—  SPECIALTY CONFERENCE  —

Infectious Disease Pathology

Case 1 - Rabies Virus Infection

Elizabeth C. Burton
Baylor University Medical Center
Dallas, TX


Click on each slide thumbnail image for an enlarged view
Clinical History
A 50 year old African American female underwent a cadaveric renal transplant for end stage renal disease secondary to hypertension and diabetes. She was readmitted (19 days post-transplantation) for an increased creatinine level, hydronephrosis and mild acute cellular rejection. Her immunosuppressive therapy was adjusted and she was discharged home four days later with a nephrostomy tube in place. Four days following discharge (27 days post-transplantation), she was readmitted to the hospital for abdominal/right flank pain that had worsened over the preceding two days. Admission laboratory data were significant for: hemoglobin of 11.2 g/dL, hematocrit of 33.7%, red cell distribution width—coefficient of variation (RDW-CV) of 16.6%, glucose level of 290 mg/dL, blood urea nitrogen (BUN) of 48 md/dL, creatinine of 1.2 mg/dL, globulin 4.0 g/dL, total bilirubin 1.0, aspartate aminotransferase (AST) 61 U/L, alanine aminotransferase (ALT) 88 U/L, and her urine culture was positive for Enterococcus faecalis. Her white blood cell count, remaining blood cell indices, and remaining chemistries were within normal limits. She underwent an appendectomy on the day following admission for presumed acute appendicitis. Postoperatively, her temperature was decreased at 95.8° Fahrenheit and a warming blanket was placed. She continued to complain of abdominal, right flank pain and itching in the right flank area. She became increasingly restless, agitated and developed seizure activity. A computed tomography (CT) scan of the head was obtained and showed no evidence of acute abnormality. An electroencephalogram (EEG) study was significant for mild to moderate generalized slowing. Over the next 36 to 48 hours, she became increasingly disoriented with hallucinations and developed hypotension, bradycardia and right bundle branch block requiring temporary pacemaker placement. She was transferred to the intensive care unit where she developed respiratory failure and was intubated (postoperative day 3). Her cyclosporine level increased to 809 ng/mL (reference 150-400 ng/mL) and her cyclosporine was held. Over the next few days, she became increasingly obtunded with increased respiratory/oral secretions, continuous eye movements and facial grimacing. By postoperative day 7, she continued to have seizure activity and a repeat CT scan of the head again showed no acute abnormalities. Within the next 8 hours, she progressed to coma with absent brainstem reflexes. A repeat CT scan was now significant for generalized edema with brainstem herniation. A "do not resuscitate order" was placed and the following morning, she developed ventricular arrythmias/asystole and died.

Autopsy Findings
Autopsy examination was significant for an organized hematoma surrounding the pelvic transplant kidney which had patent and intact vascular and ureteral anastomoses and microscopic evidence of acute tubular injury, early microangiopathy, non-specific chronic interstitial nephritis, and perihilar neuritis. The native kidneys were remarkable for end stage renal disease. There was also acute bilateral bronchopneumonia with early organization and a mild bilateral lymphocytic adrenalitis. Gross examination of the central nervous system (CNS) found marked cerebral edema with bilateral tonsillar and transtentorial herniation. Selected microscopic images from the CNS are below.


Case 1 - Figure 1 - Leptomeninges with a mild mononuclear infiltrate.
Case 1 - Figure 2 - Brainstem with perivascular lymphocytic cuffing and microglial nodules
Case 1 - Figure 3 - Lymphocytic perivascular cuffing



Case 1 - Figure 4 - Neuronophagia
Case 1 - Figure 5 - Purkinje cells with Negri bodies
Case 1 - Figure 6 - Purdinje cells with Negri bodies



Case 1 - Figure 7 - Inflammatory process in the spinal cord
Case 1 - Figure 8 - Mononuclear infiltrate in the dorsal root ganglion.

