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Subclinical Central Nervous System Infection with JC Virus in Patients with AIDS E. Byrd Quinlivan, Melanie Norris, Thomas W. Bouldin, Kinuko Suzuki, Rick Meeker, Marilyn S. Smith, Colin HaU, and Shannon Kenney
Departments of Medicine. Neurology. Pathology. and Microbiology and Immunology; UNC Lineberger Comprehensive Cancer Center: AIDS Neurologic Center. University ofNorth Carolina at Chapel Hill
lC virus (lCY) is a human polyomavirus originally recovered from a patient with progressive multifocalleukoencephalopathy (PML) [I]. Subsequently, antibodies to lCV have been found in 70% of the US adult population. with seroconversion occurring most commonly in childhood and early adolescence [2]. Primary infection with lCY has not been associated with any specific syndrome. Reactivation and shedding of lCY in the urine of pregnant women and organ transplant recipients without clinical sequelae has been reported [3-6]. In patients with depressed cellular immune function, including those with AIDS, lCY has been shown to be responsible for the development of PML, a severe subacute demyelinating disease of the central nervous system (CNS) [7]. Symptomatic impairment of neurologic function, which may include motor or sensory function, speech, vision, or mentation, develops rapidly, and death usually occurs within 3-6 months after the onset of symptoms [7]. Due to the dramatic increase in the prevalence ofPML during the AIDS epidemic, lCY infection can be expected to become a significant cause of morbidity and mortality, with a marked economic impact [8-10]. The development of PML years after primary infection
Received 19 December 1991; revised 26 February 1992. Informed consent was obtained from all patients who provided peripheral blood samples and from families of patients on whom autopsies were done. in accordance with the Committee on the Protection of the Rights of Human Subjects. University of North Carolina at Chapel Hill. Financial support: National Institutes of Health (Al-07l80 [to E.B.Q.), NS-26680. AI-25868, and CA-O1229). Reprints or correspondence: Dr. E. Byrd Quinlivan, Room 210 UNC Lineberger CCe. CB# 7295, University of North Carolina at Chapel Hill, Chapel HilL NC 27599-7295.
The Journal of Infectious Diseases 1992;166:80-5 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6601-0012$01.00
with lCY implies that the disease may result from reactivation of latent virus, although a primary infection at the time ofimmunodeficiency has not yet been excluded. The demonstration of lCV in renal tissue and in the urine ofasymptomatic patients suggests that lCY remains latent in the kidneys [3, II]. There is conflicting evidence regarding whether or not the CNS is also a site of latency. It has been proposed that infection of the CNS occurs at the time of reactivation [7], but some investigators have found lCV in the brain tissue of elderly patients without clinically evident PML [12]. Other investigators report the absence of lCY in brains of both human immunodeficiency virus (HIV)-positive (+) and -negative C) patients without neurologic disease or with other neurologic diseases (such as multiple sclerosis) using a variety of methods [II, 13, 14]. Since previous efforts leave the issue of CNS latency unresolved and since the polymerase chain reaction technique (PCR) is quite sensitive, we chose this method to examine autopsy brain specimens for the presence of lCY in HIY+ patients.
Materials and Methods Collection of brain specimens for gross and microscopic examination. Brains from 13 HIV+ (type I) patients and 12 HIV- control patients were studied for the presence of lev DNA. Brains taken at autopsy from patients without clinical or pathologic evidence of HIV infection or PML were classified as HIY- and PML- and served as controls. Patients with positive HIV serologies served as the HIV+ study group. All patients in the study group but not the two medical examiner cases, for which clinical information was limited, had evidence of AIDS. Brains were coronally sectioned after fixation for ;:,.2 weeks in 10% buffered formalin. Paraffin-embedded blocks of tissue from posterior frontal cerebral cortex and white matter, hippocampus, basal ganglia. pons, medulla, and cerebellum were sec-
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Immunocompromised patients, particularly those with AIDS, develop progressive multifocal leukoencephalopathy (PML) due to central nervous system infection with JC virus (JCV). It is unknown whether JCV infection in the central nervous system can occur in the absence of PML symptoms. To address this question, autopsy specimens from patients with AIDS were examined. The brains of a group of patients without AIDS or central nervous system disease were also examined. JCV DNA was detected by the polymerase chain reaction in brain tissue from 4 (31%) of 13 human immunodeficiency virus (HIV)-positive patients. JCV was also detected in 1 elderly HIV -negative patient but not in the 11 other control brains. JCV was not detected in 22 myocardial specimens obtained at autopsy from HIV-negative patients nor 10 peripheral blood specimens from HIV -positive patients. The presence of JCV in brains of patients without clinically evident PML suggests that JCV may be present in the central nervous system without clinical disease.
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probe (lCY nucleotide 4871-4903: 5'-TCCCCACCTTTATCAGGGTGGAGTTCTTTGCAT-3') that recognized the lCY sequence within the 160-bp PCR product. All experiments contained "solution only" samples with Taq DNA polymerase as negative controls. Tissue processing and PCR reaction mixture setup took place in a separate laboratory. removed from the location of the amplification process and any lCY clones. All PCR workspace and nondisposable equipment were routinely cleaned with a I M solution of HCl or a dilute solution of chlorine-based bleach. Tissue samples were considered to contain lCY DNA if DNA was amplified in two separate experiments. PCR and Southern blot analysis were done on all samples at least twice. Clinical history. Each patient chart was reviewed, with particular attention to the cause of death, any history or clinical evidence of neurologic disease. and the results of recent neurologic studies. The evaluator was blinded to the results of the PCR evaluation at the time of the review.
