Antibodies to antigen A60 in cerebrospinal fluid from patients with tuberculous meningitis

Cerebrospinal fluid (CSF) anti-mycobacterial antigen 60 (A60) IgM, IgG and IgA in patients affected by meningitis of different etiologies were assayed as a rapid diagnostic test in cases of tuberculous meningitis. A commercial EIA was used to test 127 CSF samples classified as follows: tuberculous meningitis (n=27 CSF samples from 16 patients, 6 of them with AIDS), pyogenic meningitis (n=13), non-tuberculous aseptic meningitis (n=43) and 44 normal CSF samples (16 of them from HIV-positive patients, 8 of whom had extraneurological tuberculosis). Anti-A60 IgM was positive only in two cases (1 tuberculous meningitis and 1 self-resolving aseptic meningitis). Positive CSF anti-A60 IgG and IgA were observed in eight and nine out of 16 patients with tuberculous meningitis, but only in four and five out of 13 samples studied prior to or in the first ten days of treatment, respectively. Most of the patients with false-positive IgG and IgA (16 %) had pyogenic meningitis, but without intrathecal synthesis of antibodies. In patients with aseptic meningitis, the finding of CSF anti-A60 IgG plus IgA, initially or during follow-up, can be used as a diagnostic criterion for tuberculous meningitis, with a specificity of 100 %, a positive predictive value of 1, and a negative predictive value of 0.81. However, its sensitivity is only 50 % in immunocompetent patients and 16 % in patients with AIDS.     

Analysis of tuberculous meningitis cases by an immunoblotting assay based on a mycobacterial antigen complex.

Tuberculous meningitis cases were analyzed by an immunoblotting test based on Mycobacterium bovis BCG antigen complex A60. Anti-A60 immunoglobulin G (IgG) in cerebrospinal fluid (CSF) allowed early diagnosis, and concentrations decreased after recovery. In primary meningitis forms, anti-A60 IgGs were intrathecally synthesized and specific oligoclonal IgGs were present in CSF. In meningeal complications of pulmonary tuberculosis, there were matching titers of anti-A60 IgG in blood and CSF (mirror pattern). Correlation between CSF-restricted patterns and CSF pleocytosis was shown.     

Immunodiagnosis of tuberculous meningitis: detection of antibody reactivity to antigens of Mycobacterium tuberculosis and Cysticercus cellulosae in cerebrospinal fluid tuberculous meningitis patients by ELISA

Enzyme-linked immunosorbent assay (ELISA) was standardized and evaluated for detection of antibody response in cerebrospinal fluid (CSF) to antigens of Mycobacterium tuberculosis and Cysticercus cellulosae. Sonicated extracts of heat killed M. tuberculosis H37Rv and C. cellulosae were prepared and used in ELISA to detect respective antibody response in CSFs for a definitive diagnosis as to tuberculous meningitis (TBM)/neurocysticercosis (NCC). ELISA was performed in a total of 201 CSF samples, which include Group I: chronic infections of the central nervous system (CNS) with possible diagnosis of TBM, tuberculoma, or NCC (n = 70), and Group II: control group of patients with infectious neurological (n = 19), non-infectious neurological (n = 82), and non-infectious non-neurological conditions, i.e., spinal anaesthesia CSFs (n = 30). Specificity in this study was 99.9% and no true cross-reactivity between antimycobacterial antibodies and C. cellulosae antigens and vice-versa was observed. However, in 17.14% of CSFs (12/70), both antimycobacterial and anticysticercal antibodies were detected, 50% of these cases were diagnosed as TBM. But none of the proven NCC cases showed presence of antimycobacterial antibodies. Results of this study would indicate that it would be beneficial if both antibody and antigen responses are detected in CSFs to infectious aetiologies such as M. tuberculosis, C. cellulosae, and C. neoformans in order to enhance the diagnostic accuracy and proper management, as these diseases are highly endemic in underdeveloped and developing countries.     

