Tetrahydrobiopterin deficiency in human rabies

Summary

Rabies is a fatal viral encephalitis characterized by a clinically acute and progressive course. With rare exceptions, there is a discrepancy between clinical outcome and frank histological alterations in rabies. Investigators have postulated that rabies virus may modify neurotransmission through occupancy of cellular receptors or alteration of ion channels. We took advantage of these observations to improvise a successful therapy for rabies. The Milwaukee protocol (www.mcw.edu/rabies) was further modified to treat two German patients. We measured pterins and monoamine neurotransmitter metabolites in the CSF of patients with rabies by HPLC with electrochemical or fluorescent detection. We report loss of tetrahydrobiopterin (BH₄) and associated pathological decrease of dopaminergic and serotoninergic neurotransmission in three successive patients with rabies. CSF levels of BH₄ and neurotransmitter metabolites increased in two patients who were supplemented. Our findings support the long-standing speculation of modified neurotransmission in the pathogenesis of rabies, but by another mechanism. Brain turnover of dopamine and serotonin is reduced following rabies-acquired BH₄ deficiency. Neuronal nitric oxide synthase is BH₄-dependent and may also be involved, possibly causing cerebrovascular insufficiency in one patient. This work must be carefully replicated in animal models and future patients. We are cautiously optimistic at the prospect of readily available, metabolically specific, enteral therapy for rabies.

Introduction

Rabies is a fatal viral encephalitis characterized by a clinically acute and progressive course over 5–7 days. Signs can include fever, altered alertness, agitation, insomnia, hallucinations, focal paraesthesias or myoclonus, phobic spasms, disturbances of blood pressure, heart rhythm and water balance, seizures, flaccid paralysis, coma and death (Jackson 2007). Electroencephalograms (EEG) are characterized by generalized slowing that progresses to loss of cortical electrical activity. In patients with early demise without major complications of medical care, brain histology generally shows rabies virus antigens by immunofluorescence or detection of Negri bodies, but little necrosis, apoptosis or inflammation (Iwasaki and Tobita 2002). Early brain imaging by computed tomography or magnetic resonance imaging (MRI) may appear normal, consistent with autopsy findings (Laothamatas et al 2003).

We took advantage of the discrepancy between clinical outcome and histological blandness to improvise a successful therapy for rabies. We induced coma to suppress neurological dysfunction, applied anti-viral drugs in an attempt to minimize agent replication, and relied on the normal development of an immune response to clear rabies virus infection (Willoughby et al 2005). The Milwaukee protocol (MP) was then modified to treat two German patients.

In these three patients, we identified in addition a severe acquired defect of tetrahydrobiopterin (BH₄) metabolism that was restricted to the central nervous system. The deficit in BH₄ metabolism may partially explain features of rabies. It is in principle treatable by oral supplementation, as evidenced by neurochemical responses in two patients.

Methods

Case report methodology.

We measured pterins and monoamine neurotransmitter metabolites in the CSF of patients with rabies by HPLC with electrochemical or fluorescent detection (Blau et al 2001).

Results

Case 1

Our index patient’s exposure to a bat, clinical presentation, course, and rehabilitation have been reported previously (Hu et al 2007; Willoughby et al 2005). Sedation and analgesia were tapered on hospital day (HD) 9. The 15-year-old girl was initially fully paretic and unresponsive to noises, oculocephalic manoeuvres, corneal stimulation and pain. She rapidly recovered sensation and movement but displayed a sequence of rapidly evolving movement disorders over 52 days of in-patient rehabilitation. She did not initially receive medication for her movement disorders because of her general improvement and the rapid evolution of the disorders, which precluded assessment of benefit or adverse effect from new therapies. By HD40, her progress in gross motor skills, ambulation and conditioning was excellent but her facial and buccolingual movement disorders compromised oral communication and feeding. A constellation of increased signal in the basal ganglia and midbrain by fluid-attenuated inversion-recovery (FLAIR) MRI, cardiomyopathy, pancreatitis, and mild lactic acidosis was present early in her disease, but then persisted despite otherwise rapid rehabilitation.