Results lCV in the CNS and peripheral blood of HIV+ patients. Using the PCR technique, we found that sections of 4 (31 %) of the 13 brains of HIY+ patients were positive for lC virus (table 1). Although a number of pathologic changes were present in these brains, no specific findings correlated with the presence or absence of lCY, and there was no evidence of PML in any of these patients. Three of the 4 brains had lCY in the region of the hypothalamus, and 2 of the 4 brains also had lCY in the region of the caudate-putamen. However, the sample size is too small to assess whether this localization has significance. Review of the premortem history did not reveal any clinical characteristics that correlated with the presence of lCY by PCR in these patients (table 1). We did not detect lCY in 10 peripheral blood specimens from HIY+ patients (data not shown). PCR and Southern blot analysis demonstrated c-K-ras in all specimens except from the hypothalamus region of one patient (data not shown). A Southern blot analysis that demonstrated the PCR products in one of the brains in which lCY was detected (a 34year-old black man) is shown in figure 1. The PCR product hybridized to the internal probe with equal intensity as did the positive control, which contained a dilution of lCY plasmid equal to 6 X 10- 15 g in the PCR reaction. This sensitivity is about equal to the detection of one copy of lCY per 500 cells. For comparison, Southern blot analysis of a biopsy specimen from a patient with PML demonstrated that the number of lCY genomes in brains with PML was much greater than that seen in the reaction with the 6 X 10- 15 g lCY plasmid DNA (figure 2). lCV in the CNS and myocardium of HIV- patients. We compared our abilities to detect lCY in the brains ofHIY+ or HIY- patients. HIY- patients were those who had no evidence of HIY risk factors or HIY infection (by clinical history or autopsy findings). One of 12 control brains contained
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tioned at 6 mm, stained with solochrome-eosin, and specifically screened for changes of PML, cytomegalovirus-type intranuclear inclusions, encephalitis, meningitis, and lymphoma. Specimen collection for DNA extraction. Tissue sections (0.5-5.0 mm') of formalin-fixed brain from the 13 HIY+ and 12 HIY- patients were obtained from the cortex, caudate-putamen, globus pallidus, hippocampus, and hypothalamus regions. Formalin-fixed brain biopsy specimens from a patient with clinical and pathologic evidence of PML were used as a comparison of lCY copy number. Myocardial tissue taken at autopsy from 22 patients without clinical or pathologic evidence of HIY infection or PML was used as a negative control to exclude laboratory contamination of the samples. Peripheral blood specimens from 10 AIDS patients were used as a control for the possibility that positive results were actually due to the presence of lCY in the blood within the cerebral vessels rather than within the brain parenchyma. The mononuclear cell fraction of the fresh blood sample was harvested by processing the sample through a lymphocyte separation medium. DNA extraction. Samples of brain tissue were incubated in 0.5 M TRIS, 0.02 M EDTA, 0.01 M NaCl, 1% SDS, and 200 JLg/ml proteinase K at 48°C for 24-72 h interrupted by aggressive vortexing and two washes in PBS. Phenol extraction, chloroform purification, and ethanol precipitation were done [15]. Pelleted DNA was resuspended in sterile water and stored at - 20°e. PCR was done on all samples for detection of c-K -ras with primers (5'-ATGACTGAATATAAACTTGT-3' and 5'CTCTATTGTTGGATCATATT-3') to verify the quality of the DNA for amplification. PCR amplification was done in a 100-JLl reaction mixture of I ng of each primer, 2 JLg of sample DNA, 91 mM each dinucleotide phosphate, 0.04% Triton X, 3.6 units of Taq polymerase (Promega, Madison, WI, 16.6 mM (NH 4hS04, 67 mMTRIS-HCI (pH 8.8), 6.7 mMMgCI 2 , and 10 mM {3-mercaptoethanol in a thermal cycler (Perkin-Elmer, Emeryville, CA) for 42 cycles: cycle I: 95°C for 5 min, 55°C for I min, 70°C for I min; cycles 2-41: 94°C for I min, 55°C for I min, 70°C for 1 min; cycle 42: 94°C for I min, 55°C for 1 min, 70°C for 20 min. The PCR products were subjected to electrophoresis through a 2.5% agarose gel and transferred to a nylon filter for hybridization. The filter was hybridized at 48-50°C overnight with 50% formamide, 0.1% SDS, lOX Denhardt's solution 5X and standard saline citrate, and 50 mM NaH 2P0 4/NaHP04 to an oligonucleotide probe (5'-GGAGCTGGTGGCGTAGGCAA-3') that recognized the c-K-ras sequence within the IIO-bp PCR product. The probe was end-labeled with [')'-32P]adenosine triphosphate by means ofT4 polynucleotide kinase; the results were visualized by autoradiography. PCR and Southern blot analysis. PCR was done with primers to the T antigen region of lCY (lCY nucleotide 48514870: 5'-TAAAAGATCCTTCTCTTCATCTTGTCTTCG-3'; lCY nucleotide 5003-4984: 5'-ATTGGGAATTCTGCTGAAT AGGGAATCC-3'). PCR and Southern blot analysis were done as above with the following changes in 42 cycles of amplification done in a DNA thermal cycler (Coy, Ann Arbor, MI: cycle I: 94°C for 5 min, 65°C for 1 min, 70°C for 2 min; cycles 2-41: 94°C for I min, 65°C for 1 min, 70°C for I min; cycle 42: 94°C for I min, 65°C for I min. 70°C for 20 min. The filter was then hybridized at 42-45°C overnight to an oligonucleotide
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Table 1. Detection of JC virus (JCY) in brains of HIY+ patients. CNS region ofJCV detection
39. white . male 60. white . male 41. white . male 47 . white . male 34. black . male
CPo HI. HY CP.GP HY Negative HY
31. black. male
Negative
40 . black. 31. black. 42 . white. 40. black.
female male male male
Negative Negative Negative Negative
39. black. male 38. black. female 27. white. male
Negative Negative Negative
Pathologic findings
CNS disease by clinical history
None None Multinucleate cells Brain perivascular chronic inflammation. white matter pallor Multinucleate cells. meningeal perivascular chronic inflammation. CMV-type nuclear inclusions. white matter pallor Multinucleate cells. meningeal perivascular chronic inflammation White matter pallor Microglial nodules Microglial nodules. CMV-type nuclear inclusions Multinucleate cells. brain perivascular chronic inflammation. CMV-type nuclear inclus ions. CNS cryptococcosis, CNS lymphoma Meningeal perivascular chronic inflammation Multinucleate cells. CNS lymphoma Perivascular chronic inflammation. microglial nodules. CMV-type nuclear inclusions. white matter pallor. CNS lymphoma
Hepatic encephalopathy Cryptococcal meningitis (inactive) Cytomegalovirus (CMV) retinitis Dementia Dementia
ME case' Mild dementia None Cryptococcal meningitis (inactive). CMV retinitis Cryptococcal meningitis (inactive)
ME case' CMV retinitis CMV retinitis
NOTE. Negative; JCV not detected by polymerase chain reaction technique (PCR) or detected in only one experiment; otherwise JCV detected by PCR in two or more separate experiments. CPo caudate putamen; GP. globus pallidus : HI. hippocampus; HY. hypothalamus; CNS. central nervous system. * Medical examiner case . autopsy limited to CNS.
detectable lCY (table 2) . This 79-year-old man, the oldest patient in our series, died from cancer of the hypopharynx, chronic lung disease, and a myocardial infarction. The diagnosis of cancer was made shortly before the patient's death and he refused all therapy. There was no evidence that he
6x1 a-13 g JCV clone I 6x10- 14 g JCV clone I 6x1 a- 15 g JCV clone I 6x1 a- 16 g JCV clone Negative control: Solutions only
IC I CP I GP I HI I HY Figure 1. Detection ofJC virus (lCY) by polymerasechain reaction technique (PCR) and Southern blot analysis. DNA extracted from regions of cortex (C). caudate-putamen (CP), globus pallidus (OP), hippocamus (HI). and hypothalamus (HY) were subjected to PCR and Southern blot analysis with JCY. Serial dilutions of a genomic clone of lCY were included as positive controls. Sample without DNA was included as negative control (solutions only).
received any immunosuppressive therapy. In addition, there was no evidence clinically or pathologically that he had PML. We did not detect lCY in any of22 autopsy specimens of cardiac tissue from HIY- patients (data not shown). c-Kras was successfully detected by PCR followed by Southern blot hybridization in all specimens (data not shown).