A study of Mycobacterium tuberculosis antigen and antibody in cerebrospinal fluid and blood in tuberculous meningitis

Radioimmunoassay (RIA) techniques have been evaluated to detect specific tubercular antigen (TB Ag) and antitubercular antibody (TB Ab) in CSF and serum of patients with tuberculous meningitis (TBM). A solid-phase RIA using H37RV sonicate antigen of Mycobacterium tuberculosis, anti-BCG antibody, and staphylococcal protein A was standardized. TB Ag and TB Ab levels were noted to be significantly elevated in cerebrospinal fluid (CSF) as well in circulating immune complexes (CIC) isolated from serum samples of TBM patients as compared to control group (P less than 0.01). Detectability of disease by demonstrating elevated TB Ag and/or TB Ab levels in either CSF or CIC or both was 95%. There was no correlation between individual levels of TB Ag and TB Ab in CSF and in circulation. A follow-up study in patient over a period of 4-12 weeks revealed that TB antigen and/or TB Ab persisted in the majority of the cases for several weeks despite chemotherapy.     

Rapid diagnosis of influenza infection of NP antigen using an immunocapture ELISA test

An immunocapture ELISA test for the diagnosis of human and animal influenza A and/or B is described. A monoclonal anti-nucleoprotein (NP) antibody was used to capture the NP antigen and the captured antigen was detected by an anti-NP polyclonal rabbit antiserum. Compared with the usual diagnostic method by cultivation in embryonated eggs, this test had a high specificity (97%) and sensitivity when used for diagnosis using clinical nasopharyngeal samples obtained from patients and animals. Immunocapture ELISA permitted an easier reading than the indirect immunofluorescence technique. It also permitted diagnosis in frozen samples (-20 degrees C) or in infected LLCMK2 cells mixed with uninfected nasopharyngeal cells and kept at 20 degrees C for one week. This test can be carried out in 3 h.     

Evaluation of ELISA using A60 antigen from Mycobacterium bovis BCG: specific IGG and IGM in mycobacterial infections

The authors have evaluated an ELISA (A60-Tb, Anda biologicals) allowing the detection of specific IgG and IgM against A60 antigen from Mycobacterium bovis BCG during mycobacterial infections. This study included sera from 110 african subjects and from 71 French subjects distributed in 4 clinical groups: 55 tuberculous patients (I), 41 leprous patients (II), 33 pneumopathies (III) and 52 healthy subjects (IV). Serological results were compared taking as reference for the diagnosis of tuberculosis the positivity of culture and/or that of a direct examination, and for leprosy the positivity of a direct examination associated either with a Mitsuda's reaction or with an histopathological examination. IgG were found to be more discriminative than IgM. Considering together the results of groups I and II, the authors found a sensitivity of 95.8 p. cent and a specificity of 75.3 p. cent with threshold of 200 U/ml for specific IgG. Anti-A60 antigen antibodies obtained for groups I and II were significantly higher (IgG: p less than 0.0001; IgM: p less than 0.001) than those observed in other groups. African subjects presented IgG titers higher than those obtained by French subjects (p less than 0.0001). IgM response was more frequent among group II (97.6 p. cent) than group I (21.8 p. cent). However, IgG (26.9 p. cent) and IgM titers (30.8 p. cent) were detected among group IV. This test would allow a control of therapeutic efficacy with an additional interest for classifying borderline forms of leprosy.     