Histologic findings
H&E: Examination of the leptomeninges (figure 1) found a mild mononuclear infiltrate. Sections from the brainstem (figures 2, 3, 4) found perivascular lymphocytic cuffing, neuronophagia and microglial nodules. Sections from the cerebellum (figures 5 and 6) found clusters of Purkinje cells containing eosinophilic cytoplasmic inclusions (Negri bodies). A section from the spinal cord (figure 7) found mononuclear inflammatory infiltrates, perivascular lymphocytic cuffing, microglial nodules and neuronophagia with both gray and white matter involvement. A section from the dorsal root ganglion found an extensive mononuclear inflammatory cell infiltrate and occasional Negri bodies within the dorsal root ganglion cells.

Diagnosis: Rabies virus infection

Additional clinical information
This patient was one of three patients who received a solid organ from a common donor that was infected with rabies. A fourth liver transplant recipient had received a vascular graft from the same donor. The donor had unknowingly been infected with the rabies virus and died at an outside hospital from other suspected causes and did not undergo an autopsy. The three solid organ recipients had all presented within 30 days of their transplant procedures with signs and symptoms of progressive encephalitis that progressed to coma and death. The fourth patient who received the vascular segment had a protracted course complicated by wound dehiscence, abdominal infections, hepatic artery stenosis—necessitating two additional returns to the operating room, seizures, rapid neurologic deterioration, coma and died without ever leaving the hospital following her transplantation procedure. Clinically, the differential diagnosis in the four transplant recipients included toxic encephalopathy, herpes encephalitis, and West Nile virus infection.

Laboratory evaluation
Antemortem diagnosis of rabies infection rests on demonstration of viral nucleic acid and/or viral antigen in skin, saliva, cerebrospinal fluid, or corneal impressions (representing sites infected through centrifugal spread of the virus from the brain downward). Skin can be evaluated for rabies antigen using fluorescent antibody techniques (the optimal skin biopsy is a 5-mm punch biopsy from the nape of the neck above the hairline where there is dense innervation surrounding hair follicles). Serum and CSF can be evaluated for the detection of rabies virus IgG and IgM antibodies. CSF, saliva, and tissue (skin or brain) can be evaluated for rabies virus RNA by RT-PCR. Brain biopsy, although not commonly done can be assessed for rabies by multiple methods including viral isolation, fluorescent antibody testing, immunoperoxidase staining, in situ hybridization for rabies virus RNA, or detection of rabies virus RNA by RT-PCR amplification.

Histopathology of rabies encephalomyelitis
Microscopic findings do not necessarily parallel the severity of clinical disease, differ from case to case and generally are subtle or inconspicuous. Negri bodies can provide microscopic evidence of rabies infection but the sensitivity is low. Negri bodies appear as single or multiple 1-20 µm glassy eosinophilic round, oval, or spindle-shaped cytoplasmic masses and are most commonly found in the pyramidal cells in Ammon's horn of the hippocampus and the Purkinje cells of the cerebellum. Pyknotic or chromatolytic neurons can be seen throughout the CNS most frequently in the brainstem and periaqueductal gray matter. These neurons generally do not harbor Negri bodies but are positive for viral antigens by immunostaining and may be accompanied by an inflammatory reaction. As in other encephalitides, perivascular cuffing and parenchymal collections of predominantly mononuclear cells, neuronophagia and glial nodules can be identified.

Additional testing on transplant cases
(performed at the CDC)

  1. Direct fluorescent antibody testing found rabies virus antigen in Vero E6 cell cultures inoculated with CSF and 10% homogenates of brain, spinal cord and kidney and CNS tissues from all four recipients.
  2. Suckling mice inoculations resulted in neurologic abnormalities and death, 7-8 days post inoculation.
  3. Electron microscopy of the mouse and human CNS tissue found viral particles with the characteristic bullet shape of rabies virus nucleocapsids.
  4. Immunohistochemical stains for rabies antigen found intracytoplasmic rabies viral antigen in neurons from the CNS, nerves from the transplanted kidneys, liver, and arterial grafts and in renal tubular epithelium, smooth muscle, histiocytes, and vascular endothelium.
  5. Serologic testing found IgM and IgG antibodies reactive to rabies in the donor's serum and three out of four of the recipients collected antemortem (only IgG antibodies were present in the patient that received the arterial segment). Neither IgM nor IgG antibodies were present in the serum of one of the kidney recipients.
  6. Viral identification using antigenic typing found a previously characterized rabies virus variant associated with insectivorous bats.

