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logic differences, it is impossible to accurately compare the relative sensitivities of these primers, but they all seem to be specific. Acknowledgment
We thank Kate Gross for manuscript preparation. References
6. Feldman S. Epp E. Detection of viremia during incubation of varicella. J Pediatr 1979;94:746-8. 7. Ozaki T. Ichikawa T. Matsui Y. et al. Viremic phase in nonimmunocompromised children with varicella. J Pediatr 1984;104:85-7. 8. Ozaki T. Ichikawa T. Matsui Y. et al. Lymphocyte-associated viremia in varicella. J Med Virol 1986; 19:249-53. 9. Vonsover A. Leventon-Kriss S. Langer A. et al. Detection of varicellazoster virus in lymphocytes by DNA hybridization. J Med Virol 1987;21: 57-66. 10. Kido S. Ozaki T. Asada H. et al. Detection of varicella-zoster virus (VZV) DNA in clinical samples from patients with VZV by the polymerase chain reaction. J Clin Microbiol 1991;29:76-9. II. Ozaki T. Miwata H. Matsui Y. Varicella-zoster virus DNA in throat swabs. Arch Dis Child 1990;65:333-4. 12. Baird RE. Daly p. Sawyer MH. Varicella arthritis diagnosed by polymerase chain reaction. Pediatr Infect Dis J 1991;10:950-2. 13. Koropchak CM. Graham G. Palmer J. et al. Investigation of varicellazoster virus infection by polymerase chain reaction in the immunocompromised host with acute varicella. J Infect Dis 1991;163: 101621. 14. Asano Y. Itakura N. Hiroishi Y. et al. Viremia is present in incubation period in nonimmunocompromised children with varicella. J Pediatr 1985; I 06:69-71. 15. Dankner WM. McCutchan JA. Richman DO. Hirata K. Spector SA. Localization of human cytomegalovirus in peripheral blood leukocytes by in situ hybridization. J Infect Dis 1990; 161:31-6.
Detection of Human Immunodeficiency Virus Type 2 in Brain Tissue Dominic E. Dwyer, Sophie Matheron, Serge Bakchine, Jean-Marie Bechet, Luc Montagnier, and Rosemay Vazeux
Unite d'Oncologie Virale, lnstitut Pasteur. and Service des Maladies Infectieuses et Tropicales, Hiipital Bichat-Claude Bernard. and Service de Neurologie et Neuropsychologie, Hiipital de la Salpetriere, Paris. France
Infection due to human immunodeficiency virus (HIV) type 2 is believed to cause a clinical picture similar to that of HIV-1, although extensive data are not available. In 2 patients with West African exposure and neurologic symptoms, HIV-2 was detected in the central nervous system using DNA and RNA polymerase chain reaction, in situ hybridization, and immunohistology. In the first patient, the neurologic disease was most likely due to productive infection with HIV-2. In the second, a combination of neuropathologic abnormalities (including the presence of HIV-2) explained the clinical features. Thus HIV-2, like HIV-l, can be readily detected in brain tissue in patients with neurologic abnormalities, although the exact role ofHIV-2 in pathogenesis of AIDS-associated neurologic disease requires further study.
Human immunodeficiency virus (HIV) type 2 was originally isolated from West African patients and has been recognized since 1985 as a cause ofAIDS [1]. Although there have not been extensive longitudinal studies of HIV-2-induced
Received 16 December 1991; revised 10 March 1992. Presented in part: VI International Conference on AIDS. San Francisco. June 1990 (abstract FA 324). Reprints or correspondence (present address): Dr. Dominic E. Dwyer. Department of Virology, ICPMR, Westmead Hospital. Westmead, NSW, 2145, Australia.
The Journal of Infectious Diseases 1992;166:888-91 © 1992 by The Universityof Chicago. All rights reserved. 0022-1899/92/6604-0029$01.00
disease, it is known that HIV-2-infected patients may have a clinical presentation similar to HIV-L-infected patients, including primary infection, an asymptomatic period, AIDSrelated complex, and AIDS [2-4]. However, there have been suggestions that HIV-2-infected patients may have a longer asymptomatic period [5-7], that they deteriorate clinically more slowly than Hlv-L-infected patients, and that asymptomatic patients have fewer immunologic disturbances than those with HIV-l [8]. It has also been suggested in a community-based prevalence study in Guinea-Bissau, West Africa, that HIV-2 may not be as readily transmitted as HIV-I, the incubation period may be longer, vertical transmission may be rare, and HIV-2 may be less virulent [9].