We investigated a potential mitochondrial disorder. Formal pedigree analysis did not identify metabolic, psychiatric or neurological disorders in her family. A metabolic evaluation was performed on HD44, including lumbar puncture. Magnetic resonance proton spectroscopy showed uniformly decreased N-acetyl aspartate (NAA), independent of localized FLAIR intensities and without brain volume loss by MRI. NAA is routinely interpreted as a marker for neurons but is localized to mitochondria (Heales et al 1999). Coenzyme Q₁₀ (4.3 mg/kg per day), levocarnitine (5 mg/kg per day), riboflavin (1.5 mg/kg per day), thiamine (1.5 mg/kg per day), ascorbate (7.7 mg/kg per day) and tocopherol (3 U/kg per day) were administered empirically. Her relative tachycardia (rate > 100) abated by HD53 while receiving this therapy.

Cerebrospinal fluid (CSF) demonstrated high concentrations of neopterin, undetectable BH₄, decreased homovanillic acid (HVA, the major metabolite of dopamine) and 5-hydroxyindoleacetic acid (5-HIAA, the major metabolite of serotonin; Table 1). Serum amino acid profile included normal phenylalanine. Empirical therapy with BH₄ (Schircks Laboratories, Basel, Switzerland) was begun on HD53 at 2 mg/kg body weight per day, advancing to 20 mg/kg per day of BH₄ by HD59. Her speech intelligibility improved from 25% on HD58 (BH₄ 10 mg/kg per day) to 75% on HD60 (BH₄ 20 mg/kg per day), associated with an acceleration of most remaining rehabilitation skills. The patient was discharged home on HD76, three months ahead of projections by the rehabilitation team before BH₄ was supplemented.

Table 1 Neurotransmitters and pterins in CSF of three human patients with rabies virus infection

Given the temporal association with her marked clinical response, BH₄ supplementation and the mitochondrial cocktail were continued over the first 6 months of her rehabilitation. Repeat lumbar puncture was performed on the equivalent of HD220, showing normal BH₄ levels, without BH₄ supplementation for the preceding 33 days. A much later neurological and neurochemical assessment has been reported (Hu et al 2007).

Case 2

A 46-year-old man received cadaveric kidney and pancreas transplantations on 31 December 2004 from a 26-year-old donor who died of cardiac arrest and hypoxic encephalitis (Mattner et al 2007). On 10 February 2005, he developed obstipation and hydrophobia and was hospitalized again (HD1). The patient worsened, with panic attacks, hypersalivation, swallowing difficulties, facial seizures and respiratory distress. The transplant-associated rabies outbreak was diagnosed on 14 February (HD4). In retrospect, the donor had been exposed to a dog in India in October 2004. Examination of the donor’s brain confirmed rabies.

Immune suppression, consisting of anti-thymocyte-globulin (ATG) and mycophenolate mofetil was stopped and ciclosporin was reduced stepwise over four days. VeroRab rabies vaccine was given on HD 4, 7, 14, 22 and 26; BeriRab human rabies immunoglobulin (RIG) was started on HD4 and administered daily at 27 mg/kg. He was intubated on HD6, and deep sedation was induced with ketamine and midazolam. Antiviral treatment with ribavirin was started on HD6 (22 mg/kg four times daily over 3 days, then 11 mg/kg 3 times daily). Interferon alfa was given subcutaneously every three days from HD12 and IV amantadine was added on HD 12. Phenobarbital was added on HD14, yielding controlled burst suppression in episodes of 10–25 s by EEG.

The patient lost brainstem reflexes on HD 14 as anticipated. (Hantson et al 1993). MRI on HD14 and HD20 showed mild T2 signal in brainstem and grey substance of the medulla oblongata, while CSF pleocytosis increased between HD19 and HD22. The transplanted organs functioned well despite there being no immunosuppressive therapy apart from prednisolone 15 mg daily.

The patient’s course was complicated by an episode of cardiac asystole on HD12 that could be managed with low-dose catecholamines and a temporary pacemaker device. (Schankin et al 2005). He developed cyclical episodes of severe diabetes insipidus from HD17 onward, with diuresis of 8–12 L/day requiring desmopressin (DDAVP) substitution.

Rabies virus serology used to determine the duration of the MP was hampered by restrictions on international air transport and confounded by administration of RIG. The RT-PCR assays for rabies virus amplicons from two reference laboratories were regularly negative in CSF, saliva, urine and skin biopsy, while assays from a third reference laboratory were positive in saliva on HD6 and HD12 and in CSF on HD16 and HD19 but negative on HD22.