Discussion PML, a previously rare disease, occurs in up to 4% of patients with HIY infection. Although the possibility that PML results from primary infection of immunocompromised patients has yet not been rigorously excluded, it has been assumed that the disease is a result of reactivation of lCY that has been present in a latent form in the kidneys since initial infection in childhood [7]. lCY may be transported from the kidney to the brain by peripheral blood lymphocytes [16]. However, it is also possible that PML results from reactivation of previously latent CNS infection in immunocompromised hosts. We show evidence here that lCY may be present in the brains of patients with HIY infection but without evidence ofPML. In addition, one elderly patient with head and neck carcinoma among the 12 HIY- patients had lCY detectable by PCR. No specific clinical or pathologic findings were associated with the detection of lCY, and the lCY
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Patients; age. race. sex
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CNS Infection with JCY in AIDS Patients
JID 1992;166 (July)
I I I
10x10- 15g JCV clone
I I
6x10 - 16 g JCV clone 6x1 0-1 7 g JVC clone I 6x1 0- 18 g JVC clone I
I
I I
I I
Negative control : Solutions only
} Bx
detected most likely represents latent or subclinical infection. Although PCR analysis is not strictly quantitative, our analysis of a brain from a patient with PML suggested that the level of lCY DNA in the asymptomatic patients is likely to be lower than in patients with PML (cf. figures I and 2). Other investigators have postulated that lower levels of lCY
Table 2.
Detection of JC virus (JeY) in brains of HIY- patients.
Patients: age. race. sex
CNS region of JCY detection
23. whit e. male
Negative
49 . black. male 46 . black . female 15. black . female
Negative Negative Negative
41. 27. 49 . 27.
Negative Negative Negative Negative
white. male black . male black. male Hispanic. female
16. black. female 59. white . male 43. white. female
Negative Negative Negative
79. white . male
CP
Pathologic finding s Brain and meningeal perivascular chronic inflamm ation None None Meningeal perivascular chronic inflammation None None None Meningeal perivascular chronic inflammation None None Brain and meningeal perivascula r chronic inflammation None
CNS disease by clinical history None None None None Down's syndrome None None None None None None None
NOTE. Negative : JCV not detected by polymerase chain reaction technique (PCR) or detected in only one experiment: otherwise detected by PCR in two or more experiments. CPocaudate putamen; CNS. cen tral nervous system .
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Figure 2. Southern blot analysis of brain biopsy specimen from patient with AIDS. DNA extracted from brain biopsy of patient with progressive multifocalleukoencephalopathy was subjected to polymerase chain reaction and Southern blot analysiswith JC virus (JCY) probe. Serial dilutions of genomic clone of JCY were included as positive controls. Sample without DNA was included as negative control (solutions only).
in autopsy specimens than in biopsy specimens in patients with PML may be due to degradation of the DNA during processing [13]. However, our observation that lCY is less abundant in asymptomatic patients than in the PML biopsy specimen is unlikely to be due to degradation of DNA, because we extracted the DNA rather than using a lysed-cell extract and amplified the proto-oncogene c-K-ras in all but one of our samples, indicating that the extraction was successful. One potential source oferror in this work is the presence of blood contamination of brain tissue . AIDS patients, who have a high incidence of syndromes associated with viral reactivation [17], might be expected to have a high incidence of identifiable virus in circulating blood cells . Indeed, lCY has been found in B cells of 2 patients with PML (I with AIDS and one without any known underlying disease) [ 16] and in I AIDS patient without PML [18]. PCR methods are so sensitive that the presence of lCY-containing blood cells could give false-positive results during the evaluation of brain tissue . Two procedures were undertaken to prevent this, and the results make it unlikely that our findings are due to blood contamination. First, we were unable to detect lCY in the mononuclear cell fraction of peripheral blood samples taken from a group of 10 AIDS patients (data not shown). Second, in none ofthe liver specimens from 3 of the 4 AIDS patients and the I HIY- patient who had lCY detected in their brains could we detect lCY (data not shown). Due to the vascular nature of the liver, we believe it served as an adequate control for possible blood contamination of tissue specimens, although the ideal control would have been pe-
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of PML. However, our results do not distinguish between reactivated latent CNS lCY infection and primary lCY infection in immunocompromised hosts. A prospective serologic study would be useful to determine if only AIDS patients who are initially seronegative for lCY are at risk for developing PML. It is interesting to speculate whether the subjects with positive PCR results would have developed overt disease had they survived, that is, whether the presence of lCY in brain is a predictor offuture PML. It is also interesting to consider the possible clinical spectrum of CNS infection with lCY. There certainly may be marked variability in the duration and progression ofPML. Berger et al. has drawn attention to the fact that some patients may have a particularly long course [8], and we have had similar experience (unpublished data). Two patients reported by Mori et al. with asymptomatic lCY CNS infection died with neurologic illness (dementia and Parkinson's disease), and in 3 of their 4 patients the lCY was detected near areas of tiny foci of demyelination. In the AIDS patients reported here, a number of neurologic conditions (clinical and pathologic) not typical of PML were present (table 1), but these did not correlate with the presence or absence of lCY. In summary, we have detected lCY in autopsy brain specimens of 4 of 13 HIY+ patients and I of 12 HIY- patients. We have therefore established that lCY can frequently be found by PCR in the brains of AIDS patients who do not have clinical evidence of PML. Whether this is a truely latent infection (rather than early reactivation or subclinical primary infection), whether it is a predictor of the future development ofPML, and whether it may playa role in the development of other neurologic diseases are all ideas that require further investigation.
Acknowledgments
We thank lames N. Hayward for providing workspace for PCR in a lCV-free environment and Edison Liu for assistance with PCR.
