- Email: [email protected]
ELISA in tuberculous meningitis: using two mycobacterial antigens does not improve the diagnostic yield compared to either antigen alone
Serodiagnosis
and Immunotherapy
in Infectious Disease (1990) 4, 173-I
8I
ELISA in tnberculous meningitis: using two mycobacterial antigens does not improve the diagnostic yield compared to either antigen alone Sawsan M. Al-Ramahi’, George F. Araj’*, Tulsi D. Chugh’ and Raad A. Shaki?
of ‘Microbiology
Drpartments
and ‘Medicine, Faculty of Medicine, P.O. Box 24923, Kuwait 13110
Kuwait
Universit?!.
The prevention of the devastating sequelae of tuberculous meningitis (TBM) requires the availability of a reliable test for the rapid diagnosis of this condition. This study was carried out by enzyme-linked immunosorbent assay (ELISA) to measure antimycobacterial immunoglobulin (Ig) G, IgM and IgA in the cerebrospinal fluid (CSF) of I1 patients with culture-proven TBM using a whole cell sonicate of heat-killed M. tuberculosis antigen (MTb) and Bacilli-CalmetteG&in (BCG) antigens.The findings were compared to controls consisting of 13 CSF specimens from patients with proven pyogenic meningitis (PM) and 20 control CSF specimens from individuals with normal findings. The ELISA sensitivity, specificity, positive predictive and negative predictive values for IgG were 73%, 91%, 73% and 91%, respectively. against both antigens. The optical density results for IgM and IgA in the CSF of patients with TBM and controlsweretoo low againstboth antigensto be considered in any analysis. Although ELISA for detection of anti-mycobacterialantibodiesin the CSF provides improvement over conventional methods of microscopy and
culture. this approachstill needsconsiderablerefinementto makeit suitedfor routine clinical
use.
&words: tuberculousmeningitis,ELISA. gens. diagnosis.
antibody
detection.
mycobacterial
anti-
Introduction Tuberculosis remains a major public health problem in developing countries, and in the last few years it has emerged as a significant problem in developed countries’. Tuberculous meningitis (TBM) is a seriouscomplication of extrapulmonary tuberculosis characterized by insidious onset with high morbidity and mortality in children and adults, thus, necessitating prompt diagnosis to initiate early treatment and proper management’*. Clinically, patients with TBM present with fever, headache, confusion, seizures or convulsions. Laboratory investigations of cerebrospinal fluid (CSF) usually show a mild or moderate elevation of protein, decreasedor normal sugar and moderate lymphocytosis5,“. These clinical and laboratory findings, in TBM, are non-specific and * Author
to whom
correspondence
should
be addressed 173
0888&0786/90/030173+09$03.00/0
IF 1990 Academic PressLimited
174
S. M. Al-Ramahiet ul.
could be mimicked by a variety of infectious and non-infectious CNS disorders; such as syphilis7, brucellosis’, cryptococcosis’, viruses’” and cancer”. The conventional laboratory tests, of microscopy and culture are also not helpful. Although microscopy for TBM is relatively quick and inexpensive, it lacks sensitivity as it detects mycobacteria in only 10% to 40% of cases’?. The growth of M. tuberculosis from the CSF provides definitive diagnosis. Culture, however, is time-consuming and of low yield, being positive in only 15% to 20% of all TBM cases in most tropical countries and increases to 42% to 75% in developed countries”?,“. Subsequently, immunological tests such as latex agglutination’3, radioimmunoassay (RIA)‘5.‘6 and enzyme-linked immunosorbent assay (ELISA)‘7-20 as well as non-immunological tests, such as radioactive bromide partition”, adenosine deaminase’3.2Z and electron capture gas liquid chromatography testsZ3, have been attempted for rapid diagnosis of this disease. Generally, the non-immunological tests are complex, require sophisticated equipment and have a variable sensitivity and specificity. So far, none of these is being used as a routine test in clinical laboratories. Among the immunological tests, the ELISA has been considered the most practical test for detecting mycobacterial antibodies and antigens in the CSF of patients with TBM12. A variety of antigens were used. Most studies tested one antigen that included purified protein derivative (PPD)17, Bacilli-Calmette Gutrin (BCG)‘8,20, M. tuberculosis antigen 513or sonicated M. tuberculosisH,,Rv (MTb) antigen15. Only one study tested a combination of PPD, BCG and MTb antigens I9. The class of antibody looked for was almost exclusively immunoglobulin (Ig) G antibody. This study was undertaken to evaluate the usefulness of ELISA in detecting mycobacterial specific IgG, IgM and IgA in the CSF of patients with TBM and controls against both BCG and M. tuberculosissonicate (MTb) antigens as a rapid aid to support a clinical diagnosis of TBM in a developing country.
Materials and methods Study population and specimens
A total included patients patients
of 44 CSF specimens from an equal number of individuals were used. These 11 CSF specimens, collected during the second to third week of disease, from with culture-proven TBM and 13 CSF control specimens (non-TBM) from with culture-proven micro-organisms other than mycobacteria: Brucella meli-
tensis (1 CSF), Listeria monocytogenes (l), Neisseria meningitidis (l), Streptococcus pneumoniae(3), Streptococcus pyogenes (l), Streptococcus agalactiae (2) and Haemophilus infuenzae (4). In addition, 20 CSF specimens obtained from cases suspected of
neurological problems but with essentially normal cerebrospinal fluid findings of white cell count, protein and sugar content, as well as negative in culture for micro-organisms were also included as the normal control group. All CSF specimens were stored at - 70°C until tested. Mycobacterial antigens
Two antigens were used: a commercial whole cell BCG antigen (Japan BCG Laboratory, Tokyo) and an in-house prepared heat-killed sonicated M. tuberculosis H,Rv strain (MTb) antigen. Briefly, the freeze dried BCG antigen was resuspendedin physiological
ELISA
in tuberculous
meningitis
175
saline to give a stock solution of 1 mg ml-‘. The stock solution was aliquoted and stored at - 20°C until used. For MTb antigen, the M. tuberculosis was grown on Lowenstein-m Jensen medium for 6 weeks. The bacterial growth was suspended in filter sterilized distilled water, to a density equivalent to McFarland standard no. 3. The suspension was autoclaved and somcated for 45 min (amplitude 21 microns). This stock solution of MTb antigen was aliquoted and stored at - 20°C until used. Enzyme-linked
immunosorbent
assay