Downloaded from http://jid.oxfordjournals.org/ at University of Sussex on September 5, 2015
I. Davison AJ. Scott JE. The complete DNA sequence of varicella-zoster virus. J Gen Virol 1986;67: 1759-816. 2. Mahalingam R. Well ish M. Wolf' W, et al. Latent varicella-zoster viral DNA in human trigeminal and thoracic ganglia. N Engl J Med 1990;323:627-31. 3. Trilifajova J. Bryndova D. Rye M. Isolation of varicella-zoster virus from pharyngeal and nasal swabs in varicella patients. J Hyg Epidemiol Microbiol Immunol 1984;28:201-6. 4. Ozaki T. Matsui Y. Asano Y. Okuno T. Yamanishi K. Takahashi M. Study of virus isolation from pharyngeal swabs in children with varicella. Am J Dis Child 1989; 143: 1448-50. 5. Asano Y. Itakura N. Hiroishi Y. et al. Viral replication and immunologic responses in children naturally infected with varicella-zoster virus and in varicella vaccine recipients. J Infect Dis 1985; 152:8638.
1ID 1992; 166 (October)
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Clinical neurologic dysfunction is commonly recognized in HIV-I-infected patients, and 80%-90% of AIDS patients will have neuropathologic abnormalities at autopsy. These include direct involvement by HIV-I, manifesting most commonly as AIDS encephalopathy or AIDS dementia complex but also as vacuolar myelopathy, aseptic meningitis and peripheral neuropathy, opportunistic infections, and tumors [10, 11]. There have been only occasional clinical descriptions of HIV-2-induced central nervous system (CNS) disease, mostly in conjunction with opportunistic infections [2, 4, 12]. We present evidence of direct neurologic involvement by HIV-2 in 2 patients, using DNA and RNA polymerase chain reaction (PCR), in situ hybridization, and immunohistology.
Patient J. A 27-year-old woman (born in Mozambique but spent many years in Senegal) was first diagnosed as having HIV2-associated AIDS in January 1987, when she presented with Pneumocystis carinii pneumonia 2 months after delivering a normal infant. Her T41ymphocyte count was 34/mm3 • Over the next 10 months she was noted to be severely depressed, with impaired cognitive function (but no specific neurologic abnormalities on physical examination). Sixteen months after the diagnosis of P. carinii pneumonia, she was diagnosed as having cytomegalovirus and toxoplasmosis retinitis that resolved with treatment; 22 months later she was readmitted with worsening confusion, aggression, and a decreased level of conciousness against the background of a depressive syndrome. On this admission there was no clinical evidence of a CNS opportunistic infection, no progression of the previously noted retinitis, and computed tomography (CT) revealed cortical atrophy. Cerebrospinal fluid (CSF) examination was normal, and cultures ofCSF and blood were negative for fungi, bacteria, and viruses. Results of routine biochemical and liver function tests were normal. A mild pancytopenia was confirmed by bone marrow examination, and staining of the marrow for microorganisms was negative. The patient remained hospitalized for 5 months before dying of presumed septicemia. Patient 2. A 67-year-old French heterosexual man (with a history of frequent and prolonged travel in Africa) had a history
Figure 1. Ethidium bromide-stained agarose gel and autoradiograph of products of DNA polymerase chain reaction using env primers. Samples from: lane I, patient 1; lanes 2 and 5, patient 2 (separate samples);lanes 3 and 6,2 HIV-I-infected patients; lanes 4 and 7, 2 uninfected patients; lane 8, 250 ng of DNA, HIV-2-infected CEM cells; lane 9, 250 ng of DNA, HIV-I-infected CEM cells; lane 10, 250 ng of DNA, uninfected CEM cells; lane 11, no DNA.
of 18 months of slowly progressive memory decline, increased emotional lability, and occasional episodes of confusion with delusions. Results of physical examination at the onset of this decline were initially reported as normal, and investigations (including HIV-l but not HIV-2 serology) were normal except for a CT scan showing mild frontal cortical atrophy. In the absence of any obvious cause for this neurologic decline, a clinical diagnosis of Alzheimer's dementia was made. His last admission was precipitated by a 4-day history of confusion, and clinical examination revealed a febrile (38.7°C), drowsy, and disorientated man with bradykinesia, a mild diffuse rigidity (but no tremor), and frontal lobe dysfunction including bilateral grasp reflexes, slowed mentation, diminished verbal fluency, and behavioral disinhibition. Investigation showed HIV-2 antibodies in the blood and CSF, HIV-2 antigenemia, and a T41ymphocyte count of 68/mm 3 . CSF analysis revealed two mononuclear cells and a mildly elevated protein level with oligoclonal IgG bands. Cultures of CSF and blood and serologic testing for other infectious agents (including cytomegalovirus, Toxoplasma species, Pneumocystis species, herpes simplex virus, syphilis, and hepatitis B virus) were negative. There was no evidence of a toxic cause for this presentation, and results of routine