Early viral brain invasion in iatrogenic human immunodeficiency virus infection L.E. Davis, MD; B.L. Hjelle, MD; V.E. Miller, PhD; D.L. Palmer, MD; A.L. Llewellyn, MD; T.L. Merlin, MD; S.A. Young, PhD; R.G. Mills; W. Wachsman, MD, PhD; a n d C.A. Wiley, MD, PhD

Article abstract-We report a 68-year-old man who received an IV inoculation of WBCs for an indium radionuclide scan containing 600 to 700 tissue culture infectious doses of human immunodeficiency virus type 1 (HIV-1) from an HIV-1-infected individual. The recipient immediately received zidovudine, then was switched to dideoxyinosine and interferon-a, but died of hepatorenal syndrome and hepatic encephalopathy 15 days later. HIV-1 cultures were positive from the recipient's blood on day 14 but not days 0, 1,and 8. At autopsy, cultures of parietal lobe isolated HIV-1. HIV-1 nucleic acid was present in several brain areas, but not in several other organs, by two independent laboratories using the polymerase chain reaction. The brain showed mild perivascular cuffing and a mild lymphocytic meningitis, but there was no evidence of glial nodules, giant cells, or white matter abnormalities. HIV-1 gp41 viral antigen was seen by immunoperoxidase staining in rare infiltrating cells within perivascular and subpial spaces. Thus, HIV-1 was isolated from brain 15 days after mistaken HIV-1 inoculation arid 1day after virus was first recovered from blood. NEUROLOGY 1992;42:1736-1739

How soon after the primary infection human immunodeficiency virus type 1 (HIV-1) crosses t h e blood-brain barrier to invade the CNS is presently unknown. Existing information suggests HIV- 1 can i n v a d e t h e meninges w i t h i n m o n t h s of p r i m a r y infection, with acute viral meningitis as an initial clinical manifestation of the infection. l z 2 In m a c a q u e s i n t r a v e n o u s l y infected w i t h a related lentivirus, simian immunodeficiency v i r u s (SIV), v i r a l replication occurred i n brain p a r e n c h y m a within 7 days.3 Knowing w h e n HIV-1 enters the CNS may be important in choosing antiretroviral drugs that cross the blood-brain barrier. We took t h e opportunity t o investigate a nonHIV-infected patient who accidentally received an inoculum of HIV-1-infected WBCs. Within 15 days of the infusion error, the recipient developed viral and histologic evidence of an HIV-1 infection of the brain. Methods. Case report. The recipient of the HIV-1 inoculum was a 68-year-old man who was hospitalized for evaluation of left hip pain and fever. He had no prior risk factors for HIV-1 infection. He had received a prosthetic

left hip joint 12 years earlier. Thirty years earlier, he had experienced a gunshot wound to his left frontal lobe. The patient had a history of severe alcoholism, hepatic cirrhosis, abdominal ascites, anemia, and pancytopenia. A bone marrow aspiration 1 year earlier was consistent with changes of chronic alcoholism. On examination, the recipient was alert and oriented but had a mild dementia. He had a right Babinski sign and a moderate peripheral neuropathy with diminished appreciation of light touch, temperature, vibration, and position sense in the feet and fingertips. An anterior left thigh abscess was identified, incised, and drained. Antibiotics were given. A radiolabeled indium 111 P"In) WBC scan was ordered to determine whether osteomyelitis of the left femur was also present. Through an error in patient identification, the recipient received 5 ml IV of "'In WBCs that had originated from an HIV-1-infected patient with the same last name. The error was recognized immediately and the recipient was informed. With his consent, he was given his first dose of zidovudine, 400 mg orally, within 45 minutes of the infusion error. The recipient then received 200 mg of zidovudine orally every 4 hours for 3 days. He was then switched to dideoxyinosine (ddI), 250 mg orally twice a day. In addition to the ddI, the recipient received