The ELISA technique used for the determination of mycobacterial specific IgG, IgM and IgA in CSF specimens was essentially that described by Engvall & Perlmannz4 with optimization of test conditions. Briefly, 96-well microtiter plates (Nunc-Immuno Plate I, Denmark) were coated with 50 l.d of 0.05 M sodium carbonate-bicarbonate buffer, pH 9.6, containing predetermined optimum concentrations of BCG and MTb antigens. Separate plates for each antigen and immunoglobulin class were used. The plates were incubated overnight at room temperature, in humid boxes, rinsed three times with distilled water and soaked for 10 min in phosphate-buffered saline, pH 7.4, containing 0.05% Tween-20 (PBST). After blotting, 50 pl CSF specimens from patients and controls, diluted I:25 in PBST, were added, in duplicates, to appropriate wells and the plates were incubated for 2 h at 37°C. After washing and blotting, 50 pl of a 1:lOOO dilution (in PBST) of alkaline phosphatase conjugated goat anti-human IgG, IgM and IgA (SIGMA chemical company, St. Louis, MO, USA) were individually added to designated wells and incubated for 2 h at 37°C. The washing procedure was repeated and then, 50 ~1 of phosphatase substrate, p-nitrophenyl phosphate (SIGMA), was added to each well and the plates were incubated for 45 min at 37°C. Immediately, the plates were read (optical density, OD) with 405 nm filter using Titertik multiskan spectrophotometer (Flow Laboratory, Scotland). In each run, the same known positive and negative CSF specimens were included. The OD for these specimens was permitted to vary within 10% in each run, otherwise, the whole test was repeated. The cut-off reading for positive (elevated) results was determined at an OD reading equal to or above those OD readings of 90% of all control specimens. Statistical analysis The sensitivity, specificity, positive predictive (PPV) and negative predictive (NPV) values of the assays were calculated as previously reported25. Student’s t-test was used to compare mean OD values between TBM patients and controls. Probabilities of ~0.05 were considered statistically significant. Results
The distribution of the individual OD values of IgG antibody against BCG and MTb antigens in CSF of patients and controls are shown in Figures 1 and 2. The OD of IgG anti-BCG in CSF of TBM patients (Figure 1) ranged from 0.057 to 0.233 (mean f SD = 0.159 f 0.061). However, the OD readings of the non-TBM group ranged from 0.014 to 0.204 (O-096 f 0.065) while all the OD readings of the normal control group were below 0.050 (O-015 f 0.010). Although, there was a considerable overlap in the distribution of OD values for TBM and non-TBM patients, the geometric mean level
176
S. M. Al-Ramahi
et al.
0.25 .
.
.
0.20 g 0.15 2 -----TJ 0 ; O.lO-
. . . . ” . --..---------.
-
0
0.05 -
. .
. . . . . . . .
-------
-_____________
:
.
A .
.
l
:
: . I:
:
.
. o-00
TBM patients (n=ll)
.
Non-TBM
.
. .I
Normals
(n=l3) (n=20) Figure 1. Distribution of optical density (OD 405 nm) values of IgG against BCG in CSF of patients with tuberculous meningitis (TBM), non-TBM and normal controls. Horizontal line represents the cut-off value and the vertical line represents the OD mean + standard deviation for the group.
of the OD values for IgG antibody to BCG and MTb antigens was significantly higher in CSF of TBM patients than that in non-TBM patients (P = O-025 and P = 0.016, respectively). No overlap was observed in the distribution of OD values for TBM patients and those with normal CSF. The mean OD values for IgG antibody to BCG or MTb antigens in TBM patients were significantly higher than that found in this control group (P < 0.0001 and P < 0.002, respectively). The OD of IgG anti-MTb in CSF of TBM patients (Figure 2) ranged from 0.057 to 089 (0.365 f 0.273). On the other hand, the OD values in the CSF of non-TBM patients ranged from 0.012 to O-370 (0.122 f 0.133) and those for normal controls ranged from 0.002 to 0.080 (0.027 f 0.021).
Figure 2. Distribution of optical density (OD 405 nm) values of IgG against MTb antigen in CSF of patients with tuberculous meningitis (TBM), non-TBM and normal controls. Horizontal line represents the cut-off OD value and the vertical line represents the OD mean + standard deviation for the group.
ELISA
in tuberculous
177
meningitis
The OD values of IgM and IgA antibodies against BCG and MTb antigens for TBM patients and controls were too low (OD < 0.050) to be presented graphically or analysed. Based on the cut-off OD readings for positive (elevated) results that included OD equal or above 90% of specimensfrom both control groups, the discriminative cut-off OD reading for IgG antibody to BCG and MTb antigens were 0.130 and 0.150, respectively. Table 1 presents the elevated anti-BCG and anti-MTb results in the study population basedon thesecut-off values. Anti-BCG and anti-MTb were elevated in 73% and 23% of patients with TBM and non-TBM. respectively. None of the CSF specimens from the normal controls showed elevated IgG. The positive and negative results with MTb and BCG antigens were identical in CSF specimensfrom patients with TBM. The false-positive results encountered against both antigens were in two CSF specimens;one from a patient with L. monocytogenes and the other with S. pneumoniae infection. In addition. one CSF, each. with S. agalactiae and S. pneumoniae infection gave falsepositive results against BCG and MTb antigens, respectively. The sensitivity, specificity. positive predictive (PPV) and negative predictive values (NPV) for the elevated IgG in CSF of TBM patients were 73%, 91%. 73% and 91%, respectively, for both BCG and MTb antigens. Discussion
The presenceof antigen-specific antibodies and lymphocytes in the CSF of patients with CNS infections has been attributed to the local activation of immune response rather than the passivediffusion from the bloodz6,z7.For example, patients with TBM showed increased PPD induced transformation of CSF lymphocytes compared with CSF lymphocytes from patients with aseptic meningitislx. Other reports have shown greater PPD proliferative responseof CSF lymphocytes than the responseof peripheral blood lymphocytes from patients with TBM29.‘0. Kalish et al.” have demonstrated the local production of IgG anti-PPD in CSF which increasedduring early stagesin patients with TBM then decreased as the patient improved clinically and as the CSF cell count, protein level and glucose level returned to normal. Similarly an intrathecal synthesis of oligoclonal IgG has been reported in the CNS of patients with TBM’O. Thus, determination of anti-mycobacterial antibodies in the CSF is a justifiable approach in the rapid diagnosis of patients with TBM. The present ELISA study, with two antigens, shows promising findings since it was able to detect elevated IgG antibodies to MTb and BCG antigens in a high percentage of Table 1. Frequency of elevated IgG anti-BCG tuberculous meningitis (TBM). ___Group No. tested
and MTb non-TBM
in CSF specimens from patients with and normal controls
No. (96) of elevated IgG to BCG antigen
MTb
antigen -,-~ ~~
TBM Non-TBM Normal controls
11 13 20
8(73) 3(23) 0
803) 3(23) 0
either BCG or MTb ~~~~ -W73)
323) 0
178
S. M. Al-Ramahi
et al.