hematologic, biochemical, and liver and thyroid function tests were normal, as were vitamin B9 and B12 levels. The patient died 9 weeks later of bronchopneumonia. Histopathologic examination. CNS tissue was obtained at autopsy from both patients for histopathologic examination with hematoxylin-eosin, Nissl's, Wolcke, Bodian, Grocott-Gomori, Perls', and Gram's stains. Samples from cerebral hemispheres, basal ganglia, brain stem, and cerebellum were taken, subdivided, and stored at -80 a C. CNS samples from HIV-I-infected and -uninfected patients were also obtained at autopsy and used as negative controls for immunohistology and in situ hybridization. Brain tissue was not cultured for viruses. Pc.R. Total DNA and RNA were extracted simultaneously from samples of white matter and basal ganglia from each patient by cesium chloride purification. HIV-2-specific primer pairs in the long terminal repeat (LTR) (5'-GCCCTGGGAGGTTCTCTCC-3', 5'-GACCAGGCGGCGACTAGGAGAGAT-3' [187 bpJ), gag (5'-TGGCGCCTGAACAGGGAC-3', 5'TGCCCACACAATATGTTTTA-3' [323 bpD, and env regions (5'-TATAGGCCTGTTTTCTCTTCCCC-3', 5'-ATCAGCTGGCGGATCAGGAAATG-3' [169 bpD from HIV-2RoD were used for DNA PCR. Primers for RNA PCR were in the second
Downloaded from http://jid.oxfordjournals.org/ at University of Sussex on September 5, 2015
Materials and Methods
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tients using the env primers is shown in figure 1). peR to detect tat mRNA was positive in two separate samples from patient 1. In situ hybridization using HIV-2 gag probes in multiple brain sections showed strong signals in cells disseminated in the parenchyma and perivascular regions (figure 2). Patient 2. Histopathologic examination showed no evidence ofHI V subacute encephalitis, but some neurofibrillary
Downloaded from http://jid.oxfordjournals.org/ at University of Sussex on September 5, 2015
(5'-GAAATCCTCTCTCAGCTATACC-3') and third exons (antisense env primer above [313 bp]) of the tat mRNA coding sequence. With a modification of Saiki et al.'s method [13], PCR was done on 2 J.Lg of extracted DNA or eDNA from the reverse transcription of 2 J.Lg of RNA. Aliquots were run on ethidium bromide-stained agarose gels, transferred, and hybridized with 32P-end-labeled probes (LTR probe 5'-TCTGCCCAGCACCGGCCAAGTGC-3', gag probe 5'-GGCCCGGCGGAAAGAAAAA-3', env and tat probes 5'-ATATATGCTATCGGCCAGGGCCA-3'). Negative controls included both DNA and RNA from uninfected and HIV-I-infected brain tissue extracted simultaneously with the patient samples and reactions containing no template DNA. A positive control using 250 ng of DNA or RNA extracted from HIV-2-infected CEM cells was included. Immunohistologic examination. Tissue sections were deposited on neoprene-coated glass slides, acetone-fixed, and stored at -20°C for immunologic staining and in situ hybridization. We studied at least 10 tissue blocks from each patient, with samples checked for HIV-2 antigens using monoclonal antibodies directed against the p 18 and p25 viral antigens (gift of B. Parekh, Bio-Rad, Richmond, CA). Toxoplasma, cytomegalovirus, herpes simplex viruses 1 and 2, and IC virus antigens were examined in each sample by immunologic staining as previously described [14]. All tissue blocks were screened for the presence of monocyte/macrophage/microglial abnormalities using antibodies directed against CD68 (KiM?; Behring, Marburg, Germany). Monocytes and Band T lymphocytes were detected using antibodies directed against CD 14, CD 19, CD3, CD8, and CD4 molecules (Becton Dickinson, Mountain View, CA), and CNS cells were identified using antibodies against astrocytes (GFAP; Amersham, Buckinghamshire, UK), neurons (D. Paulin, Institut Pasteur), and endothelial cells (Factor VIII; Dakopatts, Carpinteria, CA). In situ hybridization. HIV-2 gag probes were labeled by in vitro transcription with 35S-labeled UTP and 35S-labeled ATP, and in situ hybridization was done as previously described [14]. Samples from HIV-2-uninfected patients were included as negative controls; HIV-2-infected CEM cells and brain tissue from simian immunodeficiency virus-infected rhesus macaques were used as positive controls.
110 1992; 166 (October)
Results Patient 1. Histopathologic examination of brain tissue revealed diffuse demyelination with macrophage infiltration in perivascular and periventricular areas and swelling ofneurons and oligodendrocytes. Occasional clusters of Toxoplasma cysts without surrounding inflammation were seen in the occipital cortex and near the lateral ventricles. Immunohistology using monoclonal antibodies to HIV-2 pl8 and p24 antigens was weakly positive, but no Toxoplasma species, cytomegalovirus, herpes simplex virus, IC virus, or HIV-1 were detected by this technique. DNA PCR on a single sample of brain tissue was positive using all three primer pairs, with a further two separate samples positive using only the env primers (an example of the DNA PCR for both pa-
Figure 2. In situ hybridization. A, patient I; B, patient 2; C, uninfected control.
