A 3-year-old boy was admitted with recurrent febrile illnesses and neurological symptoms. The first illness, at 21 months of age, manifested as a high-grade fever with non-paroxysmal cough, respiratory distress, and a generalized macular rash, present over one week. The perinatal period and development were normal. Computed tomography scan of the chest showed nodules in the bilateral lower lobe with necrotic conglomerating mediastinal lymph nodes suggestive of infective etiology. A clinical diagnosis of probable measles with pneumonia was considered. At 24 months of age, he again presented to our center with a febrile illness. Examination showed the absence of tonsils and peripheral lymph nodes, and presence of a BCG scar. Family history revealed that two of his maternal uncles had died in early childhood due to an undiagnosed infection. A clinical diagnosis of X-linked agammaglobulinemia (XLA) was considered and confirmed by specific investigations as per the diagnostic criteria proposed by European Society for Immuno-deficiencies [1]. Monthly intravenous immunoglobulin (IVIg) (400 mg/kg) was initiated, and he showed symptomatic improvement. At 26 months of age, he developed left-sided focal motor status epilepticus and left-sided hemiparesis without associated fever, altered sensorium, cranial nerve involvement, or features of raised intracranial pressure. Investigations are shown in Table I. Magnetic resonance imaging (MRI) of the brain showed focal signal changes (details in the section on investigations). A clinico-radiological diagnosis of XLA with probable focal enteroviral encephalitis was considered. Intravenous acyclovir (30 mg/kg/d for 15 days) and high-dose IVIg (1g/kg) were given. He recovered with residual left hemiparesis. He was readmitted a month later with persistent vomiting, intermittent lethargy, redness of eyes with watery discharge, and brief intercurrent seizures over the past one week. He had been on regular monthly IVIg replacements, and trough IgG level was 600 mg/dl. He continued to have altered sensorium and residual left hemiparesis with radiological progression of the focal encephalitic changes (details in the section on investi-gations). He had two further admissions, one and six months later, with persistent left-sided, focal seizures and residual left hemiparesis. By 34 months of age, he developed subacute neurological deterioration (progressive lethargy and reduced interaction), visual deterioration, and recurrent right-sided focal tonic-clonic seizures followed by right hemiparesis. At this time, examination showed weight 10 kg (-2 to -3 z), length 90 cm (-1 to -2 z) and head circumference 48 cm (-1 to -2 z). His Glasgow Coma Scale was 11 (E3M5V3-4), and pupils were 2mm bilaterally equal and reacting to light. He had chronic left-sided motor weakness with lower limb spasticity, acute-onset right-sided weakness with diminished tone, brisk deep tendon reflexes and bilateral extensor plantar response. Rest of the systemic examination was unremarkable.

Investigations (Table I): XLA being a prototype primary humoral immunodeficiency disorder, T cells and their subsets were not tested in the blood or on the brain sections at autopsy. Stool sample was negative for both polio and non-polio viruses. Baseline MRI of the brain at the onset of seizures showed presence of a right-sided, frontal, subcortical white matter lesion, which showed gliosis later. New lesions appeared in bilateral occipital and parietal subcortical white matter, and the thalamus, suggesting a progressive, subcortical, multifocal involvement (Fig. 1A-H). MR spectroscopy revealed reduced NAA with a small lactate peak.

Fig. 1 Sequential axial flair MR images of the patient: Baseline MRI a) shows the presence of frontal subcortical white matter lesion (white arrow). MRI after 24 days b) shows the progression of frontal subcortical white matter lesion (black arrow). Interval MRI after one month, c,d) shows gliosis in the right frontal subcortical lesion (white arrowheads) and appearance of bilateral occipital subcortical lesions (black arrowheads). Interval MRI after 1.5 months (e,f) shows gliosis in the frontal (white star) and occipital (black stars) subcortical lesions. Interval MRI after five months (g,h) shows the appearance of new parietal subcortical (curved black arrow) and thalamus (curved white arrow) lesions. No evidence of any diffusion-weighted abnormality, susceptibility-weighted abnorma-lity, contrast enhancement, or angiographic abnormality was noted in all the MRI examinations.

Course and Management: Multiple anti-epileptic drugs (phenytoin, valproate, levetiracetam, phenobarbitone, midazolam infusion) were sequentially given for right-sided epilepsia partialis continua. He was intubated and ventilated for worsening sensorium. Intravenous acyclovir and high dose IVIg (2g/kg total dose) were restarted. Brain biopsy was deferred due to the poor general condition of the patient. Newer therapies such as pleconaril and pocapavir were considered but could not be tried due to resource-constraints. The patient further developed high-grade fever secondary to nosocomial infection and/or aspiration pneumonia. Intravenous antimicrobials were sequentially upgraded (ceftriaxone, meropenem, vancomycin). He suffered a cardiac arrest on day 19 of hospital stay and could not be revived.