References I. Padgett BL, Walker DL, ZuRhein GM, Eckroade RJ, Dessel BH. Cultivation of pap ova-like virus from human brain with progressive multifocal leukoencephalopathy. Lancet 1971; I: 1257-60. 2. Padgett BL, Walker DL. Prevalence ofantibodies in human sera against JC virus, an isolate from a case ofprogressive multifocalleukoencephalopathy. J Infect Dis 1973;127:467-70. 3. Arthur RR. Shah KV. Occurrence and significance of papovaviruses BK and JC in the urine. Prog Med Virol 1989;36:42-61. 4. F1regstad T, Sundsfjord A, Arthur RR, Pedersen M. Traavik T. Subramani S. Amplification and sequencing of the control regions of BK and JC virus from human urine by polymerase chain reaction. Virology 1991;180:553-60. 5. Myers C Frisque RJ, Arthur RR. Direct isolation and characterization
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ripheral blood samples from all patients in the study group. In one additional AIDS patient (a 47-year-old white man), lCY was detected all of three times in the liver specimen but only once in the cortex and hippocampus regions of the CNS. We classified this patient as negative as we were not able to amplify lCY from any region of the CNS more than once and the liver specimen was consistently positive. However, it is possible that lCY was actually present in the brain tissue of this patient. Particular care was taken to avoid laboratory contamination, a common source of error with PCR. All of our experiments contained solution-only samples as negative controls. Tissue processing and PCR reaction mixture setup took place in a laboratory removed from the location of the amplification process and any lCY clones, and all PCR workspace and nondisposable equipment were routinely cleaned. In addition, we required that before any specimen was considered positive, lCY was detected on at least two occasions. As a further control, autopsy tissue from 10 hearts from HIYpatients was evaluated using the same techniques, and none demonstrated lCY. The question ofCNS latency of lCY in non-HIY-infected patients has been studied but not resolved [19]. Telenti et al. [13] looked for lCY by PCR in the brains of 17 neurologically intact patients and 4 patients with neurologic conditions. Only one patient, with herpes encephalitis, showed a very weak detection of lCY, and this was attributed to contamination. In contrast, Mori et al. [12], using in situ hybridization and immunocytochemistry, systematically examined the brains of 10 elderly patients without PML or known immunosuppressive conditions and found lCY in 4. They postulated these findings may be due to the advanced ages of the patients and the chronic nature of the diseases present at the time of death. Elderly patients have also been reported to have an increased incidence of lCY shedding into the urine, presumably from a renal source [20]. In keeping with this, the one HIY- patient in whom we detected lCY was 79 years old. With this in mind, the finding of such a high incidence of apparently asymptomatic lCY CNS infection in our HIY+ population may not be surprising. Imrnunocompromised patients and AIDS patients in particular are known to have a general increase in viral disease. Other investigators, at the most recent International Conference on AIDS, have reported finding lCY in the CNS of AIDS patients without clinical evidence of PML [21]. Since the reported incidence of PML is higher in those with AIDS than in any other patient population (although neither population has been studied prospectively) [8-10], it is reasonable that AIDS patients may also have a higher frequency of subclinical (or latent) lCY CNS infection. Our findings add further support to the idea that lCY may be present in the brains of patients without clinical evidence
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6.
7. 8.
9.
10.
II.
13.
of JC virus from urine samples of renal and bone marrow transplant patients. J Virol 1989;63:4445-9. Yogo Y. Kitamura T. Sugimoto C. et at. Isolation of a possible archetypal JC virus DNA sequence from nonimmunocompromised individuals. J Virol 1990;54:3139-43. Greenlee JE. Progressive multifocal leukoencephalopathy. Curr Clin Top Infect Dis 1989;10:140-56. Berger JR. Kaszovitz B. Post J. Dickinson G. Progressive multifocal leukoencephalopathy associated with human immunodeficiency virus infection. Ann Intern Med 1987; 107:78-87. Krupp LB. Lipton RB. Swerdlow ML. Leeds NE. L1ena J. Progressive multifocal leukoencephalopathy: clinical and radiographic features. Ann Neurol 1985; 17:344-9. Levy RM. Bredesen DE. Rosenblum ML. Neurological manifestations of the acquired immunodeficiency syndrome (AIDS): experience at UCSF and review of the literature. J Neurosurg 1985;62:475-95. Chesters PM. Padgett BL. Walker DL. Persistance of DNA sequences of BK virus and JC virus in normal human tissues and diseased tissues. J Infect Dis 1983;147:676-84. Mori M. Kurata H. Tajima M. Shimada H. JC virus detection by in situ hybridization in brain tissue from elderly patients. Ann Neural 1991 ;29:428-32. Telenti A. Aksamit AJ Jr. Proper J. Smith TF. Detection of JC virus DNA by polymerase chain reaction in patients with progressive multifocal leukoencephalopathy. J Infect Dis 1990; 162:858-61.
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14. Wiley CA, Grafe M. Kennedy C. Nelson JA. Human immunodeficiency virus (HIV) and JC virus in acquired immune deficiency syndrome (AIDS) patients with progressive multifocalleukoencephalopathy. Acta Neuropathol 1988;76:338-46. 15. Goeiz SE. Hamilton SR. Vogelstein B. Purification of DNA from formaldehyde fixed and paraffin embedded human tissue. Biochem Biophys Res Comm 1985;130:118-26. 16. Houff SA, Major EO, Katz DA, et at. Involvement of JC virus-infected mononuclear cells from the bone marrow and spleen in the pathogenesis of progressive multifocal leukoencephalopathy. N Engl J Med 1988;318:301-5. 17. DeVita VT. Hellman S. Rosenberg SA. AIDS: etiology. diagnosis. treatment. and prevention. 2nd ed. Philadelphia: JB Lippincott. 1988:474. 18. Berger JR. Tornatore C. Major EO. et at. Relapsing and remitting human immunodeficiency virus-associated leukoencephalopathy. Ann Neurol 1992;31 :34-8. 19. Lipton HL. Is JC virus latent in brain? Ann Neurol 1991;29:433-4. 20. Kitamura T. Aso Y. Kuniyoshi N. Hara K. Yogo Y. High incidence of urinary JC virus excretion in nonimmunosuppressed older patients. J Infect Dis 1990; 161: 1128-33. 21. Ingrand D. Morat H. Sazdovitch V. Meharzi J. Huraux JM. Incidence and clinical significance of polyomaviruses in brain tissue from patients with HIV infection [abstract M.B.2032]. In: Program and abstracts: VII International Conference on AIDS (Florence). 1991.
