JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1992, p. 3286-3287
Vol. 30, No. 12
0095-1137/92/123286-02$02.00/0 Copyright C 1992, American Society for Microbiology
Early Diagnosis of Toxoplasmic Encephalitis in AIDS Patients by Dot Blot Hybridization Analysis PZSENNY,l CARLOS ZALA,2 ROBERTO GONZALEZ,2 H. PERELMULTER,2 AND JUAN C. GARBERIl* Centro de Investigaciones Medicas, Albert Einstein Fundacion CIMAE, Luis Viale 2831, 1416 Buenos Aires, and Hospital F. J. Muniz, UTI-ID, Buenos Aires,2 Argentina SERGIO ANGEL,' ESTER MAERO,1 JORGE C. BLANCO,' VIVIANA
Received 20 July 1992/Accepted 24 September 1992
Central nervous system toxoplasmosis is a life-threatening infection with a mortality rate of higher than 60%. An early and rapid diagnosis is important for effective treatment of the disease. A new approach for detection of cerebral toxoplasmosis is described here. DNAs extracted from cells in cerebrospinal fluid samples (0.3 to 0.8 ml) of patients suspected of having cerebral toxoplasmosis were analyzed by a dot blot hybridization technique. A highly repetitive DNA sequence of Toxoplasma gondii (ABGTg4) was nonisotopically labelled with digoxigenin-dUTP and used as a specific DNA probe. Four of six patients analyzed gave positive signals in our hybridization assay. Two of them recovered with pyrimethamine-sulfadiazine, a drug recommended for treatment of toxoplasmosis. The other two patients with positive signals died soon after diagnosis. Patients with negative signals were found to suffer from mycobacterial infection (patient 1) or varicella-zoster virus infection (patient 6).
About 30% of AIDS patients who are seropositive for Toxoplasma gondii will ultimately develop toxoplasmic encephalitis (6). Diagnosis of cerebral toxoplasmosis in these patients remains a problem. Currently, diagnosis relies on a compatible clinical presentation, serological evidence of exposure to T. gondii, and the discovery of cerebral lesions through computer tomography (CT) (10). The diagnosis is confirmed if clinical and tomographic responses with specific treatment are observed within 20 days (10). However, this strategy has an important drawback: the high incidence of bone marrow toxicity with anti-T. gondii drugs (9). Therefore, accurate and rapid diagnosis of toxoplasmosis infection is essential for improved prognosis. Serological parameters for the diagnosis of reactivated infection remain uncertain (7, 9). In these cases, direct detection of the parasite is desirable. Here, we report direct diagnosis of active human cerebral toxoplasmosis by a dot blot hybridization technique. Five human immunodeficiency virus-positive patients suspected of having cerebral toxoplasmosis and one patient who was a negative control were studied as follows. Patient 1 was 25 years old and an intravenous drug user. She developed headaches, horizontal nystagmus, and mild ataxia. Results of CT scanning were normal, and her cerebrospinal fluid (CSF) showed mild pleocytosis and a slight increase in the level of protein. The serum immunoglobulin G (IgG) antibody titer against T. gondii by indirect immunofluorescence (IFI) was 1/64, and the CD4 cell count of peripheral blood mononuclear cells was 315/mm3. Patient 2 was 21 years old and an intravenous drug user with no previous opportunistic infection. Seven days before admission, the patient began to suffer from headaches, vomiting, fever, and photophobia. Neurological examination showed gait disturbance. CT scanning showed a focal hypodense lesion at the level of the cerebellum (IFI IgG antibody titer, 1/512; CD4 cell count, 294/mm3). Patient 3 was a 43-year-old homosexual who suffered from seizures and right hemiparesis. CT scanning *
