Key-words:
Acquired immunodeficiency syndrome - brain biopsy - demyelination - DNA test - human
immunodeficiency virus - JC virus - progressive multifocal leukoencephalopathy
Introduction
Progressive multifocal leukoencephalopathy (PML) is an infectious demyelinating disease
of the brain, caused by the human JC polyomavirus (JCV).[[1]] The JCV is a small ubiquitous DNA polyomavirus with a 5.13 Kb circular enclosed
double-stranded DNA. It is a neurotropic virus that infects only humans.[[1]] JCV was first isolated in 1971 from the brain of a patient with Hodgkin's disease
who died of PML and was named based on the patient's initials.[[1]],[[2]] In the first two decades after its initial description, PML remained a rather rare
disease, occurring mainly in patients with hematopathies, solid organ malignancies,
and inflammatory disorders, as well as in organ transplant recipients. This changed
drastically in the 1980s, in the advent of the human immunodeficiency virus (HIV)
epidemic. PML was soon recognized as a major opportunistic infection of acquired immunodeficiency
syndrome (AIDS), occurring in up to 5% of patients, and did not decrease significantly
despite advances in antiretroviral treatment.[[1]] Interests in PML increased further by withdrawal of natalizumab (Tysabri ®) from
the marketplace. The drug, which was promising in the treatment of multiple sclerosis
(MS) and Crohn's disease, was withdrawn after the report of two cases of PML in MS
patients and a patient with Crohn's disease.[[1]] Several classes of immunomodulatory medications for autoimmune diseases have been
reportedly associated with PML, including natalizumab for MS and Crohn's disease,[[3]] rituximab for lupus,[[4]] and efalizumab for psoriasis.[[5]]
The diagnosis of PML is suggested by clinical and neuroimaging features and is further
established by demonstrating the presence of JCV DNA in the cerebrospinal fluid (CSF)
using polymerase chain reaction (PCR).[[6]],[[7]] Although CSF PCR for JCV is highly specific (92%–99%) and sensitive (74%–93%),
false negatives do occur.[[8]],[[9]],[[10]],[[11]] If ascertaining the diagnosis of PML remains elusive, a brain biopsy should be
undertaken.[[10]] This remains the gold standard for the diagnosis of PML, specifically when clinical
and neuroradiological features are suggestive of PML despite negative JCV CSF PCR
analysis.[[10]],[[11]] We describe a case of PML with false-negative CSF JCV PCR, which reinforces the
importance of obtaining histologic confirmation when clinicoradiological findings
are suggestive of PML, even with initially normal CSF studies.
Case Report
A 44-year-old male patient presented with dysphasia, confusion, and memory impairment
that developed following a generalized seizure. He had been living in Jakarta, Indonesia,
for the last 6 years. He was diagnosed as AIDS during a routine health examination
in 2011. However, he willingly did not seek any medical treatment. Two months prior
to presentation, an unremitting diarrhea developed and he returned to his hometown
for medical evaluation. A diagnosis of cytomegalovirus (CMV) colitis was made through
colonoscopic biopsy. The titer of reverse transcriptase-PCR (RT-PCR) against HIV at
the time of admission was 126,629 copies/ml and absolute count of CD4 was 10/UL. Ganciclovir
(antiviral agent for CMV) and highly active antiretroviral therapy (HAART, comprising
lopinavir, ritonavir, tenofovir, and emtricitabine) were started. After recovery from
CMV colitis, his condition was stable. Despite the improvement of his diarrhea, a
generalized seizure followed by dysphasia and memory impairment developed 2 months
later. He denied any homosexual behavior and his medical history was unremarkable,
including transfusion or drug addiction.
Magnetic resonance imaging (MRI) of the brain revealed multiple confluent subcortical
lesions with high signal intensity on T2-weighted image, involving bilateral frontal,
right temporal and parietal lobes, and left thalamic and subcapsular regions [[Figure 1]]a and [[Figure 1]]b. Irregular marginal enhancement on gadolinium was evident [[Figure 1]]c. Laboratory findings were as follows: hemoglobin of 15.1 g/dL, white blood cell
(WBC) count of 4.62 × 109/L, platelet count of 293 × 109/L, erythrocyte sedimentation
ratio of 27 mm/h, and C-reactive protein level of 0.17 mg/dL. CSF examination showed
WBC count of 2 cells/UL, red blood cell count of 1 cell/UL, protein level of 33.5
mg/dL, glucose of 51 mg/dL, and chloride of 124 mEq/L. HIV titer was 80,148 copies/mL
and CD4 count was 35/UL. The results of the CSF PCR examination for toxoplasmosis,
tuberculosis, and polyoma viruses including BK and JCV were negative.
Figure 1: Magnetic resonance imaging and intraoperative findings in progressive multifocal
leukoencephalopathy. (a) A T2-weighted axial image showing multiple convoluted irregular
cortical and subcortical lesions in bilateral frontal and parietal lobes. (b) A T2-weighted
axial image showing small irregular high signal in the left thalamus. (c) An enhanced
T1-weighted axial image showing irregular marginal subcortical enhancement in multiple
frontal lesions. (d) An intraoperative photograph taken after removal of a portion
of middle frontal gyrus for brain biopsy. The subcortical white matter and surrounding
gyri seem normal in gross inspection
To confirm the pathology of the brain lesion, a brain biopsy was requested. Under
three-dimensional MRI guidance, small craniotomy of the left frontal was performed.