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degeneration and senile plaques were observed in the gray matter with Lewy bodies in the substantia nigra (features of both Alzheimer's and Parkinson's diseases). There were no microglial nodules and only minimal perivascular infiltration. Immunohistologic tests for HIV-2 and other infectious agents were negative. However, HIV-2 DNA PCR using three primer pairs was positive in two separate samples, with two of a further four samples positive using only the env primers. PCR for tat mRNA was negative in five samples. In situ hybridization for H1V-2 showed positive signals in cells in perivascular regions.
Discussion
Africa, more studies using these techniques are required to further delineate the disease spectrum of this virus.
Acknowledgment
We thank C. Marche from Hopital Bichat-Claude Bernard, Paris, for doing the autopsy on patient I.
References I. Clavel F. Guetard D. Brun-Vezinet F. et al. Isolation ofa new human retrovirus from West African patients with AIDS. Science 1986;233:343-6. 2. Clave I F, Mansinho K. Chamaret S. et al. Human immunodeficiency virus type 2 infection associated with AIDS in West Africa. N Engl J Med 1987;316:1180-5. 3. Besnier J, Barin F, Baillou A. et al. Symptomatic HIV-2 primary infection [letter]. Lancet 1990; I:798. 4. Brun-Vezinet F. Rey MA, Katlama C, et aI. Lymphadenopathy associated virus type 2 in AIDS and AIDS-related complex. Lancet 1987; I: 128-32. 5. Ancelle R. Bletry 0, Baglin AC, et al. Long incubation period for HIV2 infection [letter]. Lancet 1987; I:688-9. 6. Dufoort G. Courouce AM, Ancelle-Park R, Bletry O. No clinical signs 14 years after HIV-2 transmission via blood transfusion [letter]. Lancet 1988;2: 510. 7. Gody M, Ouattara SA. De The G. Clinical experience of AIDS in relation to HIV-I and HIV-2 infection in a rural hospital in Ivory Coast. Africa. AIDS 1988;2:433-6. 8. Marlink RG. Ricard D. M'Boup S. et al. Clinical hematologic and immunologic cross-sectional evaluation of individuals exposed to the human immunodeficiency virus type-2. AIDS Res Hum Retroviruses 1988;4: 137-48. 9. Poulsen AG. Kuinesdal B. Aaby P. et al. Prevalence of and mortality from human immunodeficiency virus type 2 in Bissau. West Africa. Lancet 1989;1:827-30. 10. Price RW, Brew B. Sidtis J, et al. The brain in AIDS: central nervous system HIV-I infection and AIDS dementia complex. Science 1988;239:586-92. II. de la Monte SM. Ho DO, Schoeley RT. et al. Subacute encephalomyelitis of AIDS and its relation to HTLV-III infection. Neurology 1987;37:562-9. 12. Hormigo A, Bravo-Marques JM. Souza-Ramalho P, et al. Uveorneningitis in a human immunodeficiency type 2 seropositive patient. Ann Neurol 1988;23:308-10. 13. Saiki RK. Gelfand DH. Stoffel S, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988;239:487-91. 14. Vazeux R, Brousse N, Jarry A, et al. AIDS subacute encephalitis: identification of HIV infected cells. Am J PathoI1987;126:403-10.
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These cases confirm that HIV-2, like HIV-1, can infect the CNS, and that this neurologic involvement is readily detected and localized by PCR and in situ hybridization. In patient 1, the clinical, histopathologic, and in situ hybridization findings were consistent with an HIV-2-associated encephalopathy, with active viral replication suggested by the detection of both viral DNA and mRNA. In patient 2, typical features of AIDS encephalopathy were not found on histopathologic examination, although HIV-2 genomic material was detected by PCR and in situ hybridization. The contribution ofHIV-2 infection to this patient's clinical features in view of the other histopathologic abnormalities is uncertain, but it has been noted previously in adults and children that AIDS dementia and other HIV-induced neurologic abnormalities may occur in the presence of minimal HIV replication, in the absence of CNS inflammation, or in the presence of opportunistic infection. In such patients (as in these patients), there is usually laboratory evidence of immunosuppression. Similarly, there can be histopathologic and PCR evidence of CNS involvement by HIV-l without clinical symptoms (unpublished data) [IO, 11]. Presumably, the same features may occur with HIV-2. In view of its extreme sensitivity and ease of performance, PCR is a useful technique for the assessment of HIV tissue involvement, provided that appropriate precautions are taken to avoid contamination. PCR can detect both DNA and RNA, and it lends itself more easily than does in situ hybridization to the screening of multiple tissue samples from I or many patients. As HIV-2 has now been detected in the United States, South America, Europe, and western