Enteroviral infections are known to cause difficult, persistent CNS disease in children with XLA. As opposed to other viruses, which are dealt with by cell-mediated immunity, the host response to enteroviral infections in XLA is by forming neutralizing antibodies. In one of the largest series of 36 patients of XLA seen over two decades, CNS infections constituted a significant proportion (25%) of all infections [7] with enteroviral infections including echo, polio and coxsackie being the most problematic [8]. As seen in our case, enteroviral encephalitis in XLA are described as insidious onset, slowly progressive loss of motor and cognitive mile-stones over 2-3 years, followed by spastic quadriplegia, coma with mortality in nearly 44% of cases [9, 10]. CSF samples remained negative for all enteroviruses, tested both by PCR and by viral cultures in the index case, which may be falsely negative and does not exclude the infection. Though adequate trough levels by IVIg replacement therapy may prevent bacterial infections it protect against enteroviral infections [3].

Besides enteroviruses, astrovirus, measles and herpes viruses need to be considered in XLA [11, 12]. Though, the index case had a measles-like past illness, and had been immunized with a live vaccine, measles inclusion body encephalitis was unlikely, because it is a disease of patients with depressed cell-mediated immunity with a rapid and fatal course. The clinical and radiological presentation in the index case did not favor subacute sclerosing pan-encephalitis. Moreover, the measles serology was negative and measles virus inclusion bodies were absent on the histopathology. Other than these, clinical presentations described in the anecdotal reports of other viruses such as adenovirus, influenza virus, cytomegalovirus, and John Cunningham (JC) virus [13] did not fit into our clinical scenario. Progressive multifocal encephalopathy, reported with XLA was also unlikely [14,15]. Dysregulated immunity leading to autoimmune encephalitis, abnormal immune response to drugs [2] or IVIg therapy induced progressive, chronic neuro-regression [3] were other differential diagnosis, but unlikely in this case.

Pediatric immunologist 1: In the present case, the trough level of immunoglobulins being well above 600 mg/dL, he was protected against the common bacterial pathogens, thus making bacterial infection of CNS unlikely. The most common CNS pathology in such patients is an enteroviral infection, which can occur even when the patient is on regular IVIg therapy. Therefore, this is consistent with a case of chronic enteroviral infection with XLA.

Neurologist 1: The diagnosis of chronic enteroviral infection with XLA seems most likely in this case. The early onset of gliosis in the MRI brain of this patient suggests a vascular invasion. As pointed rightly by the clinical discussant, measles inclusion body encephalitis is a fulminant infection that does not follow such a chronic indolent course. Additionally, there is no contrast enhancement in any of five sets of MRIs probably due to the lack of immunity to mount an inflammation. JC virus could be another possibility.

Virologist: Enterovirus is the most common etiology for this clinical presentation. Astrovirus, as the discussant highlighted, is also being reported. Sensitivity of detection of enterovirus by CSF PCR ranges from 75-100%. It would be ideal to take a throat swab along with CSF samples. Studies have shown that sensitivity has improved when throat swab is being examined along with CSF PCR.

Pediatric pulmonologist: Although the case strongly points towards a viral infection, yet the presence of necrotic lymph nodes in computed tomogrpahy chest and persistent mastoiditis might be suggestive of other infectious agents such as fungus or an invasive hospital-acquired infection. As the autopsy was done for brain only, infection elsewhere in the body could not be identified.

Pediatric hemato-oncologist 1: One could consider granulomatous amoebic infections such as Balamuthia, which have been previously reported from the center, although the MRI picture does not conform to it.

Pediatric immunologist 2: The patient received 4-5 doses of oral polio vaccine in routine immunization schedule, along with a dose on the national immunization day, 15 days prior to the onset of illness. Live viral vaccines are contraindicated in such patients as well as in siblings and their surroundings.

Physician: Measles seems more likely in the index case than enteroviruses as the posterior parts of the brain are more involved. The absence of enhancement and cells in the CSF and the presence of high protein and gliosis in the brain are described in measles. However, the clinical course in measles is shorter for 1-3 months, as compared to the chronic course in the index lasting nearly 12 months.

Clinical discussant: Measles inclusion body ence-phalitis is primarily a disease of cell-mediated immunity, seen more commonly in adult patients, and follow a rapid fulminant course. Subacute-chronic measles virus infection could not be completely excluded. Serological testing for measles virus was not feasible as B-cells are deficient in XLA. JC virus infection would be unusual as the virus is carried to the brain by B-cells, which are deficient in XLA patients. Polioviruses are also a type of enteroviral infections. XLA patients are prone for atypical manifestation of enteroviruses, which includes fulminant polio encephalitis as well as paralytic poliomyelitis. The abnormal chest findings on computed tomography described the lung pathology at the time of first illness when the child was admitted with a viral prodrome, bacterial pneumonia and neutropenia suggestive of an acute bacterial necrotizing pneumonia. Additionally, investigations for tuberculosis and fungal infections had been non-corroborative at that time. It would be very unusual for a mycobacterial or fungal infection to present with such CNS manifestations over several months. However, systemic infection with pneumocystis carinii is described in patients with XLA.