From the Neurology (Dr. Davis), Medical (Dr. Palmer), and Pathology Services (Drs. Llewellyn and Merlin), Albuquerque Veterans Affairs Medical Center, Albuquerque, NM, the Departments of Neurology (Dr. Davis), Pathology (Drs. Hjelle and Miller), and Microbiology (Dr. Young and R.G. Mills), University of New Mexico School of Medicine, Albuquerque, NM; the Research Service (Dr. Wachsmanl, San Diego Veterans Affairs Medical Center; and the Division of Hematology-Oncology (Dr. Wachsman) and Department of Pathology (Dr. Wiley), University of California, San Diego, CA. Supported by the Research Service, Department of Veterans Affairs, Blood Systems Research Foundation, NIH grants R01 CA55480, NS-25178 and NS27810, University of California AIDS task force grant, aiid the San Diego VAMC Research Center on AIDS and HIV infection. Dr. Wachsman is the recipient of a VA Clinical Investigation award. Received November 6, 1991. Accepted for publication in final form February 27, 1992 Address correspondence and reprint requests to Dr. Larry E. Davis, Neurology Service, Albuquerque VA Medical Center, 2100 Ridgecrest Drive SE, Albuquerque, NM 87108.

1736 NEUROLOGY 42 September 1992

Figure 1. (A) Focus of mononuclear cells around a blood vessel i n the thalamus of the recipient (hematoxylineosin, X200 before 8 8 reduction). (B) Focus of mononuclear cells in the meninges of the cerebellum of the recipient (hematoxylin eosin, X200 before 12%, reduction). f C ) Paraffin section of cerebral cortex immunocytochemically stained for H N g p 4 1 . In cerebral cortex a solitary, pericapillary cell is immunostained (red)for the transmembrane envelope protein gp41 of H N . Adjacent cortical tissue is not inflamed. (Counterstained with hematoxylin, X400 before 5% reduction.)

interferon-a, 30 million U intramuscularly, followed by 20 million U every other day. The decision to switch drugs was based on the belief t h a t the donor's HIV-1 strain might be zidovudine r e s i ~ t a n t . ~ During the hospitalization, the recipient slowly deteriorated and became more lethargic. The diminished ment a l s t a t u s was felt to r e p r e s e n t increasing hepatic encephalopathy as he developed increasing blood ammonia and alanine aminotransferase levels. He never developed headaches, meningismus, myalgias, rashes, pharyn-

gitis, or systemic adenopathy. He developed increasing anemia and required several blood transfusions. The prothrombin time was prolonged, preventing diagnostic lumbar punctures. Fourteen days after the HIV-l-infected infusion, the patient became comatose; and he died on day 15 of hepatorenal syndrome. The donor of HIV-1-infected cells was a 34-year-old HIV-1 antibody-positive homosexual male who had developed AIDS 15 months earlier. He had received zidovudine for the past 18 months. An "'In WBC scan September 1992 NEUROLOGY 42 1737

Table. HIV isolation and PCR studies of HIV-1infected WBC infusion recipient

i HN-1 detection method

Blwd Virus isolation ProviralDNAby PCR

Days after HIV-1-infected WBC infwion

I

15

0

1

8

14

autopsy

Neg Neg

Neg Neg

Neg Pas

Pas Pas

ND ND

Brain Virus isolation Proviral DNA by PCR

Pas Pos

ND Notdone.