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logic differences, it is impossible to accurately compare the relative sensitivities of these primers, but they all seem to be specific. Acknowledgment
We thank Kate Gross for manuscript preparation. References
6. Feldman S. Epp E. Detection of viremia during incubation of varicella. J Pediatr 1979;94:746-8. 7. Ozaki T. Ichikawa T. Matsui Y. et al. Viremic phase in nonimmunocompromised children with varicella. J Pediatr 1984;104:85-7. 8. Ozaki T. Ichikawa T. Matsui Y. et al. Lymphocyte-associated viremia in varicella. J Med Virol 1986; 19:249-53. 9. Vonsover A. Leventon-Kriss S. Langer A. et al. Detection of varicellazoster virus in lymphocytes by DNA hybridization. J Med Virol 1987;21: 57-66. 10. Kido S. Ozaki T. Asada H. et al. Detection of varicella-zoster virus (VZV) DNA in clinical samples from patients with VZV by the polymerase chain reaction. J Clin Microbiol 1991;29:76-9. II. Ozaki T. Miwata H. Matsui Y. Varicella-zoster virus DNA in throat swabs. Arch Dis Child 1990;65:333-4. 12. Baird RE. Daly p. Sawyer MH. Varicella arthritis diagnosed by polymerase chain reaction. Pediatr Infect Dis J 1991;10:950-2. 13. Koropchak CM. Graham G. Palmer J. et al. Investigation of varicellazoster virus infection by polymerase chain reaction in the immunocompromised host with acute varicella. J Infect Dis 1991;163: 101621. 14. Asano Y. Itakura N. Hiroishi Y. et al. Viremia is present in incubation period in nonimmunocompromised children with varicella. J Pediatr 1985; I 06:69-71. 15. Dankner WM. McCutchan JA. Richman DO. Hirata K. Spector SA. Localization of human cytomegalovirus in peripheral blood leukocytes by in situ hybridization. J Infect Dis 1990; 161:31-6.
Detection of Human Immunodeficiency Virus Type 2 in Brain Tissue Dominic E. Dwyer, Sophie Matheron, Serge Bakchine, Jean-Marie Bechet, Luc Montagnier, and Rosemay Vazeux
Unite d'Oncologie Virale, lnstitut Pasteur. and Service des Maladies Infectieuses et Tropicales, Hiipital Bichat-Claude Bernard. and Service de Neurologie et Neuropsychologie, Hiipital de la Salpetriere, Paris. France
Infection due to human immunodeficiency virus (HIV) type 2 is believed to cause a clinical picture similar to that of HIV-1, although extensive data are not available. In 2 patients with West African exposure and neurologic symptoms, HIV-2 was detected in the central nervous system using DNA and RNA polymerase chain reaction, in situ hybridization, and immunohistology. In the first patient, the neurologic disease was most likely due to productive infection with HIV-2. In the second, a combination of neuropathologic abnormalities (including the presence of HIV-2) explained the clinical features. Thus HIV-2, like HIV-l, can be readily detected in brain tissue in patients with neurologic abnormalities, although the exact role ofHIV-2 in pathogenesis of AIDS-associated neurologic disease requires further study.
Human immunodeficiency virus (HIV) type 2 was originally isolated from West African patients and has been recognized since 1985 as a cause ofAIDS [1]. Although there have not been extensive longitudinal studies of HIV-2-induced
Received 16 December 1991; revised 10 March 1992. Presented in part: VI International Conference on AIDS. San Francisco. June 1990 (abstract FA 324). Reprints or correspondence (present address): Dr. Dominic E. Dwyer. Department of Virology, ICPMR, Westmead Hospital. Westmead, NSW, 2145, Australia.
The Journal of Infectious Diseases 1992;166:888-91 © 1992 by The Universityof Chicago. All rights reserved. 0022-1899/92/6604-0029$01.00
disease, it is known that HIV-2-infected patients may have a clinical presentation similar to HIV-L-infected patients, including primary infection, an asymptomatic period, AIDSrelated complex, and AIDS [2-4]. However, there have been suggestions that HIV-2-infected patients may have a longer asymptomatic period [5-7], that they deteriorate clinically more slowly than Hlv-L-infected patients, and that asymptomatic patients have fewer immunologic disturbances than those with HIV-l [8]. It has also been suggested in a community-based prevalence study in Guinea-Bissau, West Africa, that HIV-2 may not be as readily transmitted as HIV-I, the incubation period may be longer, vertical transmission may be rare, and HIV-2 may be less virulent [9].