CSF specimensfrom patients with TBM compared to controls. The 73% sensitivity and 9 1% specificity noted in this study falls within the ranges of sensitivity (40% to 100%) and specificity (72% to 100%) reported from different parts of the world (Table 2). The IgG antibody in the present study was the most discriminative diagnostically, while the levels of IgM and IgA. in the CSF, were too low to be considered of value in the diagnosis of patients with TBM. Only two studies have reported the detection of specific IgM antibodies in the CSF of TBM patients but their assaysand data were integrated with IgG”,‘“. To our knowledge, there have been no reports on specific IgA antibodies or independent detection of IgM antibodies in the CSF of patients with TBM. The detection of specific IgM in the CSF, however, has been used in the diagnosis and monitoring of other bacterial CNS infections such as brucellosis3’ and syphilis”. In addition, detection, in CSF, of brucella-specific IgA has been shown to be of value in the diagnosis of patients with neurobrucellosis”. The reported variation in antibody assaysfor diagnosis of TBM could be, in part, due to lack of standardized methodology as shown in Table 2. For example, Kalish et al.“, used PPD antigen in an ELISA and reported 100% sensitivity and specificity in detecting IgG anti-PPD at 1:32 dilution of CSF. Chandramuki et aZ.16have also usedPPD antigen in a solid phase RIA with a monoclonal antibody, but have reported a sensitivity of 68% and specificity of 72% at 1:2 CSF dilution. On the other hand, Prabhakar & Oommeni9, showed weak positive to no reaction in five of the 10 CSF from patients with definite TBM when tested neat in ELISA, against PPD. Variation was also noted when BCG was used as an antigen in ELISA. Hernandez ef Q/.~*reported a 100% sensitivity and specificity in detecting anti-BCG antibodies in CSF dilution 1:5, while Prabhakar & Oommeni9 reported a sensitivity and specificity of 40% and 90%, respectively, at CSF dilution of I:200 and Watt et a/.“’ reported a sensitivity and specificity of 52% and 96%, respectively, at CSF dilution of 1:64. Lessvariation was noted when sonicated M. tuberculosis antigen was usedwhere Samuel et aLI reported a sensitivity of 83% and specificity of 100% using a CSF dilution of I:2 in RIA and Prabhakar & Oommeni’ reported a sensitivity of 72% and specificity of 96% with CSF Table 2. Summary of studiesin detecting anti-mycobacterialantibodiesin cerebrospinalfluid (CSF) of patientswith tuberculousmeningitis(TBM)
Assay
ELISA* RIA* ELISAt RIA* ELISA* ELISA* ELISAt ELISAl
*
IgG; tIgG
CSF of TBM (no.1
CSF dilution
Antigen
3
I:32
PPD
18
1:2
20 25
I:.5 1:2
15 10
1:20 1:200
H,,Rv BCG PPD Ai@ PPD BCG
Sensiti- Specifivity city (%I WI 100 83
100 100
100
100
68 53
72 90
d
d
Indian Mexican Indian South Africa Indian
90 92 96 91
Philippine Kuwaiti
91
29
1:64
H&v BCG
11
1:25
BCG
40 72 52 73
H,,Rv
73
+ IgM
together;
1 IgG,
IgM,
Patient population
IgA separately;
USA
6 test was considered
Reference
17 15
18 16
13 19 20
Present study
unsuitable withthisantigen.
ELISA
in tuberculousmeningitis
179
dilution l:200 in ELISA. In the present study we used a CSF dilution of 1:25 and the sensitivity and specificity were 73% and 91%. respectively, for both BCG and MTb antigens. Compared to the results from other geographic locations. the ELISA results in this study indicate that the patients in Kuwait show more similarity to the patients from the Indian subcontinent” than to the patients of the Western countries”. Although the ELISA for detection of mycobacterial antibody in the CSF shows an improvement in sensitivity and specificity over the smear and culture positivity of 5% to 350/uin TBM casesin most tropical countries”.‘“. the test has to be interpreted with caution in the light of the possible false-positive and false-negative findings (Table 2). In the present study, none of the normal control CSF specimens showed false-positive results. similar to what was reported by others’“‘7.!X. However. 23% of the CSF specimens from patients with non-TBM showed false-positive antibodies to both antigens. Similar false-positive findings were noted in patients with pyogenic meningitis (e.g. due to Huetnophilzrs and Ptzeutnococcus). from areas with a high prevalence of tuberculosis”-” . . Kuwait can be considered as an area of high TB prevalence since an average incidence rate of 42 casesper 100,000population has been reported per annum during the last few years according to the reports of the Ministry of Health. The exact reasonsfor these false-positive findings, in patients with pyogenic meningitis, remain to be elucidated. It is unlikely that this elevation in antibody response is due to cross reaction between mycobacteria and pyogenic bacteria since no positive results were obtained when we tested TBM CSF with antigens of pyogenic bacteria (data not shown). On the other hand, the high levels of IgG antibodies to BCG and MTb antigens, in such a control group. could be attributed to vaccination or to exposure to environmental M~dm*teria spp. as well as to other bacterial species(e.g. Cotyebacteria, Nocardia and Rhodococcw) which share antigens with M. tuberculosis and result in cross reactive antibodies?“. These cross reactive antibodies could have entered the CSF from the circulation due to the blood-brain barrier impairment by the inflammatory process in the CNS” and could have contributed to false-positive findings. False-negative results were encountered in the present study aswell as in other similar studies”~‘s.‘6.‘9~2”. Though the exact reasons are unknown, probable factors contributing to false-negative results include the dilution of CSF used, the effect of treatment. especially steroids, the stage of the disease, and the presence of antigen-antibody complexes in the CSF’3.‘5.‘9.Thus, the negative findings do not exclude the diagnosis of TBM. To overcome the aforementioned limitations in tests based on antibody detection, other approaches such as antigen detection assaysj7and nucleic acid probesi have been attempted and so far showed promising results. These approaches merit pursuing to evaluate their clinical efficacy and reliability in the rapid diagnosis of patients with tuberculosis”. To conclude, the useof ELISA for detecting antibodies in CSF against MTb and BCG antigens simultaneously, as a tool in the diagnosis of patients with TBM, offers improvement over conventional methods of microscopy and culture. This approach, however. still needs considerable refinement to make it suited for routine clinical use.
Acknowledgements We thank Mr Ishak Mugrabe, Mr John Zakaria and Mrs Marie Al-Haj for their
180 excellent technical assistance Supported in part by Kuwait
S. M. Al-Ramahiet al. and Dr Mustafa University grant
Abu Salim for his valuable No. MT 052 to G.F.A.
comments.
