651
DETECTION OF MYCOBACTERIAL ANTIGENS step procedurewas added, and a 1 mg/ml solution of
IN CEREBROSPINAL FLUID OF PATIENTS WITH p-nitrophenylphosphate was used as substrate. Absorbance at 405
TUBERCULOUS MENINGITIS BY ENZYME- nm was read in an ELISA reader (’Titertek’; Dynatech, Alexandria,
LINKED IMMUNOSORBENT ASSAY Virginia); BCG protein standards of 3 ng/ml, 7 ng/ml, and 14 ng/ml
gave absorbance values of of 0 -122,0 - 213, and 0 - 325, respectively.
In samples values ≥0. 20 were considered positive for the presence
EDUARDO SADA GUILLERMO M. RUIZ-PALACIOS of M tuberculosis antigens. Each sample was run in duplicate, and
YOLANDA LÓPEZ-VIDAL SERGIO PONCE DE LEÓN commercial BCG protein was used as positive control. Statistical
Department of Infectious Diseases and Teaching Division, analysis was done by the Mann Whitney U test for two samples.
Instituto Nacional de la Nutrición, Mexico
Results
Summary Culture and identification of Mycobacterium The mean absorbance of the sixteen samples from patients
tuberculosis in cerebrospinal fluid (CSF) of with tuberculous meningitis (group I) was 0.619±SD0.676
patients with tuberculous meningitis is slow, and prognosis (see figure). In all four patients with confirmed diagnosis
depends on early diagnosis and tratament. An enzyme-linked (group IA) the absorbance was above 0-20 (mean
immunosorbent assay with commercial BCG and anti-BCG 0.511±0.112). Of the twelve patients with probable
was designed to detect mycobacterial antigens in human body tuberculous meningitis (group IB), absorbance was above
fluids. Absorbance at 405 nm was measured and readings 0’ 20 in nine (mean for all twlve 0 -655±0.783). In group II,
≽0·20 were considered positive. The readings were above all samples but one had an absorbance below 0 - 20 (mean
0·20 for four patients with confirmed tuberculous meningitis 0.098±0.071); the exception was the patient with
and nine of twelve patients with probable tuberculous cryptococcal meningitis, whose sample had an absorbance of
meningitis. In all but one of ten patients with meningitis of 0’ 293. In all eleven control subjects (group III) readings were
other causes and in eleven control subjects the readings were below 0’ 20 (mean 0 -.086±0.037). The mean absorbance in
below 0·20. The specificity of the test was 95% and its group I was significantly higher than that in groups II and III
sensitivity 81· 25% (U value 4-701, p<0. 001). The specificity of the test was
95% (one false-positive out of twenty-two negative samples
Introduction
tested). There were three false-negative results among sixteen
TUBERCULOUS meningitis remains a common disease in CSF samples from patients with tuberculous meningitis,
developing countries, and the prognosis is closely related to therefore the sensitivity was 8125%.
the stage of the illness at the initiation of treatment.’Culture
and identification of Mycobacterium tuberculosis in
cerebrospinal fluid (CSF) from patients with tuberculous
meningitis is slow and tedious. Results are not available for
2-4 weeks with routine methods,2 or for 1 week with other
assays such as selected ion monitoring,3 and a rapid and
reliable test to detect mycobacterial antigens in body fluids is
therefore needed. We now report the development of an
enzyme-linked immunosorbent assay (ELISA) to detect
soluble antigens in CSF of patients with tuberculous
meningitis. Chaparas and Hendricks have shown that
M tuberculosis and BCG have the same surface antigens, as
tested by a reference antiserum;4 we therefore used antibody
against BCG in the assay.
Patients and Methods
CSF samples were obtained from thirty-eight subjects. Group I,
consisting of sixteen patients with tuberculous meningitis, was
divided into group lA, four patients with bacteriologically Study groups
confirmed disease, and group IB, twelve patients with the Detection of mycobacterial antigens in 38 CSF samples by ELISA in
presumptive diagnosis of tuberculous meningitis, supported by a patients with tuberculous meningitis and controls.
CSF glucose level of <40 mg/dl, protein >40 mg/dl, pleocytosis
with lymphocytosis, negative culture for other bacteria and fungi, Discussion
plus two or more of the following-clinical picture of tuberculous The diagnosis of tuberculous meningitis is usally made on
meningitis, positive tuberculin skin test, evidence of tuberculosis in
other sites, or a clinical response to treatment. Group II consisted of the basis of a history of exposure, clinical picture,
five patients with bacterial meningitis other than tuberculous characteristic CSF, positive tuberculin skin test, and the
meningitis, two with viral encephalitis, two with cysticercosis, one culture of M tuberculosis in CSF.7-9 However, cultures are
patient with meningeal cryptococcosis, and one with meningeal positive in only 55-75% of patients.7,8 Other methods have
tumoral infiltration. Group III consisted of eleven subjects without been used in the diagnosis of tuberculous meningitis, such as
neurological disorders who had lumbar punctures for epidural radioactive bromide partition, reactivity of adenosine
anaesthesia.