On HD35, ketamine and phenobarbital were discontinued for an isoelectric EEG. The patient remained unresponsive to external stimuli and without spontaneous motion. MRI on HD 41 showed T2 hyperintensity of the cortical grey matter, basal ganglia and subthalamic nuclei, with aggravation of all lesions by MRI two days later. Lumbar puncture on HD42 abruptly yielded little CSF, with cellular and very proteinaceous liquid. This was associated with systemic leukocytosis (29 000 cells/mm³), but bacterial meningitis was excluded. Transcranial Doppler ultrasonography (TCD) on HD44 showed increased velocity in the left middle cerebral artery (MCA) with biphasic flow in the right MCA. MR-angiography (MRA) showed no blood flow in the right MCA, the right anterior cerebral artery, and vertebral artery. HMPAO-technetium scan showed no intracranial flow. Brain biopsy, performed on HD55 showed colliquative (liquefactive) necrosis. The patient was pronounced brain dead on HD56. RT-PCR of autopsy spinal fluid was positive for rabies virus amplicons at several reference centres.

In the CSF, high concentration of neopterin and low concentration of BH₄, 5HIAA and HVA were already found after 2 weeks of illness (HD 14). During the course of the disease, these declined progressively until CSF obtained on HD39 showed undetectable BH₄ and severely decreased HVA and 5-HIAA (Table 1).

Case 3

A 55-year-old man developed fever, paraesthesia of the left hand and difficulty swallowing on 13 March 2007. He had been bitten on the same hand 6 weeks earlier in Morocco while separating his pet dog from a stray dog. He received medical care but no post-exposure prophylaxis was given. His dog was euthanized 4 weeks later for inability to stand, but had documented rabies vaccination for the past 3 years. He was hospitalized two days later (HD1) and received Rabipur rabies vaccine and RIG by the WHO-recommended ‘Essen protocol’. He was transferred to a referral hospital on HD3, where the diagnosis of rabies was confirmed by RT-PCR of saliva and corneal swab.

A modification of the MP was begun on HD4, consisting of ketamine, midazolam, amantadine and high-dose phenobarbital. The Essen protocol was continued. Ribavirin was not given because of possible immunosuppressive side-effects. Lamotrigine was given prophylactically. An intraventricular drain was placed on HD7. The patient remained without rabies antibody, and a live-attenuated rabies virus vaccine (VirBac, SAG-2 strain) was administered on HD8 and HD13 to accelerate the immune response. MRI scans, TCD and intracranial pressures remained normal. On HD21, the patient developed dysautonomia, hypersalivation, dilated and eccentric pupils and a flat EEG. Barbiturates and benzodiazepines were tapered. Auditory evoked potentials showed no brain activity and EMG reflected severe axonal neuropathy on HD24. MRI on HD29 showed generalized oedema of the cortex and basal ganglia. Multiple organ failure occurred and the patient died on HD31.

Virological assays showed transient viraemia associated with SAG-2 inoculation and transient viral amplicons in CSF associated with the trauma of ventriculostomy placement. Wild-type rabies virus RNA in the CSF and blood was otherwise not detectable until shortly before death. Rabies neutralizing antibody appeared in serum on HD14 and in CSF on HD18. Titres increased slowly until two days before the patient died.

CSF, obtained on HD14, showed high levels of neopterin and very low concentrations of BH₄, HVA and 5-HIAA. After initiation of oral BH₄ supplementation on HD14 (20 mg/kg daily), CSF analysis on HD23 revealed high BH₄ and normalized HVA and 5-HIAA (Table 1). CSF neopterin concentrations were notably higher in this patient than in patient 2, possibly owing to the accelerated vaccination strategy that was chosen.

Discussion

This constellation of BH₄ deficiency resulting in neurotransmitter deficiencies is known from rare inborn errors of metabolism and leads, when untreated, to progressive, often lethal encephalopathies characterized by mental retardation, convulsions, disturbance of tone and posture, progressive extrapyramidal movements, hypersalivation, and swallowing difficulties (Hoffmann 2006). Such metabolic patients respond to supplementation of BH₄ and additional substitution of neurotransmitter precursors such as l-dopa or 5-hydroxytryptophan.