Portions of the middle frontal gyrus and subcortical white matter showing enhancement
on MRI were taken for pathologic examination [[Figure 1]]d. The histologic examination revealed multifocal demyelinated areas with reactive
astrocytosis and perivascular lymphocytic cuffing. Several large infected oligodendrocytes
with inclusion-bearing dark nuclei or clear nucleoplasm having marginated chromatin
were present in the periphery of demyelinated area [[Figure 2]]a. The demyelinated areas revealed small round lesions with myelin breakdown in
the luxol-fast blue staining for myelin [[Figure 2]]b. Numerous macrophages had infiltrated and phagocytosis containing fragmented myelin
was apparent. The hyperchromatic, inclusion-bearing oligodendrocytes displayed high
ki-67 proliferative index [[Figure 2]]c and intense p53 nuclear immunostaining. Electron microscopy revealed infected
oligodendrocytes filled by viral particles evident as spheres measuring up to 40 nm
in diameter [[Figure 2]]d. JCV DNA was detected in brain tissue by PCR. Therefore, the final diagnosis of
PML was confirmed and HAART was continued. His neurologic status was stable despite
continued memory impairment. A generalized seizure developed 3 weeks after brain biopsy
and his mentality became stuporous. Epileptiform discharges over the left frontocentral
lobes were found on electroencephalography, and anticonvulsants (levetiracetam 2000
mg/day and valproate 900 mg/day) were added. Despite anticonvulsants and HAART, his
mentality did not improve. Recurrent seizures developed and additional anticonvulsants
were prescribed (levetiracetam 2000 mg/day, valproate 1200 mg/day, topiramate 600
mg/day, and clobazam 20 mg/day). The patient died 3 months from the onset of neurological
symptoms.
Figure 2: Histopathologic examination of progressive multifocal leukoenecephalopathy. (a) Several
large infected oligodendrocytes (arrows) with inclusion-bearing dark nuclei or clear
nucleoplasm having marginated chromatin (H and E, x400). b) Small round demyelinated
lesions with myelin breakdown (luxol-fast blue, xl00). (c) High Ki-67 proliferative
oligodendrocytes (immunohistochemistry, x200). (d) Viral particles measuring up to
40 nm spheres (EM, x80,000)
Discussion
Progressive multifocal leukoencephalopathy
PML is a demyelinating disease of the central nervous system (CNS) caused by a lytic
infection of oligodendrocytes due to the presence of the JCV.[[12]] JCV is a ubiquitous polyomavirus that infects 50% or more of the adult population
globally.[[6]] PML remains an extraordinarily rare complication of this infection in otherwise
normal persons and occurs almost exclusively in immunocompromised conditions, such
as HIV infection, lymphoid malignancies, and after organ and stem cell transplantations.[[11]],[[12]] While PML achieved prominence during the first two decades of the HIV endemic,
effective antiretroviral treatment and restitution of T-cell function have led to
PML being less prominent in this population.[[13]] HIV infection as a predisposing factor has now been supplanted by T-cell immunodeficiency
induced by a range of immune-mediated therapies, including monoclonal antibodies,
such as natalizumab, rituximab, infliximab, and efalizumab, as a major cause of PML.[[13]]
The onset of PML is insidious, with subtle changes in cognition and loss of memory
in association with a range of progressive neurological deficits.[[6]],[[13]] These are varied and include cognitive deficits, sensory deficits, hemianopsia,
aphasia, and difficulties in coordination and gait, consistent with a multifocal cerebral
pathology. A high index of suspicion is required for early diagnosis, as these clinical
features are not sufficiently distinctive to enable a definitive clinical diagnosis.[[13]] Brain imaging reveals characteristic multiple lesions in the subcortical hemispheric
white matter or the cerebellar peduncles. Contrary to the implication in the title
to this article that only cerebral white matter is involved, PML lesions also occur
in gray matter areas such as the basal ganglia or thalamus.[[13]]
No single criterion establishes the diagnosis of PML. Rather, it requires clinical,
imaging, and virologic evidences.[[6]] Many authorities considered the demonstration of JCV DNA coupled with appropriate
clinical and imaging features to be diagnostic of PML, obviating the need for a brain
biopsy.[[6]],[[14]] However, either the clinical or imaging features are unconvincing or CSF PCR is
negative or has not been performed.[[6]] In the context of diagnostic uncertainty, the diagnostic criteria of PML were suggested
by the American Association of Neurology in 2013. Definitive diagnosis of PML requires
neuropathologic demonstration of the typical pathologic triad (demyelination, bizarre
astrocytes, and enlarged oligodendroglial nuclei) coupled with the techniques to show
the presence of JCV.[[6]] The presence of clinical and imaging manifestations consistent with the demonstration
of JCV by PCR in CSF is also considered diagnostic.[[6]]
The diagnosis of PML is based on the PCR demonstration of JCV in CNS tissue and/or
CSF.[[1]] Although sampling of the CSF is usually recommended, false-negative PCR-based analysis
of JCV in CSF, as shown in our case, has been reported several times [[Table 1]].[[8]],[[9]],[[10]],[[11]] In a series of laboratory-confirmed PML patients,[[15]] the sensitivity of DNA PCR examination was approximately 80% and these false-negative
findings may have been due to low titers of JCV DNA in CSF or technical instability
at commercial laboratories.[[15]],[[16]] Therefore, two approaches to establishing the diagnosis of PML were suggested:
securing the diagnosis with tissue or, as is more commonly practiced, establishing
the diagnosis with clinical or radiographic criteria coupled with demonstrating the
presence of the virus in the CSF compartment.[[6]]
Table 1: Summary of reports regarding progressive multifocal leukoencephalopathy diagnosed
by brain biopsy followed by false-negative polymerase chain reaction assay in cerebrospinal
fluid
Conclusions
Although PML has been regarded as an incurable disease and to be associated with doomed
prognosis, potentially useful medications have been proposed for PML recently.[[11]] Therefore, the early diagnosis of this disease is essential. In case of negative
PCR in CSF study, brain biopsy would be a useful means of establishing the diagnosis
of PML.