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12.
CNS Infection with JCV in AIDS Patients
Subclinical Central Nervous System Infection with JC Virus in Patients with AIDS E. Byrd Quinlivan, Melanie Norris, Thomas W. Bouldin, Kinuko Suzuki, Rick Meeker, Marilyn S. Smith, Colin HaU, and Shannon Kenney
Departments of Medicine. Neurology. Pathology. and Microbiology and Immunology; UNC Lineberger Comprehensive Cancer Center: AIDS Neurologic Center. University ofNorth Carolina at Chapel Hill
lC virus (lCY) is a human polyomavirus originally recovered from a patient with progressive multifocalleukoencephalopathy (PML) [I]. Subsequently, antibodies to lCV have been found in 70% of the US adult population. with seroconversion occurring most commonly in childhood and early adolescence [2]. Primary infection with lCY has not been associated with any specific syndrome. Reactivation and shedding of lCY in the urine of pregnant women and organ transplant recipients without clinical sequelae has been reported [3-6]. In patients with depressed cellular immune function, including those with AIDS, lCY has been shown to be responsible for the development of PML, a severe subacute demyelinating disease of the central nervous system (CNS) [7]. Symptomatic impairment of neurologic function, which may include motor or sensory function, speech, vision, or mentation, develops rapidly, and death usually occurs within 3-6 months after the onset of symptoms [7]. Due to the dramatic increase in the prevalence ofPML during the AIDS epidemic, lCY infection can be expected to become a significant cause of morbidity and mortality, with a marked economic impact [8-10]. The development of PML years after primary infection
Received 19 December 1991; revised 26 February 1992. Informed consent was obtained from all patients who provided peripheral blood samples and from families of patients on whom autopsies were done. in accordance with the Committee on the Protection of the Rights of Human Subjects. University of North Carolina at Chapel Hill. Financial support: National Institutes of Health (Al-07l80 [to E.B.Q.), NS-26680. AI-25868, and CA-O1229). Reprints or correspondence: Dr. E. Byrd Quinlivan, Room 210 UNC Lineberger CCe. CB# 7295, University of North Carolina at Chapel Hill, Chapel HilL NC 27599-7295.
The Journal of Infectious Diseases 1992;166:80-5 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6601-0012$01.00
with lCY implies that the disease may result from reactivation of latent virus, although a primary infection at the time ofimmunodeficiency has not yet been excluded. The demonstration of lCV in renal tissue and in the urine ofasymptomatic patients suggests that lCY remains latent in the kidneys [3, II]. There is conflicting evidence regarding whether or not the CNS is also a site of latency. It has been proposed that infection of the CNS occurs at the time of reactivation [7], but some investigators have found lCV in the brain tissue of elderly patients without clinically evident PML [12]. Other investigators report the absence of lCY in brains of both human immunodeficiency virus (HIV)-positive (+) and -negative C) patients without neurologic disease or with other neurologic diseases (such as multiple sclerosis) using a variety of methods [II, 13, 14]. Since previous efforts leave the issue of CNS latency unresolved and since the polymerase chain reaction technique (PCR) is quite sensitive, we chose this method to examine autopsy brain specimens for the presence of lCY in HIY+ patients.
Materials and Methods Collection of brain specimens for gross and microscopic examination. Brains from 13 HIV+ (type I) patients and 12 HIV- control patients were studied for the presence of lev DNA. Brains taken at autopsy from patients without clinical or pathologic evidence of HIV infection or PML were classified as HIY- and PML- and served as controls. Patients with positive HIV serologies served as the HIV+ study group. All patients in the study group but not the two medical examiner cases, for which clinical information was limited, had evidence of AIDS. Brains were coronally sectioned after fixation for ;:,.2 weeks in 10% buffered formalin. Paraffin-embedded blocks of tissue from posterior frontal cerebral cortex and white matter, hippocampus, basal ganglia. pons, medulla, and cerebellum were sec-
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Immunocompromised patients, particularly those with AIDS, develop progressive multifocal leukoencephalopathy (PML) due to central nervous system infection with JC virus (JCV). It is unknown whether JCV infection in the central nervous system can occur in the absence of PML symptoms. To address this question, autopsy specimens from patients with AIDS were examined. The brains of a group of patients without AIDS or central nervous system disease were also examined. JCV DNA was detected by the polymerase chain reaction in brain tissue from 4 (31%) of 13 human immunodeficiency virus (HIV)-positive patients. JCV was also detected in 1 elderly HIV -negative patient but not in the 11 other control brains. JCV was not detected in 22 myocardial specimens obtained at autopsy from HIV-negative patients nor 10 peripheral blood specimens from HIV -positive patients. The presence of JCV in brains of patients without clinically evident PML suggests that JCV may be present in the central nervous system without clinical disease.
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probe (lCY nucleotide 4871-4903: 5'-TCCCCACCTTTATCAGGGTGGAGTTCTTTGCAT-3') that recognized the lCY sequence within the 160-bp PCR product. All experiments contained "solution only" samples with Taq DNA polymerase as negative controls. Tissue processing and PCR reaction mixture setup took place in a separate laboratory. removed from the location of the amplification process and any lCY clones. All PCR workspace and nondisposable equipment were routinely cleaned with a I M solution of HCl or a dilute solution of chlorine-based bleach. Tissue samples were considered to contain lCY DNA if DNA was amplified in two separate experiments. PCR and Southern blot analysis were done on all samples at least twice. Clinical history. Each patient chart was reviewed, with particular attention to the cause of death, any history or clinical evidence of neurologic disease. and the results of recent neurologic studies. The evaluator was blinded to the results of the PCR evaluation at the time of the review.