had been ordered to evaluate a possible osteomyelitis. Due to the mistake, the patient did not receive his ll11nlabeled WBC scan on that day. One day later, the "'In WBC scan was repeated under conditions identical to the previous preparation. At this time, a n aliquot of WBCs from the final WBC preparation was cultured for t h e presence of HIV-1 virus. Tenfold dilutions of the white cells were made and individually inoculated into susceptible peripheral blood mononuclear cell (PBMC) cultures to determine the tissue culture infectious dose (TCID) of HIV-1 present in the 5 ml i n o c ~ l u m . ~ Autopsy. The recipient had 4 liters of abdominal ascites, severe cirrhosis of the liver, splenic enlargement, and congestion and varices of the esophagus. An osteomyelitis was present in the left femur. Tissue from bone marrow, spleen, and blood were taken for HIV-1 culture. The brain weighed 1,100 grams. On gross inspection, a fibrotic scar in the left frontal cortex was seen that corresponded to his old gunshot wound. Microscopically, foci of mononuclear cells were identified around many blood vessels scattered throughout the cerebral cortex, basal ganglia, and cerebellum (figure 1A). The meninges of the occipital and temporal lobes and cerebellum demonstrated regions containing mild mononuclear cell infiltrates (figure 1B). No multinucleated giant cells were seen in the brain parenchyma or meninges. The cerebral cortex demonstrated a mild prominence of glia cells, many of which were type I1 astrocytes. A piece of relatively bloodless right parietal lobe white matter that did not contain meninges was taken for HIV1culture. Virus culture and detection. PBMCs were purified by density gradient centrifugation of patient blood and from the blood of seronegative controls, as described.6 For bone marrow and spleen cell preparations, cells were taken u p in media by t r i t u r a t i o n with a pipet, a n d mononuclear cells purified as for PBMC preparations. Mononuclear cells were cocultivated with an equal number of PBMC from a seronegative blood donor in media containing 10% interleukin-2 and anti-interferon-a antibody.5 Brain tissue was washed thoroughly of any blood and homogenized in a Dounce homogenizer; the equivalent of 50 mg of brain tissue was used per lo6 uninfected PBMC in coculture. Production of HIV-1 was monitored by biweekly assays of culture supernatant for p24 antigen as recommended by the manufacturer (DuPont NEN, North Billerica, MA). In some cases, DNA was prepared from cell cocultures for polymerase chain reaction (PCR) after p24 antigen production was demonstrated. Culture supernatants repeatedly reactive for p24 antigen were retested with a confirmatory neutralization s t e p by preincubation with anti-p24 antibody to determine that the reaction to HIV-1 p24 antigen was specific. 1738NEUROLOGY 42 September 1992

Polymerase chain reaction. DNA preparation7 and PCR were essentially as described.8 Primers designated LTR8 [5' AGA,CAA,GA(T/C),ATC,CTT,GAT,CTG,TGG] and LTR129 ( 5' AGC ,ACC,ATC,CAA,AGG,TCA,GTG,G) were selected on the basis of their high conservation in numerous HIV-1 isolates. PCR reactions were carried out in 25 ~1 volume with 4 mM MgCl,, 1 mM of each dNTP, and 1.6 nM of each primer. Amplification was for six cycles at 97", 40", and 70", then 40 cycles a t 94", 55",and 72" (all cycles 1minute in length). Amplified product was detected with the 32Pend-labeled oligonucleotide LTRPROBE ( 5 ' ACA,CAC,AAG,GCT,ACT,TCC,TGA, TTG,GCA,GAA,CTA,CAC,A) in a "dot-blot" format. As a control for the quality of DNA extracted from the blood samples, PCR was also performed to detect the glyceraldehyde-3-phosphate dehydrogenase gene.g DNA extractions from paraffin-embedded blocks of tissue were performed as described p r e v i o ~ s l y PCR .~ methods were also the same as previously described, except t h a t the following HIV-1-specific primers were added: LA8(5'-GCG,CGC,ACA,GCA,AGA,GGC,GA-3') (Proviral b a s e s 7 1 1 t o 730) a n d LA9(5'-GAC,GCT,CTC, GCA,CCC,ATC,TC-3') (Proviral bases 805 to 786). Both HIV-1 positive and HIV-1 negative DNA extracted from formalin-fixed tissues were assayed as controls. Zmmunocytochemistry and in situ hybridization for HZV-1. Immunocytochemistry for HIV-1 p24 and gp41 antigen was performed according to standard protocols.1° Slides of paraffin-embedded tissues were briefly permeabilized with 0.1% triton X-100 and proteinase K digestion, and endogenous peroxidase blocked with 3% H,O, i n absolute methanol. Blocking goat antiserum was applied for 10 minutes before addition of the mouse monoclonal anti-HIV-1 p24 antibody (Dupont) at 5 p,g/ml of 1% BSA in PBS. Sections were rinsed, and biotinylated secondary goat antimouse antibody added before avidin peroxidase complex was added a n d developed with aminoethylcarbazole. Control sections were incubated in normal mouse immunoglobulin. In situ hybridization was performed as originally described'O using 35S-labeled HIV-1-specific and plasmid probes.

