Correlation between culture of Mycobacterium tuberculosis and IgG antibody to Mycobacterium tuberculosis antigen-5 in the cerebrospinal fluid of patients with tuberculous meningitis

A retrospective study was made of the correlation between culture of Mycobacterium tuberculosis and detection of IgG antibody to M. tuberculosis antigen-5 in cerebrospinal fluid (CSF) by means of an enzyme linked immunosorbent assay (ELISA). Mycobacterium tuberculosis was cultured from the CSF in 14 of 70 patients with a clinical diagnosis of tuberculous meningitis (TBM). IgG antibody to M. tuberculosis antigen-5 was demonstrated in significant titres (80-640) in all 14 culture-positive patients. Thus, positive correlation was observed between culture of M. tuberculosis and detection of IgG antibody in the CSF. As a result of this observation, the CSF from 56 culture-negative patients with a clinical diagnosis TBM was specifically investigated for the detection of IgG antibody to M. tuberculosis antigen-5 and the findings were correlated with those of culture-positive patients. The assay was positive in 34 of 56 patients, the antibody titre ranging between 80 and 640. In the CSF of 70 patients with non-tuberculous neurological diseases, the assay was negative at a dilution of 1 in 80. Thus, detection of IgG antibody to M. tuberculosis antigen-5 by indirect ELISA carried 100% specificity and 60.7% sensitivity for a tuberculous aetiology in culture-negative patients with TBM. The results of this study suggest that indirect ELISA for IgG antibody to M. tuberculosis antigen-5 in CSF holds definite promise in diagnosis of TBM, particularly when repeated cultures of CSF are negative for M. tuberculosis.     

Interpretation of mycobacterial antibodies in the cerebrospinal fluid of adults with tuberculous meningitis

Objective Microbiological identification of Mycobacterium tuberculosis is insensitive and slow, and clinical distinction of tuberculous meningitis (TBM) from other subacute or chronic meningoenchephalitides (SACM) is difficult. Successful use of highly specific M. tuberculosis serological assays on cerebrospinal fluid has been reported, but their performance for diagnosis in a tuberculosis endemic country where they would be of most value is unclear. We sought to determine the biological basis for the uncertainty in interpretation of antibody detection in the CSF of TBM patients. Methods We identified prospectively 46 adults with SACM and explored the concordance between TBM diagnosis and detection of highly specific M. tuberculosis antibodies in CSF. The source of antibodies in CSF was explored by evaluating the correlation between antibody titres in CSF with those in serum, or with the albumin quotient. Intrathecal IgG synthesis was assessed by the IgG index. Results Positive antibody titres were more frequent among TBM patients (76%), but were also present in individuals with other SACM (59%). A positive correlation between antibody titres in CSF with those in serum, or with the albumin quotient, supported the leakage of antibodies from plasma to CSF through an increased blood–brain barrier permeability. Intrathecal IgG synthesis was only detected in 35% of the TBM cases. Conclusion Plasma antibodies likely synthesized in response to previous tuberculosis infections were a major source of mycobacterial antibodies in CSF due to leakage through an impaired blood–brain barrier. Interpretation of mycobacterial antibodies in CSF of adults for TBM, however specific, must take into account the contribution of antibodies from plasma, and hence, has questionable use for diagnosis.     

Antibody responses to the 18-kDa protein of Mycobacterium leprae in leprosy and tuberculosis patients.

The 18-kDa protein of Mycobacterium leprae, as recognized by the monoclonal antibody L5, has a restricted species distribution, being confined to M. leprae and M. habana. We have developed a solid-phase ELISA using purified, recombinant M. leprae 18-kDa protein and compared the serological responses of Nepali leprosy and tuberculosis patients and endemic control subjects to the protein and the M. leprae phenolic glycolipid-I (PGL-I). Few control subjects had anti-18-kDa antibodies. A small proportion of paucibacillary (PB) leprosy and 42% of multibacillary (MB) leprosy patients had IgG anti-M. leprae antibodies. A similar proportion (47%) of Nepali tuberculosis (TB) patients were seropositive, and IgG anti-18-kDa antibody levels were significantly higher in MB and TB patients than in control subjects. By comparison, IgM anti-PGL-I antibodies were detected in 88% of MB leprosy patients and only 7% of TB patients. The possible reasons for the 18-kDa protein seroreactivity in TB patients are discussed, and the anti-18-kDa assay is compared with other antibody assays for protein and nonprotein antigens of M. leprae. It is concluded that the sensitivity and specificity of the anti-M. leprae 18-kDa ELISA are insufficient for the assay to be of clinical utility in leprosy patients.     