Demonstration of components of antigen 85 complex in cerebrospinal fluid of tuberculous meningitis patients

Tuberculous meningitis (TBM) is the most common form of chronic infection of the central nervous system. Despite the magnitude of the problem, the general diagnostic outlook is discouraging. Specifically, there is no generally accepted early confirmative diagnosis protocol available for TBM. Various Mycobacterium tuberculosis antigens are now recognized as potential markers for diagnosis of TBM. However, their presence remains questionable, and many of these antigens are reported in the blood but not in the cerebrospinal fluid (CSF). This study identifies a specific protein marker in CSF which will be useful in early diagnosis of TBM. We have demonstrated the presence of a 30-kDa protein band in CSF of 100% (n = 5) of confirmed and 90% (n = 138) of suspected TBM patients out of 153 TBM patients. The 30-kDa band was excised from the gel, destained extensively, and digested with trypsin. The resulting peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Partially purified proteins from CSF samples of TBM were analyzed by two-dimensional polyacrylamide gel electrophoresis and Western blotting. Immunoblotting and enzyme-linked immunosorbent assay (ELISA) were performed to confirm the presence of proteins in the 30-kDa protein band. The antigen 85 (Ag 85) complex was detected in CSF of TBM patients by indirect ELISA using antibodies against Ag 85 complex. The results of this study showed the 30-kDa protein band contained MTB proteins Rv3804c (Ag85A) and Rv1886c (Ag 85B), both members of the Ag85 complex. This was also confirmed by using immunotechniques such as indirect ELISA and the dot immunobinding assay. Detection of Ag85 complex was observed in CSF of 89% (71 out of 80) of suspected TBM patients that were 30-kDa protein positive. The observed 30-kDa protein in the CSF is comprised of the MTB Ag85 complex. This protein was earlier reported to be present in the blood of patients with extra-central nervous system tuberculosis. Therefore, this finding suggests that this protein can be used as a molecular marker for any type of tuberculous infection. It also provides a more sensitive immunoassay option for the early and confirmatory diagnosis of TBM.     

Sensitivity and specificity of enzyme-linked immunosorbent assay in the detection of antigen in tuberculous meningitis cerebrospinal fluids.

A sandwich enzyme-linked immunosorbent assay was developed for its potential utility in the detection of antigen in the cerebrospinal fluid of patients with tuberculous meningitis. Cerebrospinal fluids examined included those from untreated (group Ia) and treated (group Ib) Mycobacterium tuberculosis meningitis, nonseptic central nervous conditions (group II) such as epilepsy, viral meningitis, and tetany, and nonmycobacterial septic meningitis (group III). The average levels of antigens determined and percent positive specimens, respectively, for each group were (group): Ia, 1.8 micrograms/ml and 75% positive; Ib, 0.37 microgram/ml and 36% positive; II, 0.036 microgram/ml and 100% negative; and III, 0.075 microgram/ml and 100% negative. The system developed employed hyperimmune polyclonal antibody raised against M. tuberculosis and Mycobacterium bovis BCG in burros and rabbits. Cross-reactivity by other mycobacterial species was very low; e.g., 5% for M. kansasii and less than 2% for M. intracellulare, M. avium, M. vaccae, and M. fortuitum. The test shows promise as a specific adjunct for the early diagnosis of tuberculous meningitis.     

Melanosis of the appendix: common in the paediatric age group

AIMS: The objective of this study was to assess apoptotic activity in gestational trophoblastic disease (GTD) and its prognostic value in hydatidiform mole (HM). METHODS AND RESULTS: Expression of the specific caspase cleavage site within cytokeratin 18 was assessed immunohistochemically using the monoclonal antibody M30 CytoDeath in 12 spontaneous abortions, 22 partial and 57 complete HM, eight choriocarcinoma (CCA) and 28 normal placentas. The M30 immunoreactivity occurred predominantly in the syncytiotrophoblasts. A significantly higher M30 index in HM and CCA was found when compared with normal placentas and spontaneous abortions (P < 0.001). The M30 index of those HM which spontaneously regressed was significantly higher than those HM which developed persistent disease requiring chemotherapy (P < 0.001). The M30 index correlated with another apoptotic index previously detected by TdT-mediated dUTP nick-end labelling (TUNEL) (P = 0.007) and the proliferation index assessed by the Ki67 antigen (P = 0.034). CONCLUSIONS: We conclude that apoptosis is important in the pathogenesis of GTD. Assessment of apoptotic activity in HM by the M30 index may be considered as an alternative prognostic indicator for predicting the clinical behaviour.     