References 1. Styblo K. Overview and epidemiologic assessment of the current globa] tuberculosis situation with an emphasis on control in developing countries. Rev Infect Dis 1989; 11: 339-46. 2. Tandon PN. Tuberculous meningitis (Cranial and Spinal). In: Vinken PJ, Bruyn GW, &. Hand Book of Clinical Neurology (Infections of the Nervous System, Part I). Amsterdam: 1978; 33: 195-262. 3. Weir MR, Thornton GF. Extrapulmonary tuberculosis: experience of a community hospital and review of the literature. Am J Med 1985; 79: 467-78. 4. Daniel TM. Rapid diagnosis of tuberculosis: laboratory techniques applicable in developing countries. J Infect Dis 1989; 11 (suppl): 471-8. 5. Kennedy DH, Fullon R. Tuberculous meningitis. JAMA 1979; 241: 264-8. 6. Kilpatrick ME, Girgis NI, Yassin MW, Abu EL, Ella AA. Tuberculous meningitis-clinical and laboratory review of 100 patients. J Hyg (Camb) 1986; 96: 231-8. 7. Hoston JR. Modern neurosyphilis: a partially treated chronic meningitis. Western J Med 1981; 135: 191-200. 8. Shakir RA, Al Din ASN, Araj GF, Lulu AR, Mousa AR, Saadah MA. Clinical categories of neurobrucellosis: a report on 19 cases. Brain 1987; 110: 213-23. 9. Yu YL, Lau YN, Woo E, Wong KL, Tse B. Cryptococcal infection of the nervous system. Q J Med 1988; 66: 87-96. 10. Gray JA, Moffut MAJ. Sangster G. Viral meningitis: a ten year study. Scott Med J 1969; 14: 23442. 11. Dastur HM, Desai AD. A comparative study of brain culomas and gliomas based upon 107 case records of each. Brain 1985; 88: 375-96. 12. Daniel TM. New approaches to the rapid diagnosis of tuberculous meningitis. J Infect Dis 1987; 155: 599-602. 13. Coovadia YM. Dawood A, Ellis EM, Coovadia HM, Daniel TM. Evaluation of adenosine deaminase activity and antibody to M. tuberculosis antigen 5 in cerebrospinal fluid and the radioactive bromide partition test for the early diagnosis of tuberculous meningitis. Arch Dis Child 1986; 61: 428-35. 14. Krambovitis E, McIllmuraay MB, Lock PE, Hendrickse W, Holzel H. Rapid diagnosis of tuberculous meningitis by latex particle agglutination. Lancet 1984; i: 1229-31. 15. Samuel AM, Kadival GV, lrani S. Pandya SK, Ganatra RD. A sensitive and specific method for diagnosis of tubercular meningitis. Indian J Med Res 1983; 77: 752-7. 16. Chandramuki A, Allen PRJ, Keen M, lvany J. Detection of mycobacterial antigen and antibodies in the cerebrospinal fluid of patients with tuberculous meningitis. J Med Microbial 1985; 20: 23947. 17. Kalish SB, Radin RC, Levi@ D, Zeiss CR, Phair JP. The ELISA method for IgG antibody to purified protein derivative in CSF of patients with tuberculous meningitis. Ann Intern Med 1983; 99: 63&3. 18. Hernandez R, Munoz 0, Guiscafre H. Sensitive enzyme immuno-assay for early diagnosis of tuberculous meningitis. J Clin Microbial 1984; 20: 533-5. 19. Prabhakar S, Oommen A. ELISA using mycobacterial antigens as a diagnostic aid for tuberculous meningitis. J Neurol Sci 1987; 78: 203-I 1. 20. Watt G, Zaraspe G, Bautista S, Laughlin W. Rapid diagnosis of.tubercuious meningitis by using an enzyme linked immunosorbent assay to detect mycobacterial antigen and antibody in CSF. J Infect Dis 1988; 158: 681-6. 21. Mandal BK, Evans DIK, Ironside AG, Pullan BR. Radioactive bromide partition test in differential diagnosis of tuberculous meningitis. Br Med J 1972; 4: 413-5. 22. Ribera E, Martinez-Vazquez JM, Ocana I, Segura RM, Pascual C. Activity of adenosine deaminase in cerebrospinal fluid for the diagnosis and follow-up of tuberculous meningitis in adults. J Infect Dis 1987; 155: 603-7.
ELBA
in tuberculous
meningitis
181
23. Mardh RA. Larsson L, Hoby N, Engback HC, Odham G. Tuberculostearic acid as a diagnostic marker in tuberculous meningitis. Lancet 1983; i: 367. 24. Engvall E, Perlmann P. Enzyme-linked immunosorbent assay, ELISA: III. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen coated tubes. J lmmunol 1972; 109: 129935. 25. Haynes RB. How to read a clinical journal. II. To learn about a diagnostic test. Can Med Assoc J 1981; 124: 703-10. 36. Silva CA, Rio ME, Maia-Gonsalves A. Pereira S. Palmera M, Brito MR, Cruz C. Oligoclonal gammaglobulin of cerebrospinal fluid in neurobrucellosis. Acta Neurol Stand 1980; 61: 42-8. 27. Sindic CJM, Cambiaso CL, Depre A. Laterre CE, Masson RL. The concentration of IgM in the cerebrospinal fluid of neurological patients. J Neurol Sci 1982: 55: 339-50. 28. Malashkhia YA. Med M, Geladze MG. Autoradiographic studies of cultures of cerebrospinal fluid lymphocytes in non-suppurative meningitis. Neurology 1976: 26: 1081-4. 29. Plouffe JF. Silva J, Fekety R, Baird I. Cerebrospinal fluid lymphocyte transformation in meningitis. Arch Intern Med 1979; 139: 1914. 30. Kinnman J, Fryden A, Eriksson S. Moller E, Link H. Tuberculous meningitis: immune reaction within the central nervous system. Stand J Immunol 1981; 13: 289-96. 31. Araj GF, Lulu AR. Saadah MA. Mousa AM, Strannegard I-L, Shakir RA. Rapid diagnosis of central nervous system brucellosis by ELISA. J Neuroimmunol 1986; 12: 173382. 32. Scott AT, Logan L. A specific IgM antibody test in neonatal congenital syphilis. J Pediatr 1968: 73: 242~3. 33. Minden P, McClatchy JK, Cooper R, Bardana EJ. Farr RS. Shared antigens between l~j~c~ohrr~ferirrm hovis (BCG) and other bacterial species. Science 1972; 176: 57-8. 34. Stanford JL, Wong JKC. A study of the relationship between Nocardia and Mycohacterium dicwho/k+ a typical fast growing mycobacterium. Br J Exp Path 1974; 55: 291-5. 35. Ridell M. Cross reactivity between n4. tuberculosis H,,Rv and various actinomycetes and related organisms. Tubercle 1983: 64: 211-6. 36. Ahou-Zeid C. Harboe M. Sundsten B. Cocito C. Cross-reactivity of antigens from the cytoplasm and cell walls of some Corynebacteria and Mycobacteria. J Infect Dis 1985; 151: I70 8. 37. Ramkisson A, Coovadia YM, Coovadia HM. A competition ELISA for the detection of mycobacterial antigen in tuberculous exudates. Tubercle 1988; 69: 209-12. 38. Brisson-Noel A. Lecossier D. Nassif X. Gicquel B, Lery-Frebault V, Hance AJ. Rapid diagnosis of tuberculosis by amplification of mycobacterial DNA in clinical samples. Lancet 1989: ii: 1069971.
and Immunotherapy
in Infectious Disease (1990) 4, 173-I
8I
ELISA in tnberculous meningitis: using two mycobacterial antigens does not improve the diagnostic yield compared to either antigen alone Sawsan M. Al-Ramahi’, George F. Araj’*, Tulsi D. Chugh’ and Raad A. Shaki?
of ‘Microbiology
Drpartments
and ‘Medicine, Faculty of Medicine, P.O. Box 24923, Kuwait 13110
Kuwait
Universit?!.