The ELISA method used was essentially that of Engvall and
deaminase,1I or detection of mycobacterial metabolites such
as 3- (2-ketohexyl) indoline.12 However, all of these, though
Perlmann.5 Microtitre plates (’Immulon II’; Dynatech, Alexandria,
Virginia) were coated with 100 µl of a 1/800 dilution of rabbit IgG helpful, are cumbersome and require sophisticated
anti-BCG (Dako Laboratories, New York). After overnight technology and are therefore not available in most
incubation at37°C, 100 µl samples of undiluted anda 1/2 dilution of laboratories. In contrast, ELISA is a simple and rapid method
CSF were added and incubated at 37°C for 2 h. A 1/1000 dilution of and is useful in antigen detection of several other infectious
rabbit IgG anti-BCG conjugated to alkaline phosphatase by the two- diseases.13 Our results show that it is possible to detect
652
soluble antigens of mycobacteria in CSF of patients with
tuberculous meningitis. The high sensitivity and specificity
of the test suggest that it may become a useful tool in clinical Preliminary Communication
practice.
The false-positive result in the patient with cryptococcosis ALBENDAZOLE AS A POTENTIAL TREATMENT
could be due to the antibody used in our test cross-reacting FOR HUMAN HYDATIDOSIS
with the cryptococcal polysaccharide or other structures.
Other microorganisms, such as other mycobacteria, A. G. SAIMOT A. MEULEMANS
A. C. CREMIEUX M. D. GIOVANANGELI
corynebacteria, nocardia, and even more common bacteria, B. DELAITRE
share antigens withM tuberculosis. 14,15 The explanation of the J. M. HAY
J. P. COULAUD
false-negative results may be that at the time of the lumbar
puncture some patients were already receiving treatment, Institut de Médecine et d’Epidémiologie Tropicales, Hôpital Claude
which might have lowered the soluble-antigen concentration Bernard, Paris; Laboratoire de Biophysique, UER Xavier Bichat et
in the CSF. One way to improve the test is to use monoclonal Unité INSERM U 13, Paris; Hôpital Louis Mourier, Colombes;
antibodies against M tuberculosis16 or antibodies directed Hôpital Cochin, Paris, France
against antigens 5 and 6 of M tuberculosis, which are specific
for this bacillus. 14,15 Summary The pharmacokinetics of albendazole was
The ELISA method has been used to detect mycobacterial evaluated in 11 patients with hydatid disease
who underwent surgery 12 h after the last dose of drug.
antigens in animal tissues 17 but, to our knowledge, this is the
first time that it has been used to demonstrate these antigens Albendazole and its main metabolite, albendazole-
in body fluids in human tuberculosis. This test could- have sulphoxide, were assayed in the serum from peripheral and
several clinical applications. It is possible that mycobacterial portal blood, the liver, bile, lungs, and hydatid cyst walls and
fluid. After a 10-14 mg/kg daily oral dose of albendazole,
antigens could be detected in other body fluids, simplifying concentrations of 1844±904 ng/g wet tissue of albendazole-
the diagnosis of tuberculosis. The serial determination of
antigen concentration in the fluids of patients during sulphoxide were found in the liver and 749±34 ng/g wet
treatment could be useful in follow-up and in assessing tissue in the lungs. The level in hydatid cyst fluid was
921±314 ng/ml. The drug was also excreted through the bile.
prognosis.
The same daily oral dose produced very stable blood levels
We thank Dr Javier BAez-Villaselior, Dr Agustin Ocejo, Dr Judith
after 2 to 4 days of treatment (600-1000 ng/ml). In another
Dominguez, and the personnel of the Hospital de Infectologia Centro Medico
La Raza, IMSS, Mexico, for their collaboration in obtaining samples; Dr part of the study 3 patients with liver cysts, 2 with peritoneal
Beatriz Remus de Ruiz-Palacios for reviewing the manuscript; and Ms cysts, and 5 with bone cysts received 7 mg/kg twice a day
Guadalupe Perez R. for typing. This work was supported by a research prophylactically and/or therapeutically for 30 days. In all
training scholarship granted to E. S. from Secretaria de Salubridad y cases the treatment was repeated several times after intervals
Asistencia, Mexico.
of 2 weeks. The 3 patients with liver cysts were cured
Correspondence should be addressed to G. M. R.-P., Department of
Infectious Diseases, Instituto Nacional de la Nutricion, Vasco de Quiroga 15, (probably due to treatment); there was no recurrence in the 2
Tlalpan, Mexico 14000 DF, Mexico. patients with peritoneal cysts and very slight improvement in
the 5 cases with bone cysts. The drug was clinically and
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