Brain histology from patients with rabies who succumb early in the course of disease is characterized by minimal neuronal loss or inflammation (Iwasaki and Tobita 2002). Investigators have long postulated that rabies virus might modify neurotransmission. Candidate signal transduction systems included those mediated by acetylcholine, serotonin, GABA and nitric oxide (NO), as well as voltage-dependent sodium channels and inward-rectifier potassium channels (Jackson 2003). Our findings support this long-standing speculation, but by a different mechanism that is potentially treatable with enterally bioavailable BH₄.

BH₄ is the essential cofactor for phenylalanine hydroxylase (PAH, EC 1.14.16.1), tyrosine hydroxylase (TH, EC 1.14.16.2) and tryptophan hydroxylase (TPH, 1.14.16.4) and is required for synthesis of monoamine neurotransmitters such as dopamine, adrenaline (epinephrine), noradrenaline (norepinephrine), serotonin and melatonin (Blau et al 2001). Congenital deficiencies of enzymes involved in the synthesis or recycling of BH₄ lead to defective enzyme activity of PAH, TH and TPH and therefore to disturbances of biogenic amine metabolism. BH₄ deficiencies result in overlapping clinical phenotypes but show considerable variation in presentation and phenotype related to the site of the defect within the metabolic pathway and different mutations. The clinical spectrum of neurological disease is often severe and overlaps with that of rabies (Table 2) (Blau et al 2001; Jackson 2007). BH₄ deficiency in the liver leads to hyperphenylalaninaemia. Phenylalanine levels were normal in our patient, consistent with rabies virus neurotropism and restriction of BH₄ deficiency to the central nervous system.

Table 2 Symptoms and signs for rabies and BH₄ deficiencies, from websites at the University of Zürich, Switzerland and the Centers for Disease Control and Prevention, Atlanta, Georgia, USA and authoritative reviews (references provided upon request)

Infections of the CNS are known to increase tissue concentrations of BH₄ and its precursor, neopterin. BH₄, neopterin and neurotransmitter concentrations in our three patients differ clearly from those described in children with other CNS infections. Cerebral malaria is characterized by high CSF concentrations of neopterin and BH₄ and low nitrite/nitrate (NOx) (Weiss et al 1998). CSF pterins and neurotransmitter metabolites in children with herpes simplex encephalitis, Haemophilus influenza type B meningitis and tuberculous meningitis are similar to those in malaria and all are different from those we describe in rabies (Azumagawa et al 2003).

Our patients had very low concentrations of the metabolites of dopamine and serotonin in CSF, when BH₄ in CSF was clearly decreased. BH₄ concentrations in human cerebrospinal fluid correlate with concentrations of HVA, 5-HIAA and NOx (Blau et al 2001). Hence the findings in our patients support the postulated dysfunction of neurotransmission in patients with rabies, not owing to receptor or ion channel alterations but rather by defective dopamine and serotonin biosynthesis due to BH₄ deficiency. Animal models of BH₄ deficiency indicate that dopamine production becomes more compromised than serotonin production, similarly to the CSF metabolites in our patients (Hyland et al 1996; Sumi-Ichinose et al 2001). Depletion of brain dopamine and noradrenaline, but not serotonin, has also been described after intracerebral inoculation of mice with attenuated rabies virus (Baride and Gaitonde 1980).

Supplementation of patients 1 and 3 with oral BH₄ at doses of 20 mg/kg per day resulted in rapid improvement and clear increments in BH₄, HVA and 5-HIAA concentrations. Patient 1 fortunately survived rabies infection after combined and intensive treatment (Willoughby et al 2005). Patient 2 died at HD56. BH₄ supplementation was not performed in this patient. CSF analysis at HD39 showed very low BH₄, HVA and 5-HIAA (Table 1). In congenital disorders of BH₄ metabolism, a rapidly fatal progress of disease course as in our patients is not observed. On the other hand BH₄ and neurotransmitter deficiency in the patients with rabies exceeded findings in patients with congenital disorders of BH₄ metabolism. The possible impact of extremely low levels of biopterin and dopamine was seen in homozygous 6-pyruvoyl-tetrahydrobiopterin synthase PTPS (EC 4.6.1.10)-deficient mice, who died within 48 h after birth (Sumi-Ichinose et al 2001). Repeated administration of BH₄ in combination with the neurotransmitter precursors l-Dopa and 5-hydroxytryptophan rescued the perinatal lethality with 100% survival of Pts knockout mice (Elzaouk et al 2003). Surprisingly, compound heterozygous or homozygous mutations within genes for PTPS are known for human patients, which is explained by sufficient residual enzyme activities in partial PTPS deficiency to cover the tetrahydrobiopterin requirement of tyrosine hydroxylase and tryptophan hydroxylase in brain (Thony et al 2000). Homozygous tyrosine hydroxylase inactivation led to severe dopamine deficiency that resulted in mid-gestational lethality in about 90% of the mutant embryos, apparently from cardiovascular failure. Administration of l-dopa to pregnant females resulted in complete rescue of mutant mice in utero (Zhou et al 1995). Therefore, the observed, severe neurotransmitter deficiency in patient 2 could have contributed to the fatal disease progress and may partially explain features of rabies.