The prevention of the devastating sequelae of tuberculous meningitis (TBM) requires the availability of a reliable test for the rapid diagnosis of this condition. This study was carried out by enzyme-linked immunosorbent assay (ELISA) to measure antimycobacterial immunoglobulin (Ig) G, IgM and IgA in the cerebrospinal fluid (CSF) of I1 patients with culture-proven TBM using a whole cell sonicate of heat-killed M. tuberculosis antigen (MTb) and Bacilli-CalmetteG&in (BCG) antigens.The findings were compared to controls consisting of 13 CSF specimens from patients with proven pyogenic meningitis (PM) and 20 control CSF specimens from individuals with normal findings. The ELISA sensitivity, specificity, positive predictive and negative predictive values for IgG were 73%, 91%, 73% and 91%, respectively. against both antigens. The optical density results for IgM and IgA in the CSF of patients with TBM and controlsweretoo low againstboth antigensto be considered in any analysis. Although ELISA for detection of anti-mycobacterialantibodiesin the CSF provides improvement over conventional methods of microscopy and
culture. this approachstill needsconsiderablerefinementto makeit suitedfor routine clinical
use.
&words: tuberculousmeningitis,ELISA. gens. diagnosis.
antibody
detection.
mycobacterial
anti-
Introduction Tuberculosis remains a major public health problem in developing countries, and in the last few years it has emerged as a significant problem in developed countries’. Tuberculous meningitis (TBM) is a seriouscomplication of extrapulmonary tuberculosis characterized by insidious onset with high morbidity and mortality in children and adults, thus, necessitating prompt diagnosis to initiate early treatment and proper management’*. Clinically, patients with TBM present with fever, headache, confusion, seizures or convulsions. Laboratory investigations of cerebrospinal fluid (CSF) usually show a mild or moderate elevation of protein, decreasedor normal sugar and moderate lymphocytosis5,“. These clinical and laboratory findings, in TBM, are non-specific and * Author
to whom
correspondence
should
be addressed 173
0888&0786/90/030173+09$03.00/0
IF 1990 Academic PressLimited
174
S. M. Al-Ramahiet ul.
could be mimicked by a variety of infectious and non-infectious CNS disorders; such as syphilis7, brucellosis’, cryptococcosis’, viruses’” and cancer”. The conventional laboratory tests, of microscopy and culture are also not helpful. Although microscopy for TBM is relatively quick and inexpensive, it lacks sensitivity as it detects mycobacteria in only 10% to 40% of cases’?. The growth of M. tuberculosis from the CSF provides definitive diagnosis. Culture, however, is time-consuming and of low yield, being positive in only 15% to 20% of all TBM cases in most tropical countries and increases to 42% to 75% in developed countries”?,“. Subsequently, immunological tests such as latex agglutination’3, radioimmunoassay (RIA)‘5.‘6 and enzyme-linked immunosorbent assay (ELISA)‘7-20 as well as non-immunological tests, such as radioactive bromide partition”, adenosine deaminase’3.2Z and electron capture gas liquid chromatography testsZ3, have been attempted for rapid diagnosis of this disease. Generally, the non-immunological tests are complex, require sophisticated equipment and have a variable sensitivity and specificity. So far, none of these is being used as a routine test in clinical laboratories. Among the immunological tests, the ELISA has been considered the most practical test for detecting mycobacterial antibodies and antigens in the CSF of patients with TBM12. A variety of antigens were used. Most studies tested one antigen that included purified protein derivative (PPD)17, Bacilli-Calmette Gutrin (BCG)‘8,20, M. tuberculosis antigen 513or sonicated M. tuberculosisH,,Rv (MTb) antigen15. Only one study tested a combination of PPD, BCG and MTb antigens I9. The class of antibody looked for was almost exclusively immunoglobulin (Ig) G antibody. This study was undertaken to evaluate the usefulness of ELISA in detecting mycobacterial specific IgG, IgM and IgA in the CSF of patients with TBM and controls against both BCG and M. tuberculosissonicate (MTb) antigens as a rapid aid to support a clinical diagnosis of TBM in a developing country.
Materials and methods Study population and specimens
A total included patients patients
of 44 CSF specimens from an equal number of individuals were used. These 11 CSF specimens, collected during the second to third week of disease, from with culture-proven TBM and 13 CSF control specimens (non-TBM) from with culture-proven micro-organisms other than mycobacteria: Brucella meli-
tensis (1 CSF), Listeria monocytogenes (l), Neisseria meningitidis (l), Streptococcus pneumoniae(3), Streptococcus pyogenes (l), Streptococcus agalactiae (2) and Haemophilus infuenzae (4). In addition, 20 CSF specimens obtained from cases suspected of
neurological problems but with essentially normal cerebrospinal fluid findings of white cell count, protein and sugar content, as well as negative in culture for micro-organisms were also included as the normal control group. All CSF specimens were stored at - 70°C until tested. Mycobacterial antigens
Two antigens were used: a commercial whole cell BCG antigen (Japan BCG Laboratory, Tokyo) and an in-house prepared heat-killed sonicated M. tuberculosis H,Rv strain (MTb) antigen. Briefly, the freeze dried BCG antigen was resuspendedin physiological
ELISA
in tuberculous
meningitis
175
saline to give a stock solution of 1 mg ml-‘. The stock solution was aliquoted and stored at - 20°C until used. For MTb antigen, the M. tuberculosis was grown on Lowenstein-m Jensen medium for 6 weeks. The bacterial growth was suspended in filter sterilized distilled water, to a density equivalent to McFarland standard no. 3. The suspension was autoclaved and somcated for 45 min (amplitude 21 microns). This stock solution of MTb antigen was aliquoted and stored at - 20°C until used. Enzyme-linked
immunosorbent
assay
The ELISA technique used for the determination of mycobacterial specific IgG, IgM and IgA in CSF specimens was essentially that described by Engvall & Perlmannz4 with optimization of test conditions. Briefly, 96-well microtiter plates (Nunc-Immuno Plate I, Denmark) were coated with 50 l.d of 0.05 M sodium carbonate-bicarbonate buffer, pH 9.6, containing predetermined optimum concentrations of BCG and MTb antigens. Separate plates for each antigen and immunoglobulin class were used. The plates were incubated overnight at room temperature, in humid boxes, rinsed three times with distilled water and soaked for 10 min in phosphate-buffered saline, pH 7.4, containing 0.05% Tween-20 (PBST). After blotting, 50 pl CSF specimens from patients and controls, diluted I:25 in PBST, were added, in duplicates, to appropriate wells and the plates were incubated for 2 h at 37°C. After washing and blotting, 50 pl of a 1:lOOO dilution (in PBST) of alkaline phosphatase conjugated goat anti-human IgG, IgM and IgA (SIGMA chemical company, St. Louis, MO, USA) were individually added to designated wells and incubated for 2 h at 37°C. The washing procedure was repeated and then, 50 ~1 of phosphatase substrate, p-nitrophenyl phosphate (SIGMA), was added to each well and the plates were incubated for 45 min at 37°C. Immediately, the plates were read (optical density, OD) with 405 nm filter using Titertik multiskan spectrophotometer (Flow Laboratory, Scotland). In each run, the same known positive and negative CSF specimens were included. The OD for these specimens was permitted to vary within 10% in each run, otherwise, the whole test was repeated. The cut-off reading for positive (elevated) results was determined at an OD reading equal to or above those OD readings of 90% of all control specimens. Statistical analysis The sensitivity, specificity, positive predictive (PPV) and negative predictive (NPV) values of the assays were calculated as previously reported25. Student’s t-test was used to compare mean OD values between TBM patients and controls. Probabilities of ~0.05 were considered statistically significant. Results
The distribution of the individual OD values of IgG antibody against BCG and MTb antigens in CSF of patients and controls are shown in Figures 1 and 2. The OD of IgG anti-BCG in CSF of TBM patients (Figure 1) ranged from 0.057 to 0.233 (mean f SD = 0.159 f 0.061). However, the OD readings of the non-TBM group ranged from 0.014 to 0.204 (O-096 f 0.065) while all the OD readings of the normal control group were below 0.050 (O-015 f 0.010). Although, there was a considerable overlap in the distribution of OD values for TBM and non-TBM patients, the geometric mean level
176
S. M. Al-Ramahi
et al.