抗毒蕈碱受体3多肽抗体测定在干燥综合征诊断中的意义

目的 建立酶联免疫吸附试验(ELISA)法检测抗毒蕈碱3受体多肽(M3RP)抗体的方法,探讨该抗体在干燥综合征(ss)诊断中的意义.方法 以固相合成的多肽M3RP为包被抗原,ELISA定量检测94例干燥综合征,160例其他结缔组织病及200名正常人血清中的抗M3RP抗体水平,并分析其与SS临床表现的相关性.结果 抗M3RP抗体在原发干燥综合征(pSS)、继发干燥综合征(sSS)患者中阳性率分别为84.6%和81.3%,在其他结缔组织病和正常对照组中的阳性率分别为8.8%和1%.M3RP抗体在SS患者中阳性率显著高于其他结缔组织病患者及正常对照组(P＜O.01).而且,M3RP抗体在pSS和sSS中的特异性均为91.3%.pSS患者与sSS患者抗M3RP抗体阳性率差异无统计学意义.抗M3RP抗体与SS患者临床表现和脏器受累情况无明显相关性.该抗体在抗α-胞衬蛋白、抗SSA、抗SSB和抗核抗体阴性的SS患者中抗M3RP抗体的阻性率分别为85%、89.3%、88.9%和95.2%.在pSS中,抗M3RP抗体阳性的患者血清中IgG水平明显高于抗体阴性的患者.结论 ①ELISA法可检测pSS和sSS患者血清中的抗M3RP抗体,该抗体为诊断SS的较为特异的自身抗体之一.②抗M3RP抗体对其他自身抗体阴性的SS诊断有参考意义.     

Correlation between culture of Mycobacterium tuberculosis and IgG antibody to Mycobacterium tuberculosis antigen-5 in the cerebrospinal fluid of patients with tuberculous meningitis

A retrospective study was made of the correlation between culture of Mycobacterium tuberculosis and detection of IgG antibody to M. tuberculosis antigen-5 in cerebrospinal fluid (CSF) by means of an enzyme linked immunosorbent assay (ELISA). Mycobacterium tuberculosis was cultured from the CSF in 14 of 70 patients with a clinical diagnosis of tuberculous meningitis (TBM). IgG antibody to M. tuberculosis antigen-5 was demonstrated in significant titres (80–640) in all 14 culture-positive patients. Thus, positive correlation was observed between culture of M. tuberculosis and detection of IgG antibody in the CSF. As a result of this observation, the CSF from 56 culture-negative patients with a clinical diagnosis TBM was specifically investigated for the detection of IgG antibody to M. tuberculosis antigen-5 and the findings were correlated with those of culture-positive patients. The assay was positive in 34 of 56 patients, the antibody titre ranging between 80 and 640. In the CSF of 70 patients with non-tuberculous neurological diseases, the assay was negative at a dilution of 1 in 80. Thus, detection of IgG antibody to M. tuberculosis antigen-5 by indirect ELISA carried 100% specificity and 60·7 % sensitivity for a tuberculous aetiology in culture-negative patients with TBM. The results of this study suggest that indirect ELISA for IgG antibody to M. tuberculosis antigen-5 in CSF holds definite promise in diagnosis of TBM, particularly when repeated cultures of CSF are negative for M. tuberculosis.     

Efficiency of polymerase chain reaction for the diagnosis of tuberculous meningitis

The early diagnosis of tuberculous meningitis (TBM) is very important. In this study, the efficiency of the polymerase chain reaction (PCR), one of the most reliable and sensitive DNA-based assays, was compared with conventional methods (acid-fast microscopy and culture) for the detection of M. tuberculosis in cerebrospinal fluid(CSF) specimens from patients suspected of TBM. Of the 29 CSF specimens from highly-probable TBM patients (based on clinical features), 25 were positive by PCR (86.2%), whereas only one of 29 was acid-fast microscopy (AFM) positive (3.4%), and 5 out of 29 were culture-positive (17.2%). No positive results were found by AFM, culture or PCR in the non-tuberculous control group. The results of this study indicate that the application of PCR should be extremely useful in the diagnosis of TBM.     