showed a diffuse cerebral edema and a focal hypodense lesion at the frontal and left occipital hemisphere (IFI IgG antibody titer, 1/512; CD4 cell count, 80/mm3). Patient 4 was a 22-year-old intravenous drug user with previous salmonellosis and histoplasmosis infections. At the time of admission, he presented headaches, fever, and generalized seizures and was diagnosed as having toxoplasmic retinochoroiditis (IFI IgG antibody titer, 1/32; CD4 cell count, 100/mm3). Patient 5 was a 27-year-old heterosexual and was admitted with a cerebellar syndrome. CT scanning showed a focal hypodense lesion at the level of the cerebellum (IFI IgG antibody titer, 1/64; CD4 cell count, 147/mm3). Patient 6, 33 years old, was seronegative for toxoplasmosis but suffered from a varicella-zoster virus infection. Patients 2, 3, 4, and 5 were seronegative for cryptococcus, mycobacterium, and fungi in the culture analysis. In contrast, patient 1 was seropositive for Mycobacterium tuberculosis. In order to detect the parasites present in the CSF samples of the patients suspected of having cerebral toxoplasmosis (4), we used the highly repetitive DNA sequence (ABGTg4) of T. gondii previously described by us (1, 2). ABGTg4 was nonisotopically labelled with digoxigenin-dUTP (DIG DNAlabelling kit from Boehringer Mannheim). Aliquots of CSF samples were spotted onto a nitrocellulose filter and processed as described elsewhere (5). Hybridization conditions and DNA immunodetection were as described previously (1). The presence of mycobacterium and fungi was investigated in aliquots of the same CSF samples by specific culture. Four of the six CSF samples analyzed gave positive signals after hybridization with ABGTg4 (patients 2, 3, 4, and 5) (Fig. 1). Their signals were similar to the ones given by about 5 x 104 T. gondii tachyzoites. This indicates that at least 6 x 104 parasites per ml were present in the CSF samples. The CSF samples from patients 1 and 6 as well as a negative control (a pool of human DNA from T. gondiiseronegative individuals) showed no signals. Patients 2 and 5 were treated with a combination of pyrimethamine and sulfadiazine, and the tomographic and clinical recoveries
Corresponding author. 3286
NOTES
VOL. 30, 1992
CN
R2
P3
P4
P5
CTg
P3
P6
FIG. 1. Aliquots of CFS samples (0.3 ml from patient 3 [P3], spotted twice, and 0.8 ml each from patients 1, 2, 4, 5, and 6 [P1, P2. and P4 to P6]) were spotted onto nitrocellulose filters, and cellular lysis and DNA denaturation were carried out in situ. The filters were hybridized with digoxigenin-dUTP ABGTg4 as a T. gondii-specific DNA probe. CN, 5X 1705 normal human leukocytes; Crg, 5 x 104 T. gondii tachyzoites.
completed on the 25th and 35th days of treatment, respectively. Patients 3 and 4 were treated in the same way, but they unfortunately died within 72 h of the beginning of treatment. It should be pointed out that their disease was quite advanced when the diagnosis was performed. On the other hand, patient 1 recovered after specific treatment for mycobacteriosis, confirming that T. gondii was not involved in the clinical picture. Therefore, the patient could be taken as an extra control of specificity together with patient 6. To our knowledge, this is the first report demonstrating the usefulness of T. gondii repetitive DNA sequences for the diagnosis of active cerebral toxoplasmosis in patients with AIDS. Other workers who used the polymerase chain reaction (PCR) to detect low-copy-number sequences were able to detect parasites in brain tissues from patients with AIDS (7, 8). In biopsy materials, however, it is not possible to discriminate quiescent cysts and the active tachyzoites which are truly responsible for clinical symptoms. Such differentiation may be possible by measuring the amount of PCR product or developing PCR primers specific for RNA species present only in the active-tachyzoite forms of the parasite, as stated by Holliman et al. (8). In contrast, in biological fluids such as CSF, amniotic fluid, or blood, only the tachyzoite form was found (3, 4). The presence of tachyzoites in such fluids indicates an active proliferation of the parasite. This fact allows for the use of a technique that were
3287