0.25 .
.
.
0.20 g 0.15 2 -----TJ 0 ; O.lO-
. . . . ” . --..---------.
-
0
0.05 -
. .
. . . . . . . .
-------
-_____________
:
.
A .
.
l
:
: . I:
:
.
. o-00
TBM patients (n=ll)
.
Non-TBM
.
. .I
Normals
(n=l3) (n=20) Figure 1. Distribution of optical density (OD 405 nm) values of IgG against BCG in CSF of patients with tuberculous meningitis (TBM), non-TBM and normal controls. Horizontal line represents the cut-off value and the vertical line represents the OD mean + standard deviation for the group.
of the OD values for IgG antibody to BCG and MTb antigens was significantly higher in CSF of TBM patients than that in non-TBM patients (P = O-025 and P = 0.016, respectively). No overlap was observed in the distribution of OD values for TBM patients and those with normal CSF. The mean OD values for IgG antibody to BCG or MTb antigens in TBM patients were significantly higher than that found in this control group (P < 0.0001 and P < 0.002, respectively). The OD of IgG anti-MTb in CSF of TBM patients (Figure 2) ranged from 0.057 to 089 (0.365 f 0.273). On the other hand, the OD values in the CSF of non-TBM patients ranged from 0.012 to O-370 (0.122 f 0.133) and those for normal controls ranged from 0.002 to 0.080 (0.027 f 0.021).
Figure 2. Distribution of optical density (OD 405 nm) values of IgG against MTb antigen in CSF of patients with tuberculous meningitis (TBM), non-TBM and normal controls. Horizontal line represents the cut-off OD value and the vertical line represents the OD mean + standard deviation for the group.
ELISA
in tuberculous
177
meningitis
The OD values of IgM and IgA antibodies against BCG and MTb antigens for TBM patients and controls were too low (OD < 0.050) to be presented graphically or analysed. Based on the cut-off OD readings for positive (elevated) results that included OD equal or above 90% of specimensfrom both control groups, the discriminative cut-off OD reading for IgG antibody to BCG and MTb antigens were 0.130 and 0.150, respectively. Table 1 presents the elevated anti-BCG and anti-MTb results in the study population basedon thesecut-off values. Anti-BCG and anti-MTb were elevated in 73% and 23% of patients with TBM and non-TBM. respectively. None of the CSF specimens from the normal controls showed elevated IgG. The positive and negative results with MTb and BCG antigens were identical in CSF specimensfrom patients with TBM. The false-positive results encountered against both antigens were in two CSF specimens;one from a patient with L. monocytogenes and the other with S. pneumoniae infection. In addition. one CSF, each. with S. agalactiae and S. pneumoniae infection gave falsepositive results against BCG and MTb antigens, respectively. The sensitivity, specificity. positive predictive (PPV) and negative predictive values (NPV) for the elevated IgG in CSF of TBM patients were 73%, 91%. 73% and 91%, respectively, for both BCG and MTb antigens. Discussion
The presenceof antigen-specific antibodies and lymphocytes in the CSF of patients with CNS infections has been attributed to the local activation of immune response rather than the passivediffusion from the bloodz6,z7.For example, patients with TBM showed increased PPD induced transformation of CSF lymphocytes compared with CSF lymphocytes from patients with aseptic meningitislx. Other reports have shown greater PPD proliferative responseof CSF lymphocytes than the responseof peripheral blood lymphocytes from patients with TBM29.‘0. Kalish et al.” have demonstrated the local production of IgG anti-PPD in CSF which increasedduring early stagesin patients with TBM then decreased as the patient improved clinically and as the CSF cell count, protein level and glucose level returned to normal. Similarly an intrathecal synthesis of oligoclonal IgG has been reported in the CNS of patients with TBM’O. Thus, determination of anti-mycobacterial antibodies in the CSF is a justifiable approach in the rapid diagnosis of patients with TBM. The present ELISA study, with two antigens, shows promising findings since it was able to detect elevated IgG antibodies to MTb and BCG antigens in a high percentage of Table 1. Frequency of elevated IgG anti-BCG tuberculous meningitis (TBM). ___Group No. tested
and MTb non-TBM
in CSF specimens from patients with and normal controls
No. (96) of elevated IgG to BCG antigen
MTb
antigen -,-~ ~~
TBM Non-TBM Normal controls
11 13 20
8(73) 3(23) 0
803) 3(23) 0
either BCG or MTb ~~~~ -W73)
323) 0
178
S. M. Al-Ramahi
et al.