Rapid molecular diagnosis of tuberculous meningitis using the Gen-probe Amplified Mycobacterium Tuberculosis direct test in a large Canadian public health laboratory.

OBJECTIVE: A 5-year retrospective study of the performance of the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (MTD) for detecting Mycobacterium tuberculosis complex in cerebrospinal fluid (CSF). Patient data from culture-confirmed cases of tuberculous meningitis (TBM) were also analysed. RESULTS: In total, 311 CSF specimens were tested by the MTD, of which 17 were positive. When compared with culture (gold standard), the sensitivity and specificity of the MTD test were 93.8% and 99.3%, respectively. The positive and negative predictive values for TBM were 88.2%, and 99.7%. Clinical and epidemiological information was requested for all culture-positive TBM patients. These data were used to assess the mortality rate (55.6%) and to determine common factors that could be applied as selection criteria for the appropriate testing of CSF by MTD. CONCLUSION: The study found the MTD test to be a rapid, sensitive and specific test for TBM. A history of immigration from an area endemic for tuberculosis (TB), a history of TB, symptoms of neurological deficits and the results of CSF analyses could be used to appropriately select CSF for MTD testing in order to provide a critical early diagnosis of TBM.     

Serodiagnosis of tuberculosis by radioimmunoassay

Mycobacteria antigens derived from whole cells and cell walls of M. tuberculosis and M. bovis (BCG) and soluble purified protein derivative (PPD) prepared from M. tuberculosis were used in solid phase radioimmunoassays to measure the amount of reactive IgG antibody in serums from 54 patients with active (culture-positive) tuberculosis (Group I), 6 patients with inactive (culture-negative) tuberculosis (Group II), 15 healthy subjects who were skin test positive to PPD (Group III), and 30 healthy persons who were PPD skin test negative (Group IV). Patients with active tuberculosis had statistically larger (p less than 0.001) amounts of IgG antibody to M. tuberculosis whole cells, cell walls, and PPD and to BCG whole cells and cell walls when compared with the amount of antibody in serums from healthy subjects who were PPD skin test negative. However, no significant differences were detected in the mean antibody response or frequency of positive antibody responses between patients with active disease and those in clinical remission. Moreover, significant amounts of antibody were detected in 7 to 20% of healthy, tuberculin-reactive subjects. On the basis of these results, it is unlikely that antibody assays alone will prove useful in the diagnosis of this disease.     

二种抗原检测结核分枝杆菌抗体的临床意义评价

目的　评价两种抗原检测结核分枝杆菌抗体对结核病诊断的意义。方法　以结核分枝杆菌多肽 (TB4 )和脂阿拉伯甘露糖 (LAM )为抗原 ,斑点金免疫渗滤法为检测方法对 78例临床确诊为肺TB的病人血清、15例气管炎患者血清和 5 0例正常人血清进行抗体测定 ,观察这些抗原诊断结核病的敏感性、特异性。结果　两种抗原检测结核抗体的敏感性分别为84 6 % (6 6 / 78)和 73 1% (5 7/ 78) ;特异性分别达 92 3% (6 0 / 6 5 )和 95 4 % (6 2 / 6 5 ) ,与痰涂片的符合率为 84 8%和 81 8%。各指标比较 ,经 χ2 检验表明 ,差异无显著性意义 (P >0 0 5 )。结论　提示TB4是一种敏感性高 ,特异性强的抗原 ,可用于临床查病和血清流行病学调查。     

Detection and its clinical significance of IgM antibody in cerebrospinal fluid in patients with tuberculous meningitis

Specific antibody for IgM in cerebrospinal fluid (CSF) of 45 patients with tuberculous meningitis (TBM), 33 patients with non-TBM and 51 control patients was determined by enzyme-linked immunosorbent assay, and compared with specific antibody for IgG. The sensitivity and specificity was 71.1% and 98.8% for IgM antibody, and 88.9% and 96.4% for IgG antibody respectively, but the positive rate of IgM antibody at early stage of TBM was higher than that of IgG antibody. There was a highly significant positive correlation between positive rates of the two types of antibody and contents of protein in CSF. The sensitivity and specificity was 97.8% and 98.8% respectively in detecting both the IgM and IgG antibodies in CSF simultaneously, which can be used as a supplementary method for the diagnosis of TBM.     