Results lCV in the CNS and peripheral blood of HIV+ patients. Using the PCR technique, we found that sections of 4 (31 %) of the 13 brains of HIY+ patients were positive for lC virus (table 1). Although a number of pathologic changes were present in these brains, no specific findings correlated with the presence or absence of lCY, and there was no evidence of PML in any of these patients. Three of the 4 brains had lCY in the region of the hypothalamus, and 2 of the 4 brains also had lCY in the region of the caudate-putamen. However, the sample size is too small to assess whether this localization has significance. Review of the premortem history did not reveal any clinical characteristics that correlated with the presence of lCY by PCR in these patients (table 1). We did not detect lCY in 10 peripheral blood specimens from HIY+ patients (data not shown). PCR and Southern blot analysis demonstrated c-K-ras in all specimens except from the hypothalamus region of one patient (data not shown). A Southern blot analysis that demonstrated the PCR products in one of the brains in which lCY was detected (a 34year-old black man) is shown in figure 1. The PCR product hybridized to the internal probe with equal intensity as did the positive control, which contained a dilution of lCY plasmid equal to 6 X 10- 15 g in the PCR reaction. This sensitivity is about equal to the detection of one copy of lCY per 500 cells. For comparison, Southern blot analysis of a biopsy specimen from a patient with PML demonstrated that the number of lCY genomes in brains with PML was much greater than that seen in the reaction with the 6 X 10- 15 g lCY plasmid DNA (figure 2). lCV in the CNS and myocardium of HIV- patients. We compared our abilities to detect lCY in the brains ofHIY+ or HIY- patients. HIY- patients were those who had no evidence of HIY risk factors or HIY infection (by clinical history or autopsy findings). One of 12 control brains contained
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tioned at 6 mm, stained with solochrome-eosin, and specifically screened for changes of PML, cytomegalovirus-type intranuclear inclusions, encephalitis, meningitis, and lymphoma. Specimen collection for DNA extraction. Tissue sections (0.5-5.0 mm') of formalin-fixed brain from the 13 HIY+ and 12 HIY- patients were obtained from the cortex, caudate-putamen, globus pallidus, hippocampus, and hypothalamus regions. Formalin-fixed brain biopsy specimens from a patient with clinical and pathologic evidence of PML were used as a comparison of lCY copy number. Myocardial tissue taken at autopsy from 22 patients without clinical or pathologic evidence of HIY infection or PML was used as a negative control to exclude laboratory contamination of the samples. Peripheral blood specimens from 10 AIDS patients were used as a control for the possibility that positive results were actually due to the presence of lCY in the blood within the cerebral vessels rather than within the brain parenchyma. The mononuclear cell fraction of the fresh blood sample was harvested by processing the sample through a lymphocyte separation medium. DNA extraction. Samples of brain tissue were incubated in 0.5 M TRIS, 0.02 M EDTA, 0.01 M NaCl, 1% SDS, and 200 JLg/ml proteinase K at 48°C for 24-72 h interrupted by aggressive vortexing and two washes in PBS. Phenol extraction, chloroform purification, and ethanol precipitation were done [15]. Pelleted DNA was resuspended in sterile water and stored at - 20°e. PCR was done on all samples for detection of c-K -ras with primers (5'-ATGACTGAATATAAACTTGT-3' and 5'CTCTATTGTTGGATCATATT-3') to verify the quality of the DNA for amplification. PCR amplification was done in a 100-JLl reaction mixture of I ng of each primer, 2 JLg of sample DNA, 91 mM each dinucleotide phosphate, 0.04% Triton X, 3.6 units of Taq polymerase (Promega, Madison, WI, 16.6 mM (NH 4hS04, 67 mMTRIS-HCI (pH 8.8), 6.7 mMMgCI 2 , and 10 mM {3-mercaptoethanol in a thermal cycler (Perkin-Elmer, Emeryville, CA) for 42 cycles: cycle I: 95°C for 5 min, 55°C for I min, 70°C for I min; cycles 2-41: 94°C for I min, 55°C for I min, 70°C for 1 min; cycle 42: 94°C for I min, 55°C for 1 min, 70°C for 20 min. The PCR products were subjected to electrophoresis through a 2.5% agarose gel and transferred to a nylon filter for hybridization. The filter was hybridized at 48-50°C overnight with 50% formamide, 0.1% SDS, lOX Denhardt's solution 5X and standard saline citrate, and 50 mM NaH 2P0 4/NaHP04 to an oligonucleotide probe (5'-GGAGCTGGTGGCGTAGGCAA-3') that recognized the c-K-ras sequence within the IIO-bp PCR product. The probe was end-labeled with [')'-32P]adenosine triphosphate by means ofT4 polynucleotide kinase; the results were visualized by autoradiography. PCR and Southern blot analysis. PCR was done with primers to the T antigen region of lCY (lCY nucleotide 48514870: 5'-TAAAAGATCCTTCTCTTCATCTTGTCTTCG-3'; lCY nucleotide 5003-4984: 5'-ATTGGGAATTCTGCTGAAT AGGGAATCC-3'). PCR and Southern blot analysis were done as above with the following changes in 42 cycles of amplification done in a DNA thermal cycler (Coy, Ann Arbor, MI: cycle I: 94°C for 5 min, 65°C for 1 min, 70°C for 2 min; cycles 2-41: 94°C for I min, 65°C for 1 min, 70°C for I min; cycle 42: 94°C for I min, 65°C for I min. 70°C for 20 min. The filter was then hybridized at 42-45°C overnight to an oligonucleotide
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Table 1. Detection of JC virus (JCY) in brains of HIY+ patients. CNS region ofJCV detection
39. white . male 60. white . male 41. white . male 47 . white . male 34. black . male
CPo HI. HY CP.GP HY Negative HY
31. black. male
Negative
40 . black. 31. black. 42 . white. 40. black.
female male male male
Negative Negative Negative Negative
39. black. male 38. black. female 27. white. male
Negative Negative Negative
Pathologic findings
CNS disease by clinical history
None None Multinucleate cells Brain perivascular chronic inflammation. white matter pallor Multinucleate cells. meningeal perivascular chronic inflammation. CMV-type nuclear inclusions. white matter pallor Multinucleate cells. meningeal perivascular chronic inflammation White matter pallor Microglial nodules Microglial nodules. CMV-type nuclear inclusions Multinucleate cells. brain perivascular chronic inflammation. CMV-type nuclear inclus ions. CNS cryptococcosis, CNS lymphoma Meningeal perivascular chronic inflammation Multinucleate cells. CNS lymphoma Perivascular chronic inflammation. microglial nodules. CMV-type nuclear inclusions. white matter pallor. CNS lymphoma
Hepatic encephalopathy Cryptococcal meningitis (inactive) Cytomegalovirus (CMV) retinitis Dementia Dementia
ME case' Mild dementia None Cryptococcal meningitis (inactive). CMV retinitis Cryptococcal meningitis (inactive)
ME case' CMV retinitis CMV retinitis
NOTE. Negative; JCV not detected by polymerase chain reaction technique (PCR) or detected in only one experiment; otherwise JCV detected by PCR in two or more separate experiments. CPo caudate putamen; GP. globus pallidus : HI. hippocampus; HY. hypothalamus; CNS. central nervous system. * Medical examiner case . autopsy limited to CNS.