CSF specimensfrom patients with TBM compared to controls. The 73% sensitivity and 9 1% specificity noted in this study falls within the ranges of sensitivity (40% to 100%) and specificity (72% to 100%) reported from different parts of the world (Table 2). The IgG antibody in the present study was the most discriminative diagnostically, while the levels of IgM and IgA. in the CSF, were too low to be considered of value in the diagnosis of patients with TBM. Only two studies have reported the detection of specific IgM antibodies in the CSF of TBM patients but their assaysand data were integrated with IgG”,‘“. To our knowledge, there have been no reports on specific IgA antibodies or independent detection of IgM antibodies in the CSF of patients with TBM. The detection of specific IgM in the CSF, however, has been used in the diagnosis and monitoring of other bacterial CNS infections such as brucellosis3’ and syphilis”. In addition, detection, in CSF, of brucella-specific IgA has been shown to be of value in the diagnosis of patients with neurobrucellosis”. The reported variation in antibody assaysfor diagnosis of TBM could be, in part, due to lack of standardized methodology as shown in Table 2. For example, Kalish et al.“, used PPD antigen in an ELISA and reported 100% sensitivity and specificity in detecting IgG anti-PPD at 1:32 dilution of CSF. Chandramuki et aZ.16have also usedPPD antigen in a solid phase RIA with a monoclonal antibody, but have reported a sensitivity of 68% and specificity of 72% at 1:2 CSF dilution. On the other hand, Prabhakar & Oommeni9, showed weak positive to no reaction in five of the 10 CSF from patients with definite TBM when tested neat in ELISA, against PPD. Variation was also noted when BCG was used as an antigen in ELISA. Hernandez ef Q/.~*reported a 100% sensitivity and specificity in detecting anti-BCG antibodies in CSF dilution 1:5, while Prabhakar & Oommeni9 reported a sensitivity and specificity of 40% and 90%, respectively, at CSF dilution of I:200 and Watt et a/.“’ reported a sensitivity and specificity of 52% and 96%, respectively, at CSF dilution of 1:64. Lessvariation was noted when sonicated M. tuberculosis antigen was usedwhere Samuel et aLI reported a sensitivity of 83% and specificity of 100% using a CSF dilution of I:2 in RIA and Prabhakar & Oommeni’ reported a sensitivity of 72% and specificity of 96% with CSF Table 2. Summary of studiesin detecting anti-mycobacterialantibodiesin cerebrospinalfluid (CSF) of patientswith tuberculousmeningitis(TBM)
Assay
ELISA* RIA* ELISAt RIA* ELISA* ELISA* ELISAt ELISAl
*
IgG; tIgG
CSF of TBM (no.1
CSF dilution
Antigen
3
I:32
PPD
18
1:2
20 25
I:.5 1:2
15 10
1:20 1:200
H,,Rv BCG PPD Ai@ PPD BCG
Sensiti- Specifivity city (%I WI 100 83
100 100
100
100
68 53
72 90
d
d
Indian Mexican Indian South Africa Indian
90 92 96 91
Philippine Kuwaiti
91
29
1:64
H&v BCG
11
1:25
BCG
40 72 52 73
H,,Rv
73
+ IgM
together;
1 IgG,
IgM,
Patient population
IgA separately;
USA
6 test was considered
Reference
17 15
18 16
13 19 20
Present study
unsuitable withthisantigen.
ELISA
in tuberculousmeningitis
179
dilution l:200 in ELISA. In the present study we used a CSF dilution of 1:25 and the sensitivity and specificity were 73% and 91%. respectively, for both BCG and MTb antigens. Compared to the results from other geographic locations. the ELISA results in this study indicate that the patients in Kuwait show more similarity to the patients from the Indian subcontinent” than to the patients of the Western countries”. Although the ELISA for detection of mycobacterial antibody in the CSF shows an improvement in sensitivity and specificity over the smear and culture positivity of 5% to 350/uin TBM casesin most tropical countries”.‘“. the test has to be interpreted with caution in the light of the possible false-positive and false-negative findings (Table 2). In the present study, none of the normal control CSF specimens showed false-positive results. similar to what was reported by others’“‘7.!X. However. 23% of the CSF specimens from patients with non-TBM showed false-positive antibodies to both antigens. Similar false-positive findings were noted in patients with pyogenic meningitis (e.g. due to Huetnophilzrs and Ptzeutnococcus). from areas with a high prevalence of tuberculosis”-” . . Kuwait can be considered as an area of high TB prevalence since an average incidence rate of 42 casesper 100,000population has been reported per annum during the last few years according to the reports of the Ministry of Health. The exact reasonsfor these false-positive findings, in patients with pyogenic meningitis, remain to be elucidated. It is unlikely that this elevation in antibody response is due to cross reaction between mycobacteria and pyogenic bacteria since no positive results were obtained when we tested TBM CSF with antigens of pyogenic bacteria (data not shown). On the other hand, the high levels of IgG antibodies to BCG and MTb antigens, in such a control group. could be attributed to vaccination or to exposure to environmental M~dm*teria spp. as well as to other bacterial species(e.g. Cotyebacteria, Nocardia and Rhodococcw) which share antigens with M. tuberculosis and result in cross reactive antibodies?“. These cross reactive antibodies could have entered the CSF from the circulation due to the blood-brain barrier impairment by the inflammatory process in the CNS” and could have contributed to false-positive findings. False-negative results were encountered in the present study aswell as in other similar studies”~‘s.‘6.‘9~2”. Though the exact reasons are unknown, probable factors contributing to false-negative results include the dilution of CSF used, the effect of treatment. especially steroids, the stage of the disease, and the presence of antigen-antibody complexes in the CSF’3.‘5.‘9.Thus, the negative findings do not exclude the diagnosis of TBM. To overcome the aforementioned limitations in tests based on antibody detection, other approaches such as antigen detection assaysj7and nucleic acid probesi have been attempted and so far showed promising results. These approaches merit pursuing to evaluate their clinical efficacy and reliability in the rapid diagnosis of patients with tuberculosis”. To conclude, the useof ELISA for detecting antibodies in CSF against MTb and BCG antigens simultaneously, as a tool in the diagnosis of patients with TBM, offers improvement over conventional methods of microscopy and culture. This approach, however. still needs considerable refinement to make it suited for routine clinical use.
Acknowledgements We thank Mr Ishak Mugrabe, Mr John Zakaria and Mrs Marie Al-Haj for their
180 excellent technical assistance Supported in part by Kuwait
S. M. Al-Ramahiet al. and Dr Mustafa University grant
Abu Salim for his valuable No. MT 052 to G.F.A.
comments.
