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Letters to the Editor
Detection o f m y c o b a c t e r i a l antigen by dot blot assay in the cerebrospinal fluid o f patients with t u b e r c u l o u s m e n i n g i t i s Accepted for publication 3 July I99o Sir, Tuberculous meningitis ( T B M ) is one of the most common forms of extrapulmonary tuberculosis in developing countries and is being recognised with increasing frequency, especially with the emergence of AIDS. x Rapid diagnosis of T B M i s essential in reducing the high mortality rate and the neurological sequelae associated with the disease. Direct acid-fast staining of the cerebrospinal fluid (CSF) is the only quick microbiological method generally available, but it lacks sensitivity. Recently, immunological methods based on the detection of mycobacterial antigen in the C S F have been described with satisfactory results for the rapid diagnosis of T B M f l We wish to make a preliminary report on a direct dot blot enzyme immunoassay which we have developed for the early detection of mycobacterial antigen in the C S F from patients with T B M . We have previously applied the technique, with some modifications, in the determination of serum antigen or antibody in various infectious diseases? -5 A total of 38 samples of C S F was collected from 11 patients diagnosed as having tuberculous meningitis. In eight patients, T B M was proved bacteriologically by culturing the organism from the C S F or by finding acid-fast bacilli microscopically in it. In the others, the diagnosis was made on the basis of clinical features together with characteristic changes in the C S F (lymphocytosis, an increased amount of protein, a reduced concentration of glucose and an increase in adenosine deaminase) as well as response to antituberculous treatment. In addition, 25 samples of C S F from patients with bacterial, fungal and viral meningitis were examined. T h e dot blot assay was performed on a 4 × 4 x 6 cm nitrocellulose paper sheet (BioRad) placed in a Petri dish. Undiluted specimens of C S F were spotted in 2 #1 amounts on to the nitrocellulose and stabilised by drying for Io min at room temperature. In order to block free binding sites, the nitrocellulose was incubated by shaking for 30 min at 37 °C with 3 ml phosphate-buffered saline (PBS) containing 0"25 % T w e e n 20. After removing the blocking solution, horseradish peroxidase-conjugated rabbit I g G antibody to Bacille Calmette-Gu6rin (BCG) (Dako, Denmark) diluted I in 5oo in PBS was added in 3 ml volumes before the plate was incubated on a rotator for 90 min at room temperature. After two washes with PBS containing 0"05 % T w e e n 20 and a final washing with PBS, peroxidase substrate solution (I ml 0"3 % 4-chloro-Inaphthol in anhydrous methanol plus 5 ml PBS and 3 #1 3 0 % H~O2) was added. Positive reactions appeared as well-defined blue dots against the nitrocellulose white background. In order to estimate the sensitivity of the dot blot technique, a sonicated antigen prepared from Mycobacterium tuberculosis strain H37Rv (ATCC, Ioa) according to the method of Chaparas et al., ° was tested in concentrations ranging from 5 to 5000 n g / m l in PBS. T h e amount of antigen clearly detectable by the dot blot technique was I O0 ng/ml. Mycobacterial antigen was demonstrated in all samples of C S F ( i i / I I ) from patients with T B M before treatment, while only 2 (8 %) of 25 samples from patients with non-mycobacterial meningitis gave a positive reaction. T h e false-positive reactions were related to two patients with other forms of bacterial meningitis and were likely to have been due to antigens shared between M. tuberculosis and other bacterial species. Further analysis of C S F samples, collected from seven patients with
Letters to the Editor
IO7
T B M after c h e m o t h e r a p y had begun, showed a progressive decline in the concentrations of antigen. F r o m our preliminary results, the dot blot assay for detecting mycobacterial antigen in C S F appears to be a simple and sensitive m e t h o d for the early diagnosis of T B M . It also seems to be useful in monitoring the effectiveness of antituberculous treatment. T h e dot blot procedure described here is a more rapid procedure than other methods for detecting antigen. It can be readily p e r f o r m e d in routine clinical laboratories without the use of expensive and sophisticated apparatus, such as a g a m m a counter or E L I SA plate reader. Because of its ease of performance and low cost, the dot blot assay for mycobacterial antigen is suitable for use in developing countries where tuberculous meningitis is very prevalent.