结核性脑膜炎患者脑脊液中腺苷脱氨酶和结核抗体水平研究

目的探讨结核性脑膜炎(TBM)的早期诊断方法。方法采用氨的偶联酶测定法和酶联免疫吸附实验(ELISA)检测3组病例(结脑组51例,病脑组30例,非脑膜炎组20例)脑脊液(CSF)中腺苷脱氨酶(ADA)的活性水平和脑脊液结核抗体(PPD-IgG)的阳性率。结果研究发现结脑组脑脊液中腺苷脱氨酶活性水平明显升高,与病脑组和非脑膜炎组比较有显著性差异(p<0.01)。而病脑组与非脑膜炎组比较无显著性差异(p>0.05);结核性脑膜炎早期脑脊液中结核抗体的阳性率为80.4%(其中2周内PPD-IgG阳性率为80.4%,1周内为23.5%,其他为3.9%),在随后的临床跟踪检测中为100%。结论说明共同检测脑脊液中腺苷脱氨酶的活性明显升高和结核抗体阳性可以作为结脑早期诊断的重要依据。     

Antigens, antibodies and immune complexes in cerebrospinal fluid of patients with cerebral gnathostomiasis

Methods for the detection of antigens, antibodies and immune complexes in the cerebrospinal fluid (CSF) of patients with neurological manifestations suggestive of cerebral gnathostomiasis were developed, in the hope that they may be useful in confirming the diagnosis of Gnathostoma spinigerum infection. Gnathostoma antigens were determined by a sandwich enzyme linked immunosorbent assay (ELISA) using antibodies from rabbits immunized with the excretory/secretory (ES) antigens obtained from the in vitro supernatant fluid in which the third-stage G. spinigerum larvae were maintained. With a biotin streptavidin procedure, the presence of G. spinigerum antigens as low as 2 ng in one ml of CSF could be detected. An indirect ELISA was used for the quantitation of IgG antibodies in the paired serum and CSF of these patients. A complement consumption method was used for the detection of immune complexes in the concentrated CSF specimens. Of the 11 patients with clinical signs and symptoms suggestive of having G. spinigerum infection involving the central nervous system, only one patient had antigens detected in the CSF and in this one patient no antibody could be demonstrated. One other patient had immune complexes in her CSF. All remaining patients had IgG antibodies demonstrable in the CSF specimens. These data suggest that the detection of IgG antibodies in CSF is more reliable than the other two methods in confirming the diagnosis of cerebral gnathostomiasis.     

Antibodies to nuclear antigens in polymyositis: relationship to autoimmune ''overlap syndromes'' and carcinoma.

Thirty-two patients with polymyositis were categorised into 4 groups: (1) 'pure' polymyositis, (2) dermatomyositis, (3) myositis associated with autoimmune 'overlap syndrome', and (4) those with associated malignancy. Serum from each patient was examined for a range of antinuclear antibodies. Seventeen patients had ANA detected by immunofluorescence, 18 patients had raised DNA binding (greater than 25 U/ml), of whom eight had levels greater than 50 U/ml (SI conversion: U/l = U/ml x 10(3)). Antibodies to soluble nuclear antigens were detected in 23 (72%) by 1 or more of 3 methods, and in all of these anti-RNP was the main antibody detected. Antibodies to other soluble antigens were also present in 6 sera. In 2 cases, both patients with SLE/myositis overlap, these were shown to be anti-Sm. The remaining 4 had antibodies to various protein components of the extracts, but it was not possible to demonstrate an antibody of diagnostic specificity for polymyositis. Furthermore, quantitation of anti-RNP and anti-DNA antibodies failed to define a distinct clinical entity or exclude malignant disease. High levels of anti-RNP antibodies showed an association with Raynaud's phenomenon, sclerodactyly, and pulmonary fibrosis and an inverse correlation with the rash of dermatomyositis, suggesting that this antibody may be of pathogenetic rather than diagnostic significance.     