References 1. Styblo K. Overview and epidemiologic assessment of the current globa] tuberculosis situation with an emphasis on control in developing countries. Rev Infect Dis 1989; 11: 339-46. 2. Tandon PN. Tuberculous meningitis (Cranial and Spinal). In: Vinken PJ, Bruyn GW, &. Hand Book of Clinical Neurology (Infections of the Nervous System, Part I). Amsterdam: 1978; 33: 195-262. 3. Weir MR, Thornton GF. Extrapulmonary tuberculosis: experience of a community hospital and review of the literature. Am J Med 1985; 79: 467-78. 4. Daniel TM. Rapid diagnosis of tuberculosis: laboratory techniques applicable in developing countries. J Infect Dis 1989; 11 (suppl): 471-8. 5. Kennedy DH, Fullon R. Tuberculous meningitis. JAMA 1979; 241: 264-8. 6. Kilpatrick ME, Girgis NI, Yassin MW, Abu EL, Ella AA. Tuberculous meningitis-clinical and laboratory review of 100 patients. J Hyg (Camb) 1986; 96: 231-8. 7. Hoston JR. Modern neurosyphilis: a partially treated chronic meningitis. Western J Med 1981; 135: 191-200. 8. Shakir RA, Al Din ASN, Araj GF, Lulu AR, Mousa AR, Saadah MA. Clinical categories of neurobrucellosis: a report on 19 cases. Brain 1987; 110: 213-23. 9. Yu YL, Lau YN, Woo E, Wong KL, Tse B. Cryptococcal infection of the nervous system. Q J Med 1988; 66: 87-96. 10. Gray JA, Moffut MAJ. Sangster G. Viral meningitis: a ten year study. Scott Med J 1969; 14: 23442. 11. Dastur HM, Desai AD. A comparative study of brain culomas and gliomas based upon 107 case records of each. Brain 1985; 88: 375-96. 12. Daniel TM. New approaches to the rapid diagnosis of tuberculous meningitis. J Infect Dis 1987; 155: 599-602. 13. Coovadia YM. Dawood A, Ellis EM, Coovadia HM, Daniel TM. Evaluation of adenosine deaminase activity and antibody to M. tuberculosis antigen 5 in cerebrospinal fluid and the radioactive bromide partition test for the early diagnosis of tuberculous meningitis. Arch Dis Child 1986; 61: 428-35. 14. Krambovitis E, McIllmuraay MB, Lock PE, Hendrickse W, Holzel H. Rapid diagnosis of tuberculous meningitis by latex particle agglutination. Lancet 1984; i: 1229-31. 15. Samuel AM, Kadival GV, lrani S. Pandya SK, Ganatra RD. A sensitive and specific method for diagnosis of tubercular meningitis. Indian J Med Res 1983; 77: 752-7. 16. Chandramuki A, Allen PRJ, Keen M, lvany J. Detection of mycobacterial antigen and antibodies in the cerebrospinal fluid of patients with tuberculous meningitis. J Med Microbial 1985; 20: 23947. 17. Kalish SB, Radin RC, Levi@ D, Zeiss CR, Phair JP. The ELISA method for IgG antibody to purified protein derivative in CSF of patients with tuberculous meningitis. Ann Intern Med 1983; 99: 63&3. 18. Hernandez R, Munoz 0, Guiscafre H. Sensitive enzyme immuno-assay for early diagnosis of tuberculous meningitis. J Clin Microbial 1984; 20: 533-5. 19. Prabhakar S, Oommen A. ELISA using mycobacterial antigens as a diagnostic aid for tuberculous meningitis. J Neurol Sci 1987; 78: 203-I 1. 20. Watt G, Zaraspe G, Bautista S, Laughlin W. Rapid diagnosis of.tubercuious meningitis by using an enzyme linked immunosorbent assay to detect mycobacterial antigen and antibody in CSF. J Infect Dis 1988; 158: 681-6. 21. Mandal BK, Evans DIK, Ironside AG, Pullan BR. Radioactive bromide partition test in differential diagnosis of tuberculous meningitis. Br Med J 1972; 4: 413-5. 22. Ribera E, Martinez-Vazquez JM, Ocana I, Segura RM, Pascual C. Activity of adenosine deaminase in cerebrospinal fluid for the diagnosis and follow-up of tuberculous meningitis in adults. J Infect Dis 1987; 155: 603-7.
ELBA
in tuberculous
meningitis
181
23. Mardh RA. Larsson L, Hoby N, Engback HC, Odham G. Tuberculostearic acid as a diagnostic marker in tuberculous meningitis. Lancet 1983; i: 367. 24. Engvall E, Perlmann P. Enzyme-linked immunosorbent assay, ELISA: III. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen coated tubes. J lmmunol 1972; 109: 129935. 25. Haynes RB. How to read a clinical journal. II. To learn about a diagnostic test. Can Med Assoc J 1981; 124: 703-10. 36. Silva CA, Rio ME, Maia-Gonsalves A. Pereira S. Palmera M, Brito MR, Cruz C. Oligoclonal gammaglobulin of cerebrospinal fluid in neurobrucellosis. Acta Neurol Stand 1980; 61: 42-8. 27. Sindic CJM, Cambiaso CL, Depre A. Laterre CE, Masson RL. The concentration of IgM in the cerebrospinal fluid of neurological patients. J Neurol Sci 1982: 55: 339-50. 28. Malashkhia YA. Med M, Geladze MG. Autoradiographic studies of cultures of cerebrospinal fluid lymphocytes in non-suppurative meningitis. Neurology 1976: 26: 1081-4. 29. Plouffe JF. Silva J, Fekety R, Baird I. Cerebrospinal fluid lymphocyte transformation in meningitis. Arch Intern Med 1979; 139: 1914. 30. Kinnman J, Fryden A, Eriksson S. Moller E, Link H. Tuberculous meningitis: immune reaction within the central nervous system. Stand J Immunol 1981; 13: 289-96. 31. Araj GF, Lulu AR. Saadah MA. Mousa AM, Strannegard I-L, Shakir RA. Rapid diagnosis of central nervous system brucellosis by ELISA. J Neuroimmunol 1986; 12: 173382. 32. Scott AT, Logan L. A specific IgM antibody test in neonatal congenital syphilis. J Pediatr 1968: 73: 242~3. 33. Minden P, McClatchy JK, Cooper R, Bardana EJ. Farr RS. Shared antigens between l~j~c~ohrr~ferirrm hovis (BCG) and other bacterial species. Science 1972; 176: 57-8. 34. Stanford JL, Wong JKC. A study of the relationship between Nocardia and Mycohacterium dicwho/k+ a typical fast growing mycobacterium. Br J Exp Path 1974; 55: 291-5. 35. Ridell M. Cross reactivity between n4. tuberculosis H,,Rv and various actinomycetes and related organisms. Tubercle 1983: 64: 211-6. 36. Ahou-Zeid C. Harboe M. Sundsten B. Cocito C. Cross-reactivity of antigens from the cytoplasm and cell walls of some Corynebacteria and Mycobacteria. J Infect Dis 1985; 151: I70 8. 37. Ramkisson A, Coovadia YM, Coovadia HM. A competition ELISA for the detection of mycobacterial antigen in tuberculous exudates. Tubercle 1988; 69: 209-12. 38. Brisson-Noel A. Lecossier D. Nassif X. Gicquel B, Lery-Frebault V, Hance AJ. Rapid diagnosis of tuberculosis by amplification of mycobacterial DNA in clinical samples. Lancet 1989: ii: 1069971.