detectable lCY (table 2) . This 79-year-old man, the oldest patient in our series, died from cancer of the hypopharynx, chronic lung disease, and a myocardial infarction. The diagnosis of cancer was made shortly before the patient's death and he refused all therapy. There was no evidence that he
6x1 a-13 g JCV clone I 6x10- 14 g JCV clone I 6x1 a- 15 g JCV clone I 6x1 a- 16 g JCV clone Negative control: Solutions only
IC I CP I GP I HI I HY Figure 1. Detection ofJC virus (lCY) by polymerasechain reaction technique (PCR) and Southern blot analysis. DNA extracted from regions of cortex (C). caudate-putamen (CP), globus pallidus (OP), hippocamus (HI). and hypothalamus (HY) were subjected to PCR and Southern blot analysis with JCY. Serial dilutions of a genomic clone of lCY were included as positive controls. Sample without DNA was included as negative control (solutions only).
received any immunosuppressive therapy. In addition, there was no evidence clinically or pathologically that he had PML. We did not detect lCY in any of22 autopsy specimens of cardiac tissue from HIY- patients (data not shown). c-Kras was successfully detected by PCR followed by Southern blot hybridization in all specimens (data not shown).
Discussion PML, a previously rare disease, occurs in up to 4% of patients with HIY infection. Although the possibility that PML results from primary infection of immunocompromised patients has yet not been rigorously excluded, it has been assumed that the disease is a result of reactivation of lCY that has been present in a latent form in the kidneys since initial infection in childhood [7]. lCY may be transported from the kidney to the brain by peripheral blood lymphocytes [16]. However, it is also possible that PML results from reactivation of previously latent CNS infection in immunocompromised hosts. We show evidence here that lCY may be present in the brains of patients with HIY infection but without evidence ofPML. In addition, one elderly patient with head and neck carcinoma among the 12 HIY- patients had lCY detectable by PCR. No specific clinical or pathologic findings were associated with the detection of lCY, and the lCY
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Patients; age. race. sex
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I I I
10x10- 15g JCV clone
I I
6x10 - 16 g JCV clone 6x1 0-1 7 g JVC clone I 6x1 0- 18 g JVC clone I
I
I I
I I
Negative control : Solutions only
} Bx
detected most likely represents latent or subclinical infection. Although PCR analysis is not strictly quantitative, our analysis of a brain from a patient with PML suggested that the level of lCY DNA in the asymptomatic patients is likely to be lower than in patients with PML (cf. figures I and 2). Other investigators have postulated that lower levels of lCY
Table 2.
Detection of JC virus (JeY) in brains of HIY- patients.
Patients: age. race. sex
CNS region of JCY detection
23. whit e. male
Negative
49 . black. male 46 . black . female 15. black . female
Negative Negative Negative
41. 27. 49 . 27.
Negative Negative Negative Negative
white. male black . male black. male Hispanic. female
16. black. female 59. white . male 43. white. female
Negative Negative Negative
79. white . male
CP
Pathologic finding s Brain and meningeal perivascular chronic inflamm ation None None Meningeal perivascular chronic inflammation None None None Meningeal perivascular chronic inflammation None None Brain and meningeal perivascula r chronic inflammation None
CNS disease by clinical history None None None None Down's syndrome None None None None None None None
NOTE. Negative : JCV not detected by polymerase chain reaction technique (PCR) or detected in only one experiment: otherwise detected by PCR in two or more experiments. CPocaudate putamen; CNS. cen tral nervous system .
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Figure 2. Southern blot analysis of brain biopsy specimen from patient with AIDS. DNA extracted from brain biopsy of patient with progressive multifocalleukoencephalopathy was subjected to polymerase chain reaction and Southern blot analysiswith JC virus (JCY) probe. Serial dilutions of genomic clone of JCY were included as positive controls. Sample without DNA was included as negative control (solutions only).
in autopsy specimens than in biopsy specimens in patients with PML may be due to degradation of the DNA during processing [13]. However, our observation that lCY is less abundant in asymptomatic patients than in the PML biopsy specimen is unlikely to be due to degradation of DNA, because we extracted the DNA rather than using a lysed-cell extract and amplified the proto-oncogene c-K-ras in all but one of our samples, indicating that the extraction was successful. One potential source oferror in this work is the presence of blood contamination of brain tissue . AIDS patients, who have a high incidence of syndromes associated with viral reactivation [17], might be expected to have a high incidence of identifiable virus in circulating blood cells . Indeed, lCY has been found in B cells of 2 patients with PML (I with AIDS and one without any known underlying disease) [ 16] and in I AIDS patient without PML [18]. PCR methods are so sensitive that the presence of lCY-containing blood cells could give false-positive results during the evaluation of brain tissue . Two procedures were undertaken to prevent this, and the results make it unlikely that our findings are due to blood contamination. First, we were unable to detect lCY in the mononuclear cell fraction of peripheral blood samples taken from a group of 10 AIDS patients (data not shown). Second, in none ofthe liver specimens from 3 of the 4 AIDS patients and the I HIY- patient who had lCY detected in their brains could we detect lCY (data not shown). Due to the vascular nature of the liver, we believe it served as an adequate control for possible blood contamination of tissue specimens, although the ideal control would have been pe-
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of PML. However, our results do not distinguish between reactivated latent CNS lCY infection and primary lCY infection in immunocompromised hosts. A prospective serologic study would be useful to determine if only AIDS patients who are initially seronegative for lCY are at risk for developing PML. It is interesting to speculate whether the subjects with positive PCR results would have developed overt disease had they survived, that is, whether the presence of lCY in brain is a predictor offuture PML. It is also interesting to consider the possible clinical spectrum of CNS infection with lCY. There certainly may be marked variability in the duration and progression ofPML. Berger et al. has drawn attention to the fact that some patients may have a particularly long course [8], and we have had similar experience (unpublished data). Two patients reported by Mori et al. with asymptomatic lCY CNS infection died with neurologic illness (dementia and Parkinson's disease), and in 3 of their 4 patients the lCY was detected near areas of tiny foci of demyelination. In the AIDS patients reported here, a number of neurologic conditions (clinical and pathologic) not typical of PML were present (table 1), but these did not correlate with the presence or absence of lCY. In summary, we have detected lCY in autopsy brain specimens of 4 of 13 HIY+ patients and I of 12 HIY- patients. We have therefore established that lCY can frequently be found by PCR in the brains of AIDS patients who do not have clinical evidence of PML. Whether this is a truely latent infection (rather than early reactivation or subclinical primary infection), whether it is a predictor of the future development ofPML, and whether it may playa role in the development of other neurologic diseases are all ideas that require further investigation.
Acknowledgments
We thank lames N. Hayward for providing workspace for PCR in a lCV-free environment and Edison Liu for assistance with PCR.