is less sensitive than PCR but absolutely specific for the diagnosis of acute toxoplasmosis. We think that this dot blot analysis with a nonradioactive probe in fluids such as CSF and other biological fluids could be an interesting alternative technique for the diagnosis of acute human toxoplasmosis in common clinical laboratories. Further work is currently being performed in our laboratory to assess the statistical significance of the analysis. This work was partially financed by CONICET, by the Instituto de Cooperacion Iberoamericana, and by Boehringer Argentina. We are grateful to Jorge Zorzopulos and Jose Burdman for their critical readings of the manuscript. REFERENCES 1. Angel, S. O., J. C. Blanco, E. Maero, V. Pzsenny, and J. C. Garberi. 1991. Repetitive DNA sequences of Toaoplasma gondii for development of diagnostic probes. Mem. Inst. Oswaldo Cruz Rio J. 86:483-484. 2. Blanco, J. C., S. 0. Angel, E. Maero, V. Pzsenny, P. Serpente, and J. C. Garberi. 1992. Cloning of repetitive DNA sequences from Towoplasma gondu and their usefulness for parasite detection. Am. J. Trop. Med. Hyg. 46:350-357. 3. Brooks, R. G., S. D. Sharma, and J. S. Remington. 1985. Detection of Toxoplasma gondii antigens by a dot-immunobinding technique. J. Clin. Microbiol. 21:113-116. 4. Burg, J. L., C. M. Grover, P. Pouletty, and J. C. Boothroyd. 1989. Direct and sensitive detection of a pathogenic protozoan, Toaoplasma gondri, by polymerase chain reaction. J. Clin. Microbiol. 27:1787-1792. 5. Garberi, J. C., J. C. Blanco, S. 0. Angel, V. Pzsenny, M. C. Arakelian, and A. Pastini. 1990. Toxoplasma gondiu: a rapid method for the isolation of pure tachyzoites. Preliminary characterization of its genome. Mem. Inst. Oswaldo Cruz Rio J. 85:429-434. 6. Grant, I. H., J. W. M. Gold, M. Rosenblum, D. Niedzwiecki, and D. Armstrong. 1990. Toaoplasma gondu serology in HIVinfected patients: the development of central nervous system toxoplasmosis in AIDS. AIDS 4:519-522. 7. Holliman, R. E. 1991. Clinical and diagnostic findings in 20 patients with toxoplasmosis and the acquired immune deficiency syndrome. J. Med. Microbiol. 35:14. 8. Holliman, R. E., J. D. Johnson, S. Gillespie, M. A. Johnson, S. B. Squire, and D. Savva. 1991. New methods in the diagnosis and management of cerebral toxoplasmosis associated with acquired immune deficiency syndrome. J. Infect. Dis. 22:281285. 9. Luft, B. J., and J. S. Remington. 1988. Toxoplasmic encephalitis. J. Infect. Dis. 157:1-6. 10. Pedrol, E., J. M. Gonzalez-Clemente, J. M. Gateli, J. Maliolas, J. M. Miro, F. Graus, R. Alvarez, J. M. Mercader, J. Berenguer, M. T. Jimenez de Anta, M. E. Valls, and E. Soriano. 1990. Central nervous system toxoplasmosis in AIDS patients: efficacy of an intermittent maintenance therapy. AIDS 4:511-517.
Vol. 30, No. 12
0095-1137/92/123286-02$02.00/0 Copyright C 1992, American Society for Microbiology
Early Diagnosis of Toxoplasmic Encephalitis in AIDS Patients by Dot Blot Hybridization Analysis PZSENNY,l CARLOS ZALA,2 ROBERTO GONZALEZ,2 H. PERELMULTER,2 AND JUAN C. GARBERIl* Centro de Investigaciones Medicas, Albert Einstein Fundacion CIMAE, Luis Viale 2831, 1416 Buenos Aires, and Hospital F. J. Muniz, UTI-ID, Buenos Aires,2 Argentina SERGIO ANGEL,' ESTER MAERO,1 JORGE C. BLANCO,' VIVIANA
Received 20 July 1992/Accepted 24 September 1992
Central nervous system toxoplasmosis is a life-threatening infection with a mortality rate of higher than 60%. An early and rapid diagnosis is important for effective treatment of the disease. A new approach for detection of cerebral toxoplasmosis is described here. DNAs extracted from cells in cerebrospinal fluid samples (0.3 to 0.8 ml) of patients suspected of having cerebral toxoplasmosis were analyzed by a dot blot hybridization technique. A highly repetitive DNA sequence of Toxoplasma gondii (ABGTg4) was nonisotopically labelled with digoxigenin-dUTP and used as a specific DNA probe. Four of six patients analyzed gave positive signals in our hybridization assay. Two of them recovered with pyrimethamine-sulfadiazine, a drug recommended for treatment of toxoplasmosis. The other two patients with positive signals died soon after diagnosis. Patients with negative signals were found to suffer from mycobacterial infection (patient 1) or varicella-zoster virus infection (patient 6).