C. M . Mastroianni* V. Vullo F. Paoletti A. P. Massetti F. Sorice S. Delia
Department of Infectious Diseases, University La Sapienza, Policlinico Umberto I, Viale Regina Elena 33I, oOI6I Rome, Italy * Author to whom correspondence should be addressed. References
i. Harries AD. Tuberculosis and human immunodeficiency virus infection in developing countries. Lancet I99o; 335:387-39 °. 2. Daniel TM. New approaches to the rapid diagnosis of tuberculous meningitis. J Infect Dis I987; I55 : 599-602. 3. Sorice F, Vullo V, Contini C et al. Use of dot immunobinding assay for the rapid diagnosis of human hydatidosis. Boll Ist Sieroter Milan I985; 64: 414-418. 4- Delia S, Vullo V, Mastroianni CM et al. Use of dot immunobinding assay (DIB) for the rapid diagnosis of human brucellosis. J Infect I987; I4: 88-89. 5. Vullo V, Mastroianni CM, Paoletti F et al. Detection of Salmonella typhi lipopolysaccharide (LPS) in typhoid sera by dot immunobinding assay (DIB). Microbios Letters I988; 38: 79-83. 6. Chaparas SD, Brown T, Hyman I. Antigenic relationships among species of Mycobacterium studied by fused rocked immunoelectrophoresis. Int J Syst Bacteriol I978 ; z8: 547-560.
P s o a s a b s c e s s s e c o n d a r y to b r u c e l l o s i s
Accepted for publication 9 July I99O Sir, Brucellosis usually presents with a febrile s y n d r o m e without an apparent focus of infection. However, the disease often results in complications, the most frequent being those involving bones and joints. 1-5 T h e vertebral column is one of the most c o m m o n l y involved sites. Paravertebral and epidural abscesses have been reported on m a n y occasions. 1,5 Although there have been frequent reports of psoas abscesses with other infections of the vertebrae, we have found only one as a result of brucellosis. 6 We report a patient with brucellosis and lumbar spondylitis, who developed bilateral psoas abscesses. A 54-year-old male waiter, an occasional consumer of unpasteurised milk products, complained of l u m b a r pain for a year. D u r i n g this time the patient had irregular fever, sweating, malaise and a 6 kg weight loss. Physical examination revealed lumbar
Letters to the Editor
Detection o f m y c o b a c t e r i a l antigen by dot blot assay in the cerebrospinal fluid o f patients with t u b e r c u l o u s m e n i n g i t i s Accepted for publication 3 July I99o Sir, Tuberculous meningitis ( T B M ) is one of the most common forms of extrapulmonary tuberculosis in developing countries and is being recognised with increasing frequency, especially with the emergence of AIDS. x Rapid diagnosis of T B M i s essential in reducing the high mortality rate and the neurological sequelae associated with the disease. Direct acid-fast staining of the cerebrospinal fluid (CSF) is the only quick microbiological method generally available, but it lacks sensitivity. Recently, immunological methods based on the detection of mycobacterial antigen in the C S F have been described with satisfactory results for the rapid diagnosis of T B M f l We wish to make a preliminary report on a direct dot blot enzyme immunoassay which we have developed for the early detection of mycobacterial antigen in the C S F from patients with T B M . We have previously applied the technique, with some modifications, in the determination of serum antigen or antibody in various infectious diseases? -5 A total of 38 samples of C S F was collected from 11 patients diagnosed as having tuberculous meningitis. In eight patients, T B M was proved bacteriologically by culturing the organism from the C S F or by finding acid-fast bacilli microscopically in it. In the others, the diagnosis was made on the basis of clinical features together with characteristic changes in the C S F (lymphocytosis, an increased amount of protein, a reduced concentration of glucose and an increase in adenosine deaminase) as well as response to antituberculous treatment. In addition, 25 samples of C S F from patients with bacterial, fungal and viral meningitis were examined. T h e dot blot assay was performed on a 4 × 4 x 6 cm nitrocellulose paper sheet (BioRad) placed in a Petri dish. Undiluted specimens of C S F were spotted in 2 #1 amounts on to the nitrocellulose and stabilised by drying for Io min at room temperature. In order to block free binding sites, the nitrocellulose was incubated by shaking for 30 min at 37 °C with 3 ml phosphate-buffered saline (PBS) containing 0"25 % T w e e n 20. After