Results. Serial dilution studies of the donor ll1Inlabeled WBC inoculum obtained 2 4 h o u r s after t h e infusion e r r o r from the donor were found t o contain 600 to 700 TCIDs of HIV-1 virus. A blood sample from the recipient that was taken after the infusion error, but before zidovudine, failed t o contain virus b y isolation o r P C R techniques. HIV-1 was isolated by PBMC c o c u l t u r e o n l y in c u l t u r e s f r o m d o n o r blood ( d a y O), r e c i p i e n t blood ( d a y 1 4 only), and recipient brain ( d a y 15; table). H y b r i d i z a t i o n results f r o m a m p l i f i e d D N A o b t a i n e d f r o m r e c i p i e n t P B M C and t i s s u e s o b t a i n e d at a u t o p s y f r o m the r e c i p i e n t did n o t detect proviral DNA in the recipient blood until d a y 8. It was again a p p a r e n t in the d a y 14 P B M C preparation. Provirus was easily detected b y PCR in c e l l s f r o m a u t o p s y f r o z e n brain and s p l e e n (despite o u r inability t o isolate virus from spleen cells), and an extremely w e a k signal w a s observed in bone m a r r o w mononuclear cell PCR a s s a y (figure 2). P C R performed o n paraffin blocks of gut, liver, k i d n e y , heart, lung, and s p l e e n showed n o identifiable HIV-1-specific sequences. Blocks of

Figure 2. Autoradiogram of "dot-b1ot"produced by hybridization of "2P-labeledLTRPROBE to DNAs (2gi subjected to amplification with LTR8 and LTR129 primers. Source of D N A is as indicated by the coordinates of the dot. PBMC DNAs from three HIV-seropositive controls (dots A - 1 through A-31; from donor PBMCs (dot B-1); from recipient PBMCs at day 0 (B-21, day 1 (B-31, day 8 (C-11,and day 14 (C-2);mononuclear cell D N A from recipient spleen (C-3)and bone marrow (E-2);brain homogenate (0-3);D N A from cells after 14 days of coculture with uninfected PBMC prepared from donor PBMC (D-l), day 14 recipien.t PBMC (0-2, E-31, and brain homogenate (E-1);and D N A from PBMC of three HN-seronegative controls (F-1 through F-3).

ed HIV-1 contamination from the blood, we think it was unlikely. First, the PCR signal detected in the brain was of equal intensity to the blood signal, yet blood represents about 2% of brain volume. Second, HIV-1 was not detected by PCR in other organs, such as gut, liver, and kidney, which contain as much blood as the brain does. Third, HIV-1 was successfully c u l t u r e d from t h e p a r i e t a l lobe stripped of i t s vascular meninges. Fourth, we observed multiple foci of perivascular mononuclear cuffing in the brain and rare cells immunostained for HIV-1 gp41 antigen. This unique situation offered a rare glimpse of the kinetics of tissue invasion by HIV-1 during a primary infection. We found that HIV-1 infection of the brain can occur rapidly after infusion infection. Since this patient received a large inoculum of virus intravenously, we do not know whether a similar rapid brain entry occurs following other modes of transmission, such as sexual intercourse o r fingerstick inoculation. However, our findings are similar to those of SIV, in which viral replication and perivascular infiltrates occurred in monkey brains 7 days after intravenous inoculation.3

Acknowledgments We thank Drs. Mario Kornfeld a n d Margaret Listrom for their help in t h e pathologic examination of this patient.

References 1. Chiodi F , Albert J, Olausson E, et al. Isolation frequency of

cerebral cortex, spleen, and a block containing systemic tissues, including a lymph node, did contain HIV-1 nucleic acid-specific sequences. The control housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase, was detected in the DNA from all four blood samples. Whereas immunocytochemical staining for p24 did not identify any positive staining above background, staining for gp41 showed rare infiltrating cells w i t h i n p e r i v a s c u l a r a n d s u b p i a l spaces around a remote incomplete infarction (figure 1C). In situ hybridization preparations for HIV-1-specific nucleic acids were not convincingly different from control plasmid probes.

Discussion. In spite of rapid (45 minutes after infusion error) administration of zidovudine and later administration of ddI and interferon-a, the patient still became HIV-1 infected. We first detected HIV-1 virus in the recipient's blood by PCR day 8. On day 14, HIV-1 was first isolated from blood. One day later, HIV-1 was isolated from the brain. In addition, HIV-1 nucleic acid was detected in multiple areas of the brain by PCR in two different laboratories. Although it is possible that the HIV-1 recovered from the brain represent-

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