Downloaded from http://jid.oxfordjournals.org/ at University of Sussex on September 5, 2015
I. Davison AJ. Scott JE. The complete DNA sequence of varicella-zoster virus. J Gen Virol 1986;67: 1759-816. 2. Mahalingam R. Well ish M. Wolf' W, et al. Latent varicella-zoster viral DNA in human trigeminal and thoracic ganglia. N Engl J Med 1990;323:627-31. 3. Trilifajova J. Bryndova D. Rye M. Isolation of varicella-zoster virus from pharyngeal and nasal swabs in varicella patients. J Hyg Epidemiol Microbiol Immunol 1984;28:201-6. 4. Ozaki T. Matsui Y. Asano Y. Okuno T. Yamanishi K. Takahashi M. Study of virus isolation from pharyngeal swabs in children with varicella. Am J Dis Child 1989; 143: 1448-50. 5. Asano Y. Itakura N. Hiroishi Y. et al. Viral replication and immunologic responses in children naturally infected with varicella-zoster virus and in varicella vaccine recipients. J Infect Dis 1985; 152:8638.
1ID 1992; 166 (October)
JID 1992; 166 (October)
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Clinical neurologic dysfunction is commonly recognized in HIV-I-infected patients, and 80%-90% of AIDS patients will have neuropathologic abnormalities at autopsy. These include direct involvement by HIV-I, manifesting most commonly as AIDS encephalopathy or AIDS dementia complex but also as vacuolar myelopathy, aseptic meningitis and peripheral neuropathy, opportunistic infections, and tumors [10, 11]. There have been only occasional clinical descriptions of HIV-2-induced central nervous system (CNS) disease, mostly in conjunction with opportunistic infections [2, 4, 12]. We present evidence of direct neurologic involvement by HIV-2 in 2 patients, using DNA and RNA polymerase chain reaction (PCR), in situ hybridization, and immunohistology.
Patient J. A 27-year-old woman (born in Mozambique but spent many years in Senegal) was first diagnosed as having HIV2-associated AIDS in January 1987, when she presented with Pneumocystis carinii pneumonia 2 months after delivering a normal infant. Her T41ymphocyte count was 34/mm3 • Over the next 10 months she was noted to be severely depressed, with impaired cognitive function (but no specific neurologic abnormalities on physical examination). Sixteen months after the diagnosis of P. carinii pneumonia, she was diagnosed as having cytomegalovirus and toxoplasmosis retinitis that resolved with treatment; 22 months later she was readmitted with worsening confusion, aggression, and a decreased level of conciousness against the background of a depressive syndrome. On this admission there was no clinical evidence of a CNS opportunistic infection, no progression of the previously noted retinitis, and computed tomography (CT) revealed cortical atrophy. Cerebrospinal fluid (CSF) examination was normal, and cultures ofCSF and blood were negative for fungi, bacteria, and viruses. Results of routine biochemical and liver function tests were normal. A mild pancytopenia was confirmed by bone marrow examination, and staining of the marrow for microorganisms was negative. The patient remained hospitalized for 5 months before dying of presumed septicemia. Patient 2. A 67-year-old French heterosexual man (with a history of frequent and prolonged travel in Africa) had a history
Figure 1. Ethidium bromide-stained agarose gel and autoradiograph of products of DNA polymerase chain reaction using env primers. Samples from: lane I, patient 1; lanes 2 and 5, patient 2 (separate samples);lanes 3 and 6,2 HIV-I-infected patients; lanes 4 and 7, 2 uninfected patients; lane 8, 250 ng of DNA, HIV-2-infected CEM cells; lane 9, 250 ng of DNA, HIV-I-infected CEM cells; lane 10, 250 ng of DNA, uninfected CEM cells; lane 11, no DNA.
of 18 months of slowly progressive memory decline, increased emotional lability, and occasional episodes of confusion with delusions. Results of physical examination at the onset of this decline were initially reported as normal, and investigations (including HIV-l but not HIV-2 serology) were normal except for a CT scan showing mild frontal cortical atrophy. In the absence of any obvious cause for this neurologic decline, a clinical diagnosis of Alzheimer's dementia was made. His last admission was precipitated by a 4-day history of confusion, and clinical examination revealed a febrile (38.7°C), drowsy, and disorientated man with bradykinesia, a mild diffuse rigidity (but no tremor), and frontal lobe dysfunction including bilateral grasp reflexes, slowed mentation, diminished verbal fluency, and behavioral disinhibition. Investigation showed HIV-2 antibodies in the blood and CSF, HIV-2 antigenemia, and a T41ymphocyte count of 68/mm 3 . CSF analysis revealed two mononuclear cells and a mildly elevated protein level with oligoclonal IgG bands. Cultures of CSF and blood and serologic testing for other infectious agents (including cytomegalovirus, Toxoplasma species, Pneumocystis species, herpes simplex virus, syphilis, and hepatitis B virus) were negative. There was no evidence of a toxic cause for this presentation, and results of routine