About 30% of AIDS patients who are seropositive for Toxoplasma gondii will ultimately develop toxoplasmic encephalitis (6). Diagnosis of cerebral toxoplasmosis in these patients remains a problem. Currently, diagnosis relies on a compatible clinical presentation, serological evidence of exposure to T. gondii, and the discovery of cerebral lesions through computer tomography (CT) (10). The diagnosis is confirmed if clinical and tomographic responses with specific treatment are observed within 20 days (10). However, this strategy has an important drawback: the high incidence of bone marrow toxicity with anti-T. gondii drugs (9). Therefore, accurate and rapid diagnosis of toxoplasmosis infection is essential for improved prognosis. Serological parameters for the diagnosis of reactivated infection remain uncertain (7, 9). In these cases, direct detection of the parasite is desirable. Here, we report direct diagnosis of active human cerebral toxoplasmosis by a dot blot hybridization technique. Five human immunodeficiency virus-positive patients suspected of having cerebral toxoplasmosis and one patient who was a negative control were studied as follows. Patient 1 was 25 years old and an intravenous drug user. She developed headaches, horizontal nystagmus, and mild ataxia. Results of CT scanning were normal, and her cerebrospinal fluid (CSF) showed mild pleocytosis and a slight increase in the level of protein. The serum immunoglobulin G (IgG) antibody titer against T. gondii by indirect immunofluorescence (IFI) was 1/64, and the CD4 cell count of peripheral blood mononuclear cells was 315/mm3. Patient 2 was 21 years old and an intravenous drug user with no previous opportunistic infection. Seven days before admission, the patient began to suffer from headaches, vomiting, fever, and photophobia. Neurological examination showed gait disturbance. CT scanning showed a focal hypodense lesion at the level of the cerebellum (IFI IgG antibody titer, 1/512; CD4 cell count, 294/mm3). Patient 3 was a 43-year-old homosexual who suffered from seizures and right hemiparesis. CT scanning *
showed a diffuse cerebral edema and a focal hypodense lesion at the frontal and left occipital hemisphere (IFI IgG antibody titer, 1/512; CD4 cell count, 80/mm3). Patient 4 was a 22-year-old intravenous drug user with previous salmonellosis and histoplasmosis infections. At the time of admission, he presented headaches, fever, and generalized seizures and was diagnosed as having toxoplasmic retinochoroiditis (IFI IgG antibody titer, 1/32; CD4 cell count, 100/mm3). Patient 5 was a 27-year-old heterosexual and was admitted with a cerebellar syndrome. CT scanning showed a focal hypodense lesion at the level of the cerebellum (IFI IgG antibody titer, 1/64; CD4 cell count, 147/mm3). Patient 6, 33 years old, was seronegative for toxoplasmosis but suffered from a varicella-zoster virus infection. Patients 2, 3, 4, and 5 were seronegative for cryptococcus, mycobacterium, and fungi in the culture analysis. In contrast, patient 1 was seropositive for Mycobacterium tuberculosis. In order to detect the parasites present in the CSF samples of the patients suspected of having cerebral toxoplasmosis (4), we used the highly repetitive DNA sequence (ABGTg4) of T. gondii previously described by us (1, 2). ABGTg4 was nonisotopically labelled with digoxigenin-dUTP (DIG DNAlabelling kit from Boehringer Mannheim). Aliquots of CSF samples were spotted onto a nitrocellulose filter and processed as described elsewhere (5). Hybridization conditions and DNA immunodetection were as described previously (1). The presence of mycobacterium and fungi was investigated in aliquots of the same CSF samples by specific culture. Four of the six CSF samples analyzed gave positive signals after hybridization with ABGTg4 (patients 2, 3, 4, and 5) (Fig. 1). Their signals were similar to the ones given by about 5 x 104 T. gondii tachyzoites. This indicates that at least 6 x 104 parasites per ml were present in the CSF samples. The CSF samples from patients 1 and 6 as well as a negative control (a pool of human DNA from T. gondiiseronegative individuals) showed no signals. Patients 2 and 5 were treated with a combination of pyrimethamine and sulfadiazine, and the tomographic and clinical recoveries
Corresponding author. 3286
NOTES
VOL. 30, 1992
CN
R2
P3
P4
P5
CTg
P3
P6
FIG. 1. Aliquots of CFS samples (0.3 ml from patient 3 [P3], spotted twice, and 0.8 ml each from patients 1, 2, 4, 5, and 6 [P1, P2. and P4 to P6]) were spotted onto nitrocellulose filters, and cellular lysis and DNA denaturation were carried out in situ. The filters were hybridized with digoxigenin-dUTP ABGTg4 as a T. gondii-specific DNA probe. CN, 5X 1705 normal human leukocytes; Crg, 5 x 104 T. gondii tachyzoites.