Discussion
Rabies infection results in a progressive acute encephalitis caused by RNA viruses in the family Rhabdoviridae, genus lyssavirus. Rabies is a zoonosis prevalent in wildlife and domestic animals. Human rabies accounts for greater than 35,000 deaths annually worldwide; 54 deaths (including these recent cases) have occurred from rabies in the United States since 1980. Transmission occurs primarily through the bite of an infected animal; most commonly from a bat in this country; however of the reported rabies related deaths in the US only in approximately Ľ of cases was there a definitive history of a bat bite. Although human rabies in the United States is rare, it is probably underdiagnosed owing to a lack of clinical suspicion, delays in diagnosis and decreasing autopsy numbers. Human to human transmission is rare but has been described. There have been 8 documented cases of human to human transmission of rabies through corneal transplantation and although the potential for transmission through solid organ transplantation has always existed there have been no previously reported cases in which this has occurred.

Rabies is distinguished by two forms; a classic form that includes the encephalitic (furious) or paralytic (dumb) forms and a non-classic form. The classic form is almost always associated with true rabies (genotype 1) and is associated most commonly with dog bites. The non-classic form is associated with bats. Clinical features are divided into stages including the incubation period (lasting from 7 days up to 6 years), prodrome (lasting a few days to 1 week), acute neurological phase, coma and death (which occur usually within 2 weeks of the onset of acute neurological symptoms). Once the rabies virus is transmitted, the virus incubates at the inoculation site, replicates in muscle (classic form) or dermis and epidermis (non-classic form), attaches to nerve endings and moves centripetally from the periphery to the dorsal root ganglia and then on to the central nervous system by fast axonal transport. In the CNS, the virus has a predilection for the brainstem, thalamus, basal ganglia and spinal cord where it selectively replicates intraneuronally producing an acute encephalomyelitis. After replication in the CNS, the virus then can spread centrifugally along neural pathways to multiple organ and tissue sites. The prodromal phase likely represents movement of the virus from the inoculation site to the CNS and symptoms are often non-specific and go undetected. Patients infected from the bat variant rabies more commonly present with local symptoms or neuropathic pain at the inoculation site described as burning, numbness, tingling, or itching. It is during the acute neurological phase (CNS infection) that most patients present to the hospital. Symptoms include fever and intermittent periods of hyperactivity, disorientation, confusion, and agitation that may be precipitated spontaneously or by sensory stimuli followed by progressive neurological deterioration, coma and death.

Once acute neurological symptoms occur, rabies is usually fatal. Although the case-fatality ratio is high, the disease is preventable with appropriate administration of post exposure prophylaxis (rabies immune globulin and rabies vaccine) and therefore a correct diagnosis of rabies infection should be sought. Rabies post exposure prophylaxis is recommended for all persons with a history of a bite from a rabid animal and in the case of bat rabies persons bitten, scratched or exposed to a bat unless testing of the bat is proven not to be rabid. Theoretically, human to human transmission of rabies from a non-bite exposure is possible, but there have been no documented cases occurring among health care providers. For medical personnel caring for patients with suspected or known rabies infection, barrier procedures and personal-protection equipment minimize the risk of true exposure.

References

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  2. Center for Disease Control and Prevention (CDC). Investigation of rabies infections in organ donor and transplant recipients—Alabama, Arkansas, Oklahoma, and Texas, 2004. MMWR Morb Mortal Wkly Rep. 2004;53(26):586-589.
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