References I. Padgett BL, Walker DL, ZuRhein GM, Eckroade RJ, Dessel BH. Cultivation of pap ova-like virus from human brain with progressive multifocal leukoencephalopathy. Lancet 1971; I: 1257-60. 2. Padgett BL, Walker DL. Prevalence ofantibodies in human sera against JC virus, an isolate from a case ofprogressive multifocalleukoencephalopathy. J Infect Dis 1973;127:467-70. 3. Arthur RR. Shah KV. Occurrence and significance of papovaviruses BK and JC in the urine. Prog Med Virol 1989;36:42-61. 4. F1regstad T, Sundsfjord A, Arthur RR, Pedersen M. Traavik T. Subramani S. Amplification and sequencing of the control regions of BK and JC virus from human urine by polymerase chain reaction. Virology 1991;180:553-60. 5. Myers C Frisque RJ, Arthur RR. Direct isolation and characterization
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ripheral blood samples from all patients in the study group. In one additional AIDS patient (a 47-year-old white man), lCY was detected all of three times in the liver specimen but only once in the cortex and hippocampus regions of the CNS. We classified this patient as negative as we were not able to amplify lCY from any region of the CNS more than once and the liver specimen was consistently positive. However, it is possible that lCY was actually present in the brain tissue of this patient. Particular care was taken to avoid laboratory contamination, a common source of error with PCR. All of our experiments contained solution-only samples as negative controls. Tissue processing and PCR reaction mixture setup took place in a laboratory removed from the location of the amplification process and any lCY clones, and all PCR workspace and nondisposable equipment were routinely cleaned. In addition, we required that before any specimen was considered positive, lCY was detected on at least two occasions. As a further control, autopsy tissue from 10 hearts from HIYpatients was evaluated using the same techniques, and none demonstrated lCY. The question ofCNS latency of lCY in non-HIY-infected patients has been studied but not resolved [19]. Telenti et al. [13] looked for lCY by PCR in the brains of 17 neurologically intact patients and 4 patients with neurologic conditions. Only one patient, with herpes encephalitis, showed a very weak detection of lCY, and this was attributed to contamination. In contrast, Mori et al. [12], using in situ hybridization and immunocytochemistry, systematically examined the brains of 10 elderly patients without PML or known immunosuppressive conditions and found lCY in 4. They postulated these findings may be due to the advanced ages of the patients and the chronic nature of the diseases present at the time of death. Elderly patients have also been reported to have an increased incidence of lCY shedding into the urine, presumably from a renal source [20]. In keeping with this, the one HIY- patient in whom we detected lCY was 79 years old. With this in mind, the finding of such a high incidence of apparently asymptomatic lCY CNS infection in our HIY+ population may not be surprising. Imrnunocompromised patients and AIDS patients in particular are known to have a general increase in viral disease. Other investigators, at the most recent International Conference on AIDS, have reported finding lCY in the CNS of AIDS patients without clinical evidence of PML [21]. Since the reported incidence of PML is higher in those with AIDS than in any other patient population (although neither population has been studied prospectively) [8-10], it is reasonable that AIDS patients may also have a higher frequency of subclinical (or latent) lCY CNS infection. Our findings add further support to the idea that lCY may be present in the brains of patients without clinical evidence
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of JC virus from urine samples of renal and bone marrow transplant patients. J Virol 1989;63:4445-9. Yogo Y. Kitamura T. Sugimoto C. et at. Isolation of a possible archetypal JC virus DNA sequence from nonimmunocompromised individuals. J Virol 1990;54:3139-43. Greenlee JE. Progressive multifocal leukoencephalopathy. Curr Clin Top Infect Dis 1989;10:140-56. Berger JR. Kaszovitz B. Post J. Dickinson G. Progressive multifocal leukoencephalopathy associated with human immunodeficiency virus infection. Ann Intern Med 1987; 107:78-87. Krupp LB. Lipton RB. Swerdlow ML. Leeds NE. L1ena J. Progressive multifocal leukoencephalopathy: clinical and radiographic features. Ann Neurol 1985; 17:344-9. Levy RM. Bredesen DE. Rosenblum ML. Neurological manifestations of the acquired immunodeficiency syndrome (AIDS): experience at UCSF and review of the literature. J Neurosurg 1985;62:475-95. Chesters PM. Padgett BL. Walker DL. Persistance of DNA sequences of BK virus and JC virus in normal human tissues and diseased tissues. J Infect Dis 1983;147:676-84. Mori M. Kurata H. Tajima M. Shimada H. JC virus detection by in situ hybridization in brain tissue from elderly patients. Ann Neural 1991 ;29:428-32. Telenti A. Aksamit AJ Jr. Proper J. Smith TF. Detection of JC virus DNA by polymerase chain reaction in patients with progressive multifocal leukoencephalopathy. J Infect Dis 1990; 162:858-61.
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14. Wiley CA, Grafe M. Kennedy C. Nelson JA. Human immunodeficiency virus (HIV) and JC virus in acquired immune deficiency syndrome (AIDS) patients with progressive multifocalleukoencephalopathy. Acta Neuropathol 1988;76:338-46. 15. Goeiz SE. Hamilton SR. Vogelstein B. Purification of DNA from formaldehyde fixed and paraffin embedded human tissue. Biochem Biophys Res Comm 1985;130:118-26. 16. Houff SA, Major EO, Katz DA, et at. Involvement of JC virus-infected mononuclear cells from the bone marrow and spleen in the pathogenesis of progressive multifocal leukoencephalopathy. N Engl J Med 1988;318:301-5. 17. DeVita VT. Hellman S. Rosenberg SA. AIDS: etiology. diagnosis. treatment. and prevention. 2nd ed. Philadelphia: JB Lippincott. 1988:474. 18. Berger JR. Tornatore C. Major EO. et at. Relapsing and remitting human immunodeficiency virus-associated leukoencephalopathy. Ann Neurol 1992;31 :34-8. 19. Lipton HL. Is JC virus latent in brain? Ann Neurol 1991;29:433-4. 20. Kitamura T. Aso Y. Kuniyoshi N. Hara K. Yogo Y. High incidence of urinary JC virus excretion in nonimmunosuppressed older patients. J Infect Dis 1990; 161: 1128-33. 21. Ingrand D. Morat H. Sazdovitch V. Meharzi J. Huraux JM. Incidence and clinical significance of polyomaviruses in brain tissue from patients with HIV infection [abstract M.B.2032]. In: Program and abstracts: VII International Conference on AIDS (Florence). 1991.
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