removing the blocking solution, horseradish peroxidase-conjugated rabbit I g G antibody to Bacille Calmette-Gu6rin (BCG) (Dako, Denmark) diluted I in 5oo in PBS was added in 3 ml volumes before the plate was incubated on a rotator for 90 min at room temperature. After two washes with PBS containing 0"05 % T w e e n 20 and a final washing with PBS, peroxidase substrate solution (I ml 0"3 % 4-chloro-Inaphthol in anhydrous methanol plus 5 ml PBS and 3 #1 3 0 % H~O2) was added. Positive reactions appeared as well-defined blue dots against the nitrocellulose white background. In order to estimate the sensitivity of the dot blot technique, a sonicated antigen prepared from Mycobacterium tuberculosis strain H37Rv (ATCC, Ioa) according to the method of Chaparas et al., ° was tested in concentrations ranging from 5 to 5000 n g / m l in PBS. T h e amount of antigen clearly detectable by the dot blot technique was I O0 ng/ml. Mycobacterial antigen was demonstrated in all samples of C S F ( i i / I I ) from patients with T B M before treatment, while only 2 (8 %) of 25 samples from patients with non-mycobacterial meningitis gave a positive reaction. T h e false-positive reactions were related to two patients with other forms of bacterial meningitis and were likely to have been due to antigens shared between M. tuberculosis and other bacterial species. Further analysis of C S F samples, collected from seven patients with
Letters to the Editor
IO7
T B M after c h e m o t h e r a p y had begun, showed a progressive decline in the concentrations of antigen. F r o m our preliminary results, the dot blot assay for detecting mycobacterial antigen in C S F appears to be a simple and sensitive m e t h o d for the early diagnosis of T B M . It also seems to be useful in monitoring the effectiveness of antituberculous treatment. T h e dot blot procedure described here is a more rapid procedure than other methods for detecting antigen. It can be readily p e r f o r m e d in routine clinical laboratories without the use of expensive and sophisticated apparatus, such as a g a m m a counter or E L I SA plate reader. Because of its ease of performance and low cost, the dot blot assay for mycobacterial antigen is suitable for use in developing countries where tuberculous meningitis is very prevalent.
C. M . Mastroianni* V. Vullo F. Paoletti A. P. Massetti F. Sorice S. Delia
Department of Infectious Diseases, University La Sapienza, Policlinico Umberto I, Viale Regina Elena 33I, oOI6I Rome, Italy * Author to whom correspondence should be addressed. References
i. Harries AD. Tuberculosis and human immunodeficiency virus infection in developing countries. Lancet I99o; 335:387-39 °. 2. Daniel TM. New approaches to the rapid diagnosis of tuberculous meningitis. J Infect Dis I987; I55 : 599-602. 3. Sorice F, Vullo V, Contini C et al. Use of dot immunobinding assay for the rapid diagnosis of human hydatidosis. Boll Ist Sieroter Milan I985; 64: 414-418. 4- Delia S, Vullo V, Mastroianni CM et al. Use of dot immunobinding assay (DIB) for the rapid diagnosis of human brucellosis. J Infect I987; I4: 88-89. 5. Vullo V, Mastroianni CM, Paoletti F et al. Detection of Salmonella typhi lipopolysaccharide (LPS) in typhoid sera by dot immunobinding assay (DIB). Microbios Letters I988; 38: 79-83. 6. Chaparas SD, Brown T, Hyman I. Antigenic relationships among species of Mycobacterium studied by fused rocked immunoelectrophoresis. Int J Syst Bacteriol I978 ; z8: 547-560.
P s o a s a b s c e s s s e c o n d a r y to b r u c e l l o s i s
Accepted for publication 9 July I99O Sir, Brucellosis usually presents with a febrile s y n d r o m e without an apparent focus of infection. However, the disease often results in complications, the most frequent being those involving bones and joints. 1-5 T h e vertebral column is one of the most c o m m o n l y involved sites. Paravertebral and epidural abscesses have been reported on m a n y occasions. 1,5 Although there have been frequent reports of psoas abscesses with other infections of the vertebrae, we have found only one as a result of brucellosis. 6 We report a patient with brucellosis and lumbar spondylitis, who developed bilateral psoas abscesses. A 54-year-old male waiter, an occasional consumer of unpasteurised milk products, complained of l u m b a r pain for a year. D u r i n g this time the patient had irregular fever, sweating, malaise and a 6 kg weight loss. Physical examination revealed lumbar