completed on the 25th and 35th days of treatment, respectively. Patients 3 and 4 were treated in the same way, but they unfortunately died within 72 h of the beginning of treatment. It should be pointed out that their disease was quite advanced when the diagnosis was performed. On the other hand, patient 1 recovered after specific treatment for mycobacteriosis, confirming that T. gondii was not involved in the clinical picture. Therefore, the patient could be taken as an extra control of specificity together with patient 6. To our knowledge, this is the first report demonstrating the usefulness of T. gondii repetitive DNA sequences for the diagnosis of active cerebral toxoplasmosis in patients with AIDS. Other workers who used the polymerase chain reaction (PCR) to detect low-copy-number sequences were able to detect parasites in brain tissues from patients with AIDS (7, 8). In biopsy materials, however, it is not possible to discriminate quiescent cysts and the active tachyzoites which are truly responsible for clinical symptoms. Such differentiation may be possible by measuring the amount of PCR product or developing PCR primers specific for RNA species present only in the active-tachyzoite forms of the parasite, as stated by Holliman et al. (8). In contrast, in biological fluids such as CSF, amniotic fluid, or blood, only the tachyzoite form was found (3, 4). The presence of tachyzoites in such fluids indicates an active proliferation of the parasite. This fact allows for the use of a technique that were
3287
is less sensitive than PCR but absolutely specific for the diagnosis of acute toxoplasmosis. We think that this dot blot analysis with a nonradioactive probe in fluids such as CSF and other biological fluids could be an interesting alternative technique for the diagnosis of acute human toxoplasmosis in common clinical laboratories. Further work is currently being performed in our laboratory to assess the statistical significance of the analysis. This work was partially financed by CONICET, by the Instituto de Cooperacion Iberoamericana, and by Boehringer Argentina. We are grateful to Jorge Zorzopulos and Jose Burdman for their critical readings of the manuscript. REFERENCES 1. Angel, S. O., J. C. Blanco, E. Maero, V. Pzsenny, and J. C. Garberi. 1991. Repetitive DNA sequences of Toaoplasma gondii for development of diagnostic probes. Mem. Inst. Oswaldo Cruz Rio J. 86:483-484. 2. Blanco, J. C., S. 0. Angel, E. Maero, V. Pzsenny, P. Serpente, and J. C. Garberi. 1992. Cloning of repetitive DNA sequences from Towoplasma gondu and their usefulness for parasite detection. Am. J. Trop. Med. Hyg. 46:350-357. 3. Brooks, R. G., S. D. Sharma, and J. S. Remington. 1985. Detection of Toxoplasma gondii antigens by a dot-immunobinding technique. J. Clin. Microbiol. 21:113-116. 4. Burg, J. L., C. M. Grover, P. Pouletty, and J. C. Boothroyd. 1989. Direct and sensitive detection of a pathogenic protozoan, Toaoplasma gondri, by polymerase chain reaction. J. Clin. Microbiol. 27:1787-1792. 5. Garberi, J. C., J. C. Blanco, S. 0. Angel, V. Pzsenny, M. C. Arakelian, and A. Pastini. 1990. Toxoplasma gondiu: a rapid method for the isolation of pure tachyzoites. Preliminary characterization of its genome. Mem. Inst. Oswaldo Cruz Rio J. 85:429-434. 6. Grant, I. H., J. W. M. Gold, M. Rosenblum, D. Niedzwiecki, and D. Armstrong. 1990. Toaoplasma gondu serology in HIVinfected patients: the development of central nervous system toxoplasmosis in AIDS. AIDS 4:519-522. 7. Holliman, R. E. 1991. Clinical and diagnostic findings in 20 patients with toxoplasmosis and the acquired immune deficiency syndrome. J. Med. Microbiol. 35:14. 8. Holliman, R. E., J. D. Johnson, S. Gillespie, M. A. Johnson, S. B. Squire, and D. Savva. 1991. New methods in the diagnosis and management of cerebral toxoplasmosis associated with acquired immune deficiency syndrome. J. Infect. Dis. 22:281285. 9. Luft, B. J., and J. S. Remington. 1988. Toxoplasmic encephalitis. J. Infect. Dis. 157:1-6. 10. Pedrol, E., J. M. Gonzalez-Clemente, J. M. Gateli, J. Maliolas, J. M. Miro, F. Graus, R. Alvarez, J. M. Mercader, J. Berenguer, M. T. Jimenez de Anta, M. E. Valls, and E. Soriano. 1990. Central nervous system toxoplasmosis in AIDS patients: efficacy of an intermittent maintenance therapy. AIDS 4:511-517.