hematologic, biochemical, and liver and thyroid function tests were normal, as were vitamin B9 and B12 levels. The patient died 9 weeks later of bronchopneumonia. Histopathologic examination. CNS tissue was obtained at autopsy from both patients for histopathologic examination with hematoxylin-eosin, Nissl's, Wolcke, Bodian, Grocott-Gomori, Perls', and Gram's stains. Samples from cerebral hemispheres, basal ganglia, brain stem, and cerebellum were taken, subdivided, and stored at -80 a C. CNS samples from HIV-I-infected and -uninfected patients were also obtained at autopsy and used as negative controls for immunohistology and in situ hybridization. Brain tissue was not cultured for viruses. Pc.R. Total DNA and RNA were extracted simultaneously from samples of white matter and basal ganglia from each patient by cesium chloride purification. HIV-2-specific primer pairs in the long terminal repeat (LTR) (5'-GCCCTGGGAGGTTCTCTCC-3', 5'-GACCAGGCGGCGACTAGGAGAGAT-3' [187 bpJ), gag (5'-TGGCGCCTGAACAGGGAC-3', 5'TGCCCACACAATATGTTTTA-3' [323 bpD, and env regions (5'-TATAGGCCTGTTTTCTCTTCCCC-3', 5'-ATCAGCTGGCGGATCAGGAAATG-3' [169 bpD from HIV-2RoD were used for DNA PCR. Primers for RNA PCR were in the second
Downloaded from http://jid.oxfordjournals.org/ at University of Sussex on September 5, 2015
Materials and Methods
889
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tients using the env primers is shown in figure 1). peR to detect tat mRNA was positive in two separate samples from patient 1. In situ hybridization using HIV-2 gag probes in multiple brain sections showed strong signals in cells disseminated in the parenchyma and perivascular regions (figure 2). Patient 2. Histopathologic examination showed no evidence ofHI V subacute encephalitis, but some neurofibrillary
Downloaded from http://jid.oxfordjournals.org/ at University of Sussex on September 5, 2015
(5'-GAAATCCTCTCTCAGCTATACC-3') and third exons (antisense env primer above [313 bp]) of the tat mRNA coding sequence. With a modification of Saiki et al.'s method [13], PCR was done on 2 J.Lg of extracted DNA or eDNA from the reverse transcription of 2 J.Lg of RNA. Aliquots were run on ethidium bromide-stained agarose gels, transferred, and hybridized with 32P-end-labeled probes (LTR probe 5'-TCTGCCCAGCACCGGCCAAGTGC-3', gag probe 5'-GGCCCGGCGGAAAGAAAAA-3', env and tat probes 5'-ATATATGCTATCGGCCAGGGCCA-3'). Negative controls included both DNA and RNA from uninfected and HIV-I-infected brain tissue extracted simultaneously with the patient samples and reactions containing no template DNA. A positive control using 250 ng of DNA or RNA extracted from HIV-2-infected CEM cells was included. Immunohistologic examination. Tissue sections were deposited on neoprene-coated glass slides, acetone-fixed, and stored at -20°C for immunologic staining and in situ hybridization. We studied at least 10 tissue blocks from each patient, with samples checked for HIV-2 antigens using monoclonal antibodies directed against the p 18 and p25 viral antigens (gift of B. Parekh, Bio-Rad, Richmond, CA). Toxoplasma, cytomegalovirus, herpes simplex viruses 1 and 2, and IC virus antigens were examined in each sample by immunologic staining as previously described [14]. All tissue blocks were screened for the presence of monocyte/macrophage/microglial abnormalities using antibodies directed against CD68 (KiM?; Behring, Marburg, Germany). Monocytes and Band T lymphocytes were detected using antibodies directed against CD 14, CD 19, CD3, CD8, and CD4 molecules (Becton Dickinson, Mountain View, CA), and CNS cells were identified using antibodies against astrocytes (GFAP; Amersham, Buckinghamshire, UK), neurons (D. Paulin, Institut Pasteur), and endothelial cells (Factor VIII; Dakopatts, Carpinteria, CA). In situ hybridization. HIV-2 gag probes were labeled by in vitro transcription with 35S-labeled UTP and 35S-labeled ATP, and in situ hybridization was done as previously described [14]. Samples from HIV-2-uninfected patients were included as negative controls; HIV-2-infected CEM cells and brain tissue from simian immunodeficiency virus-infected rhesus macaques were used as positive controls.
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Results Patient 1. Histopathologic examination of brain tissue revealed diffuse demyelination with macrophage infiltration in perivascular and periventricular areas and swelling ofneurons and oligodendrocytes. Occasional clusters of Toxoplasma cysts without surrounding inflammation were seen in the occipital cortex and near the lateral ventricles. Immunohistology using monoclonal antibodies to HIV-2 pl8 and p24 antigens was weakly positive, but no Toxoplasma species, cytomegalovirus, herpes simplex virus, IC virus, or HIV-1 were detected by this technique. DNA PCR on a single sample of brain tissue was positive using all three primer pairs, with a further two separate samples positive using only the env primers (an example of the DNA PCR for both pa-
Figure 2. In situ hybridization. A, patient I; B, patient 2; C, uninfected control.
