-cryptococcal IgG antibody response in patients with the acquired immunodeficiency syndrome (AIDS) after Cryptococcus neoformans infection. The reactivity of 64 sera from 16 AIDS patients with cryptococcosis and 70 from noninfected HIV-negative individuals was measured against purified capsular polysaccharide by enzyme-linked immunosorbent assay (ELISA) or whole cryptococci by flux cytometry (FACS). AIDS patients had higher titres of antibodies by ELISA than controls. FACS analysis using the infecting isolate suggested antibody production was lower when the infecting isolate was serotype D. Lack of correlation between ELISA and FACS suggest antibodies produced during infection have a different specificity than that of normals. We did not find a correlation between antibody levels and clinical course. The lack of correlation between antibody and antigen titres even after pretreatment with pronase showed the proteinaceous substance unmasked by pronase is not composed only of specific antibodies.

Genital ulcer disease (GUD) is a well documented risk factor for heterosexual transmission of the human immunodeficiency virus (HIV). In Africa, chancroid is the major GUD. The epidemiology for chancroid and Haemophilus ducreyi infection is still poorly understood, mainly because diagnostic tests for chancroid and H. ducreyi infection are not well established. Yet, culture of H. ducreyi remains the method of choice for confirming clinical diagnosis inspite of an unsatisfactory isolation rate. Various non-culture tests for detection of H. ducreyi were developed. As for other diseases, the polymerase chain reaction (PCR) test is proving its usefulness for diagnosing chancroid. However, sample handling should be optimized and criteria allowing the classification of culture negative/PCR positive patients should be established. Several simple and inexpensive diagnostic tests for use in low-resource settings are in progress. Direct microscopy of Gram-stained clinical specimens is an obvious candidate, but variations in sensitivity still limit the usefulness of this test for confirmation of clinical diagnosis. An enzyme immunoassay using a specific polyclonal antiserum, and immunofluorescence microscopy using a specific monoclonal antibody may be useful for the detection of H. ducreyi antigen. As for PCR, criteria should be established to classify patients with discordant test results. Yet, serological tests are useful only for epidemiology. Specific and sensitive antigens were identified, but their usefulness in diagnostic tests remains to be established.

Two hundred faecal specimens submitted for Clostridium difficile testing were examined for: presence of C. difficile by culture, cytotoxin activity in cell tissue culture, detection of toxin A by Premier enzyme immunoassay (EIA), detection of toxins A and B by Cytoclone EIA, and for the presence of gene sequences encoding toxins A and B by the polymerase chain reaction (PCR). Sixty-three (31.5%) were positive in one or more tests of which 33 (16.5%) were positive in all tests. Sensitivities and specificities were respectively: culture 97%, 94.5%; cytotoxin assay 94.5%, 97%; Premier 100%, 97.5%; Cytoclone 100%, 97.5% and PCR of toxin genes A and B (which gave identical results) 97% and 96.5%. EIA gave the best results achievable within one day. Culture failed to distinguish toxigenic from non-toxigenic strains, while PCR failed to prove if toxigenic strains were producing toxin in vivo.

A commercial latex agglutination test (Diarlex, Orion Diagnostics, Finland) for detecting group A rotavirus (RV) and adenovirus (AdV) in faeces was evaluated. Sixty-six faeces, of which 58 were RV or AdV positive, were examined: group A RV, 23; non-group A RV, 2; AdV of subgroup F (AdF), types 40 and 41, 22; non-F AdV, 9; RV and Ad41, 2. Eight samples were RV and AdV negative. Twenty-three (92%) of the 25 specimens containing group A RV were positive by Diarlex but none of the 41 negative ones. Of the 33 AdV positive (all types) samples, 25 (76%) were also Diarlex positive. However of the 24 containing AdF strains 23 (96%) were positive compared with only two of the nine containing non-F strains. Twenty-nine of the 33 AdV negative faeces gave no reaction by Diarlex but of the remaining four, two gave false positive and two non-specific reactions. The two samples containing RV and Ad41 were Diarlex positive for both viruses. Although the rotavirus component of the test performed better than that for detecting AdV, it is concluded that Diarlex is a user-friendly test with a possible role in the routine diagnosis of viral gastroenteritis.