Pediatric hemato-oncologist 2: As progressive multifocal leukoencephalopathy secondary to JC virus infection is common in hematological conditions treated with rituximab, a similar mechanism may be proposed for XLA patients also. The patient also had persistent microcytic, hypochromic anemia with thrombocytopenia, which could be due to an enteroviral inflammatory bowel disease or an intestinal giardiasis, which is common in XLA patients.

Pediatric immunologist 1: As part of an international collaborative study, 32 children of the institute, with XLA were screened for poliovirus. None of them had poliovirus infections. In Iran, 4% of children with XLA had poliovirus infection [16]. The negative stool samples for poliovirus make this infection unlikely in the case.

Pediatric neurologist 1: Chronic herpes encephalitis type 1 and human herpes virus-6 infection may be additional possibilities.

A partial autopsy was performed in this case. The external examination of the brain weighing 1142 grams, showed slightly congested meninges, without any exudate. A mild tonsillar herniation was noted. Blood vessels of circle of Willis and brainstem appeared normal. Bilateral parieto-occipital and temporal lobes were discolored, collapsed and soft (Fig.2). The coronal sectioning of the brain revealed softening, shrinkage and thinning of the cortical ribbons of left inferior frontal, left frontal, paramedian area above cingulate gyrus and right middle and inferior frontal gyrus (Fig. 2). Both temporal lobes and bilateral parietal cortices had similar changes with a shrunken left temporal lobe. The right occipital lobe showed cystic encephalomalacia. While the left putamen and adjacent internal capsule showed necrosis explaining his right hemiparesis, the right lentiform nucleus was normal (Fig. 2). The affected areas of the brain corresponded to the anterior, middle, and posterior cerebral artery territories indicating global hypoxia. The hippocampi, thalami and midbrain appeared normal grossly. The white matter was mostly spared. The brainstem axial cuts revealed mild congestion of the dorsal parts of the pons, with unaffected medulla and cerebellum.

Fig. 2 a) The parietal convexity is discolored and collapsed due to softening of underlying cortex of the brain; b) Coronal section of the brain shows shrinkage and softening of the cortical ribbons affecting left inferior frontal gyrus and both temporal lobes. The white matter is relative spared, c) The left putamen and internal capsule are soft and necrotic in this coronal slice. The right basal ganglia and both thalami are spared. Note that left temporal is affected more than right temporal lobe; d) Sparse lymphocytic inflammation is seen in the cortical leptomeninges; e) The affected cortical areas showed neuronal loss and necrosis and replacement by macrophages and lymphocytes; f) Reactive hypertrophied astro-cytes. The occipital cortex demonstrated foci of micro-calcification and microglial proliferation, g-h) The dentate nucleus of the cerebellum shows neuronal loss, microglial proliferation, and vascular congestion.

Microscopic examination revealed sparse meningeal infiltrates, predominantly lymphocytic (Fig. 2). The grossly affected cortical areas showed laminar and transcortical necrosis, replaced by large number of foamy macrophages admixed with few lymphocytes, accompanied by reactive glial proliferation (Fig. 2). The adjacent cortical areas showed evidence of hypoxic changes, more marked at the base of the sulci than the crests. Hippocampi showed diffuse hypoxic damage and patchy neuronal loss. The posterior parts of the occipital cortex showed extensive neuronal loss, cyst formation and calcium deposits indicating chronicity (Fig. 2). Therefore, the cerebral cortex, in nutshell, showed presence of hypoxic damage and varying degree of cortical necrosis, explained by recurrent seizures. Histological examination of the dorsal pons demonstrated neuronal loss, neuronophagia and microglial proliferation with nodule formation, highlighted by CD68 immuno-staining. The perivascular lymphocytic infiltration consisted of CD3-positive T-cells without any CD20-positive B-cells. Similar changes were noted in the midbrain, dentate nucleus of the cerebellum and grey matter around 4th ventricle with cerebellar folia being unremarkable. The dorsal motor root of vagal nucleus and the anterior horn cells of the cervical segment of the cord were affected. Immunohistochemistry for herpes simplex virus 1 and 2, cytomegalovirus, simian virus 40 (SV40), Epstein Barr and parvovirus were negative. In addition, PCR for enteroviruses was negative in the brain tissue. The post-mortem biopsy samples of brain, lung and liver tissues did not contribute any significant information.

The topography of the lesions in this brain namely the involvement of dorsal pons, dentate nucleus, medulla, part of the hypothalamus and sparing of thalamus and cortical zone favours enteroviral infection, even though PCR was negative. PCR positivity depends on multiple factors and at best gives 50% positive results. The lesions and the histological features favour a chronic enteroviral infection over JC virus. SV40 antibody, which recognizes both JC virus and BK polyoma virus failed to show any positivity. The final autopsy diagnosis was XLA with probable enteroviral encephalitis and cystic cerebral encephalomalacia.