Detection of mycobacterial antigen and antibodies in the cerebrospinal fluid of patients with tuberculous meningitis

An immunodiagnostic test for the detection of a soluble nonprotein mycobacterial antigen by reverse passive haemagglutination with IgM murine monoclonal antibody was developed. The test was used to analyse the cerebrospinal fluid of 89 patients with tuberculous meningitis (TBM) from India and 127 control subjects from India and the UK. The antigen was demonstrable in 88% of culture-positive and 73% of culture-negative TBM patients. However, it was also detected in 21% of Indian patients with pyogenic meningitis, and in 8% of Indian and 1% of UK control subjects. Antibodies binding to a soluble mycobacterial extract were detected at low titre in 68% of all subjects with TBM and in 37% of Indian cases of pyogenic meningitis. Because patients with TBM had raised levels of the antigen and of antibodies to the antigen, the possible role of immune complexes in the pathogenesis of the disease is briefly discussed.     

Comparison of slide coagglutination test and countercurrent immunoelectrophoresis for detection of group B streptococcal antigen in cerebrospinal fluid from infants with meningitis.

The usefulness of Phadebact streptococcus reagents for the detection of group B streptococcal antigen in cerebrospinal fluid was evaluated in 54 infants with meningitis and in 22 normal infants. Antigens was detected by slide coagglutination in 19 (82.6%) and by countercurrent immunoelectrophoresis in 20 (87.0%) of 23 cerebrospinal fluid specimens from infants with group B streptococcal meningitis at admission. After initiation of antimicrobial therapy, antigen could be detected in 11 of 19 (by slide coagglutination) and 7 of 18 (by countercurrent immunoelectrophoresis) cerebrospinal fluids. False-positive reactions were noted by slide coagglutination in one infant with S. bovis meningitis and one with group B streptococcal bacteremia without meningitis; none occurred with countercurrent immunoelectrophoresis. The commercial availiability, simplicity, sensitivity (82.6%), and specificity (96.4%) of the Phadebact slide coaggluatination test for detecting group B streptococcal antigen in cerebrospinal fluid suggest that it may be useful for the early and rapid diagnosis of group B streptococcal meningitis.     

Variable localization of blood group antigen in group A kidneys.

The distribution of the blood group A antigen has been examined in 7 Group A kidneys using an indirect immunoperoxidase technique. Monoclonal antibody consistently demonstrated A antigen on the endothelium of all kidneys, particularly peritubular capillary endothelial cells and also the epithelial cells of distal tubules of all but 1 case. Nine hyperimmune anti-A alloantisera gave a variable pattern of staining on different endothelial cells, but no kidney was negative. Epithelial cell staining showed considerable variation both within an individual cell and between adjacent cells. Twenty-six out of 40 alloantisera from normal Group O blood donors failed to bind to endothelial cells of one kidney which was known to show strong expression of A antigen. Absorption was completely achieved using Group A red blood cells but not with a synthetic blood group substance. The variation in reaction intensity using different antisera emphasises that there is variation in antigen expression between cells and indicates the complexity of antibodies directed against the blood group A antigen.     

Diagnostic strategy in suspected duodenal ulcer disease. A questionnaire study

In a questionnaire study of 89 Danish gastroenterologists the current diagnostic strategy in patients suspected of having duodenal ulcer disease was elucidated. A case summary concerning a patient with upper abdominal pain was presented. It was assumed that the patient had had a double-contrast barium meal examination or an upper gastrointestinal endoscopy performed. If the X-ray had revealed a deformity of the duodenal bulb, 30% of the gastroenterologists would offer the patient specific medical treatment (H2-blocking agent etc.), but a significantly higher number of gastroenterologists, 45%, (p less than 0.05) would offer specific medical treatment if a deformity was revealed at endoscopy. There was also a significant difference (p less than 0.01) between those who would offer specific treatment if X-ray (84%) or if endoscopy (100%) had revealed an ulcer. Considerable variation was found among experts in their decisions on the basis of X-ray and endoscopy in patients with suspected duodenal ulcer disease. Gastroenterologists generally rely more on endoscopic than on radiographic findings.     