Rapid diagnosis of Mycobacterium tuberculosis meningitis by enumeration of cerebrospinal fluid antigen-specific T-cells.

SETTING: Hospital in-patients with suspected tuberculous meningitis (TBM), predominantly in India. OBJECTIVE: To determine whether interferon-gamma (IFN-gamma) secreting Mycobacterium tuberculosis antigen-specific T-cells are present in the cerebrospinal fluid (CSF) of patients with TBM and to evaluate the feasibility of CSF enzyme-linked immunospot (ELISpot) for the diagnosis of active TBM. DESIGN: Prospective blinded hospital-based study. RESULTS: The overnight ELISpot assay detected M. tuberculosis antigen-specific IFN-gamma secreting T-cells in CSF from nine of 10 prospectively recruited patients with TBM, and zero of seven control patients with meningitis of other aetiology. This corresponds to a diagnostic sensitivity of 90% (95%CI 56-100) and specificity of 100% (95%CI 59-100). CONCLUSION: This pilot study demonstrates proof-of-principle for a new T-cell-based diagnostic test for TBM which is rapid, sensitive and specific.     

Specific and early detection of IgG, IgA and IgM antibodies to Mycobacterium tuberculosis 38kDa antigen in pulmonary tuberculosis

The objective was to apply the purified 38kDa protein antigen of Mycobacterium tuberculosis in ELISA to estimate the IgG, IgA and IgM antibody levels in sera and circulating immune complexes of tuberculosis patients. Sera from smear and culture positive tuberculosis patients were positive for anti 38kDa IgG, IgA and IgM antibodies, with a sensitivity of 61%, 30% and 10%, respectively, and with a specificity of 100% for IgG. The sensitivity of the test improved to a level of 68% for IgG+IgA and of 71.4% for IgG+IgA+IgM without significantly compromising the specificity (IgG of 100%, IgG+IgA of 96%, IgG+IgA+IgM of 90%). Among the smear, culture-negative but X-ray-positive cases, 60% were serum positive for IgG antibody, while in smear-negative but culture-positive cases, 54% were positive for IgG antibody. Measurement of 38kDa antibodies showed a greater than 95% sensitivity in smear and culture-positive, and smear-negative and culture-positive patients, through a combination of assays for serum IgG and circulating immune complex antibodies, while the specificity was 100%.     

Significance of mycobacterial immune complexes (IgG) in the diagnosis of tuberculous meningitis

Setting: Tuberculous meningitis (TBM) has high mortality, especially in children. Early accurate diagnosis and adequate treatment would reduce this mortality. Diagnosis of TBM remains an enigma because of low cerebrospinal fluid (CSF) culture positivity for Mycobacterium tuberculosis and weak clinical correlation with conventional immunoassays.Objective: To evaluate significance of mycobacterial immune complexes (IgG) and anti-mycobacterial antibodies in the diagnosis of TBM.Method: CSF from TBM patients and various types of other neurological (both infectious and non-infectious) and non-neurological cases was studied for the presence of IgG and anti-mycobacterial antibodies using antigen capture (by anti-BCG) and multilayered ELISA (using M. tuberculosis soluble extract), respectivelyResults: IgG in CSF could be detected in 33 of 55 (60%) and anti-mycobacterial antibodies in 30 of 55 (55%) TBM cases. Presence of IgG, anti-mycobacterial antibodies or both could be detected in 45 of 55 (82%) of the TBM cases. Excepting three of the pyogenic meningitis CSF, none of the infectious (49), non-infectious neurological cases (30) and non-neurological controls (32) showed the presence of IgG or anti-mycobacterial antibodies.Conclusion: Detection of IgG along with anti-mycobacterial antibodies aids in diagnosis of a large proportion of TBM cases.     