110 1992; 166 (October)
Concise Communications
degeneration and senile plaques were observed in the gray matter with Lewy bodies in the substantia nigra (features of both Alzheimer's and Parkinson's diseases). There were no microglial nodules and only minimal perivascular infiltration. Immunohistologic tests for HIV-2 and other infectious agents were negative. However, HIV-2 DNA PCR using three primer pairs was positive in two separate samples, with two of a further four samples positive using only the env primers. PCR for tat mRNA was negative in five samples. In situ hybridization for H1V-2 showed positive signals in cells in perivascular regions.
Discussion
Africa, more studies using these techniques are required to further delineate the disease spectrum of this virus.
Acknowledgment
We thank C. Marche from Hopital Bichat-Claude Bernard, Paris, for doing the autopsy on patient I.
References I. Clavel F. Guetard D. Brun-Vezinet F. et al. Isolation ofa new human retrovirus from West African patients with AIDS. Science 1986;233:343-6. 2. Clave I F, Mansinho K. Chamaret S. et al. Human immunodeficiency virus type 2 infection associated with AIDS in West Africa. N Engl J Med 1987;316:1180-5. 3. Besnier J, Barin F, Baillou A. et al. Symptomatic HIV-2 primary infection [letter]. Lancet 1990; I:798. 4. Brun-Vezinet F. Rey MA, Katlama C, et aI. Lymphadenopathy associated virus type 2 in AIDS and AIDS-related complex. Lancet 1987; I: 128-32. 5. Ancelle R. Bletry 0, Baglin AC, et al. Long incubation period for HIV2 infection [letter]. Lancet 1987; I:688-9. 6. Dufoort G. Courouce AM, Ancelle-Park R, Bletry O. No clinical signs 14 years after HIV-2 transmission via blood transfusion [letter]. Lancet 1988;2: 510. 7. Gody M, Ouattara SA. De The G. Clinical experience of AIDS in relation to HIV-I and HIV-2 infection in a rural hospital in Ivory Coast. Africa. AIDS 1988;2:433-6. 8. Marlink RG. Ricard D. M'Boup S. et al. Clinical hematologic and immunologic cross-sectional evaluation of individuals exposed to the human immunodeficiency virus type-2. AIDS Res Hum Retroviruses 1988;4: 137-48. 9. Poulsen AG. Kuinesdal B. Aaby P. et al. Prevalence of and mortality from human immunodeficiency virus type 2 in Bissau. West Africa. Lancet 1989;1:827-30. 10. Price RW, Brew B. Sidtis J, et al. The brain in AIDS: central nervous system HIV-I infection and AIDS dementia complex. Science 1988;239:586-92. II. de la Monte SM. Ho DO, Schoeley RT. et al. Subacute encephalomyelitis of AIDS and its relation to HTLV-III infection. Neurology 1987;37:562-9. 12. Hormigo A, Bravo-Marques JM. Souza-Ramalho P, et al. Uveorneningitis in a human immunodeficiency type 2 seropositive patient. Ann Neurol 1988;23:308-10. 13. Saiki RK. Gelfand DH. Stoffel S, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988;239:487-91. 14. Vazeux R, Brousse N, Jarry A, et al. AIDS subacute encephalitis: identification of HIV infected cells. Am J PathoI1987;126:403-10.
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These cases confirm that HIV-2, like HIV-1, can infect the CNS, and that this neurologic involvement is readily detected and localized by PCR and in situ hybridization. In patient 1, the clinical, histopathologic, and in situ hybridization findings were consistent with an HIV-2-associated encephalopathy, with active viral replication suggested by the detection of both viral DNA and mRNA. In patient 2, typical features of AIDS encephalopathy were not found on histopathologic examination, although HIV-2 genomic material was detected by PCR and in situ hybridization. The contribution ofHIV-2 infection to this patient's clinical features in view of the other histopathologic abnormalities is uncertain, but it has been noted previously in adults and children that AIDS dementia and other HIV-induced neurologic abnormalities may occur in the presence of minimal HIV replication, in the absence of CNS inflammation, or in the presence of opportunistic infection. In such patients (as in these patients), there is usually laboratory evidence of immunosuppression. Similarly, there can be histopathologic and PCR evidence of CNS involvement by HIV-l without clinical symptoms (unpublished data) [IO, 11]. Presumably, the same features may occur with HIV-2. In view of its extreme sensitivity and ease of performance, PCR is a useful technique for the assessment of HIV tissue involvement, provided that appropriate precautions are taken to avoid contamination. PCR can detect both DNA and RNA, and it lends itself more easily than does in situ hybridization to the screening of multiple tissue samples from I or many patients. As HIV-2 has now been detected in the United States, South America, Europe, and western
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