Diagnostic accuracy of droplet digital PCR analysis of cerebrospinal fluid for tuberculous meningitis in adult patients

ObjectivesTuberculous meningitis (TBM) is difficult to diagnose. Digital PCR (dPCR) is a novel method which can quantify trace nucleic acids. This study sought to evaluate the diagnostic accuracy of dPCR analysis of cerebrospinal fluid (CSF) for TBM.MethodsWe collected CSF specimens from hospitalized TBM and non-TBM patients. Total CSF DNA was purified and the concentrations of Mycobacterium tuberculosis insert sequence 6110 (IS6110) and gyrase subunit B (gyrB) were quantified using droplet dPCR. The receiver operating characteristic curves of dPCR were established and the diagnostic performances were obtained. We also compared the sensitivity of dPCR with routine diagnostic tests.ResultsA total of 101 patients were recruited, 68 of whom suffered from TBM (26 definite, 34 probable and eight possible TBM) and 33 from non-TBM. The sensitivity of IS6110-dPCR assay for total TBM was higher than that of gyrB-dPCR assay (57.4% (44.8–69.3%) vs. 22.1% (12.9–33.8%)), and there was no significant difference for specificity between them (97.0% (84.2–99.9%) vs. 100% (89.4–100.0%)). The sensitivity of IS6110-dPCR in definite TBM was higher than that in probable and possible TBM (73.1% vs. 52.9% and 25.0%, respectively). IS6110-dPCR assay showed a higher sensitivity than smear microscopy (53.3% vs. 6.7%), mycobacterial culture (50.0% vs. 12.5%), IS6110-quantitative PCR (53.1% vs. 21.9%) and Xpert MTB/RIF (70.4% vs. 29.6%). Long anti-tuberculosis treatment time was found to be significantly associated with negative dPCR results.ConclusionCSF IS6110-dPCR assay is a rapid and sensitive molecular test, which has the potential to be used to enhance the diagnosis of TBM.     

Alpha-interferon responses in cerebrospinal fluid of patients with suspected meningitis.

Cerebrospinal fluid from 100 patients with clinically diagnosed meningitis was examined for alpha-interferon. In the laboratory four patient groups were identified: bacterial meningitis (n = 12), viral meningitis (n = 15), normal cerebrospinal fluid (n = 57) and abnormal cerebrospinal fluid (n = 16). A further 14 patients with cerebrospinal fluid shunts but no abnormality in the cerebrospinal fluid provided a control group for alpha-interferon determinations. The group with viral meningitis and the group with abnormal cerebrospinal fluid had significantly higher alpha-interferon concentrations (p less than 0.001) when compared with those of the three other groups. This assay had great predictive value in determining those patients with abnormal cerebrospinal fluid who did not have a bacterial cause of meningitis. As the groups with abnormal cerebrospinal fluid and viral meningitis had a similar spread in alpha-interferon values it is likely that both reflect viral infection of the central nervous system.     

Diagnosis of tuberculous meningitis by enzyme-linked immunosorbent assay to detect mycobacterial antigen and antibody in cerebrospinal fluid

Enzymed-linked immunosorbent assay (ELISA) was used to detect mycobacterial antigen and antimycobacterial antibody in cerebrospinal fluid (CSF) specimens of 50 patients with tuberculous meningitis (TBM) and 50 patients with non-tuberculous neurological diseases (control group). The assay gave no false negative results in 10 culture-positive patients with TBM. Detection of mycobacterial antigen in CSF is more sensitive and specific for the diagnosis of TBM than detection of antibody. ELISA should be considered as one of the alternative methods in the laboratory diagnosis of TBM, particularly in culturenegative patients with TBM.