Antibodies to recombinant decorin-binding proteins A and B in the cerebrospinal fluid of patients with Lyme neuroborreliosis

Cerebrospinal fluid (CSF) and serum samples from 34 patients with proven neuroborreliosis (NB) and 22 patients with suspected neuroborreliosis (SNB) from Finland were analysed for antibodies to decorin-binding proteins A (DbpA) and B (DbpB). Antibodies to recombinant protein antigens originating from Borrelia burgdorferi sensu stricto, B. afzelii, or B. garinii species were studied by enzyme-linked immunosorbent assay (ELISA). Of the 34 patients with NB, 100% of the CSF and 88% of the serum samples had IgG antibodies to 1 to 3 variants of DbpA and 79% of the CSF and 70% of the serum samples were positive for 1 to 3 DbpB variants. Antibodies to DbpB seemed to be associated with lymphocytic pleocytosis in the CSF and short duration of the disease, whereas antibodies to DbpA in the CSF were observed irrespective of the duration of the disease and lymphocytic pleocytosis. Among the variant antigens, CSF reactivity was mainly with the DbpB from B. garinii, whereas positivity with the DbpA from B. afzelii or B. garinii predominated. The results suggest that CSF antibodies to DbpB might be useful as a marker of active infection whereas antibodies to DbpA seem to persist a long time after acute phases of NB.     

Identification of Taenia solium antigens in cerebrospinal fluid and larval antigens from patients with neurocysticercosis

Two antigens (190 and 230 kDa) of the larvae of Taenia solium were detected in the cerebrospinal fluid (CSF) of 14 of 18 patients in which neurocysticercosis was suspected. Nine of these were confirmed by histopathology. Seven antigens were detected in cyst fluid of T. solium larvae removed from the brains of 6 infected patients. Two of these antigens had the same Mr as the T. solium antigens detected in the CSF. Polyacrylamide gel electrophoresis and electro-immunoblotting analyses were used for the identification and characterization. Antibodies in rabbit anti-larval antiserum and antibodies in sera of infected individuals recognized the same larval antigens in the larval cyst fluids and in the CSF of infected patients.     

The detection by immunoassay of antibody to mycobacterial antigens and mycobacterial antigens in bronchoalveolar lavage fluid from patients with tuberculosis and control subjects

Bronchoalveolar lavage (BAL) samples from patients with pulmonary tuberculosis and from control subjects were studied using direct and inhibition enzyme-linked immunosorbent assay (ELISA) for the detection of IgG and IgA antibody to Mycobacterium tuberculosis culture filtrate and purified antigen 5 and for the detection of mycobacterial antigens. The IgG and IgA antibodies were found in the majority of samples from tuberculous patients. The IgG titers were higher than were IgA titers. Substantial nonspecificity was observed for antibodies to crude M tuberculosis culture filtrate. Antibodies to purified M tuberculosis antigen 5 were more specific. The detection of antibody in BAL did not offer advantages over previously described serodiagnostic assays. Antigen was found in some BAL samples from tuberculous patients. Greater specificity was observed with an assay based on purified antigen 5 than on crude mycobacterial culture filtrate.     

13种分支杆菌菌体抗原成份分析及应用

目的分析比较结核分支杆菌与其它分支杆菌菌体多肽抗原的共性和个性，寻找与结核病诊断相关的多肽抗原。方法采用ＳＤＳ－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）和免疫印迹技术分析多肽抗原，并提取特异性抗原用于５７５例结核患者、４５０名正常人和２５０例非结核患者的抗体检测。结果结核分支杆菌、牛结核分支杆菌菌体多肽成份不仅具有相似的多肽区带，而且各主要抗原多肽具有相似的免疫原性。主要多肽成份分子量为７６０００、６５０００和２５０００等，其中分子量为７６０００、６５０００和２５０００多肽成份是１３种分支杆菌的共同抗原。提纯的特异性抗原成份用于结核病的诊断，其敏感性为８９．７％，特异性为９５．７％。结论分支杆菌菌体多肽成份经ＳＤＳ－ＰＡＧＥ分析，区带之间差异较大，提示可以用于分支杆菌菌种的鉴定。结核分支杆菌Ｈ３７Ｒｖ株特异性抗原的抗体检测，对肺外结核、菌阴结核患者的辅助诊断具有较高的应用价值。