It is difficult to interpret the sequence of events in patient 3, who, like the survivor, received BH₄ supplementation with normalization of HVA and 5-HIAA. This patient nevertheless progressed to a catastrophic event on HD21 despite normalized neurochemistry. This patient also received a sequence of rabies vaccines designed to accelerate the immune response, which may accelerate rabies disease in animal models. This case cannot elucidate whether BH₄ supplementation was helpful.

Need for further studies

Our report includes only three patients with rabies, with numerous complex interventions and variations of the MP. Loss of BH₄ and pathological decrease of dopaminergic and serotoninergic neurotransmission are consistent findings in all three patients. The symptom complex in rabies is not fully mimicked by congenital enzyme defects associated with BH₄ deficiency (Table 2). Congenital BH₄ deficiency begins in utero and might induce compensatory metabolic pathways during development (Hyland et al 1996). Furthermore, most reported mutations are missense mutations allowing for some, although low, residual enzyme activities. As a consequence dopaminergic and serotoninergic neurotransmission appeared more severely compromised in our three patients than in patients with enzyme deficiencies. Congenital BH₄ deficiencies do not lead to isoelectric EEG, global strokes or peripheral neuropathy, so additional mechanisms must be operative. Rabies virus has specific tropism in the brain, including competitive antagonism of nicotinic acetylcholine receptors, so additional mechanisms are likely.

The mechanisms behind the acquired deficiency of BH₄ in rabies are unknown. The biochemical picture suggests a severe defect in the biosynthesis of BH₄. All enzymes in the biosynthetic (GTP cyclohydrolase I (GTPCH), EC 3.5.4.16), 6-pyruvoyl-tetrahydropterin synthase (PTPS, EC 4.6.1.10), sepiapterin reductase (SR, E 1.1.1.153) and recycling pathways (dihydropteridine reductase (DHPR), EC 1.6.99.7) must be considered, but mechanistic studies are lacking. Elevated ratios of BH₂ to BH₄ reported in cases 2 and 3 suggest excess oxidation of BH₄, either through catalytic consumption or by interaction with superoxide, peroxynitrite and other reactive oxygen species. Elevated ratios of BH₂ to BH₄ are predictive of endothelial dysfunction (Vasquez-Vivar et al 2002). Paradoxical vasoconstriction or complete deficiency of BH₄, an essential cofactor of all isoforms of nitric oxide synthase (EC 1.14.13.39), may explain the generalized hypoperfusion through most intracranial arteries of our second patient on HD44, confirmed by TCD, MRA and HMPAO brain scan. Generalized asphyxia or ischaemia are common causes of BH₄ and monoamine neurotransmitter deficiencies, but would not explain improvements in neurotransmitter metabolites in cases 1 and 3 after BH₄ supplementation or the excellent clinical outcome in case 1. Animal models and autopsy series should be considered to answer these questions.

The potential for functional recovery, in the absence of clear cytopathic damage to the brain in rabies, has been demonstrated once and has additional precedent in congenital BH₄ deficiencies. Supplementation with BH₄ and monoamine neurotransmitter precursors may result in prominent clinical improvement even after months to years of static encephalopathy or neurological deterioration (Dudesek et al 2001). We are therefore cautiously optimistic at the prospect of readily available, metabolically specific therapy for rabies in the form of oral BH₄. In addition to BH₄, oral supplementation with precursors of the monoamine neurotransmitters (l-dopa and 5-HT) as well as monoamine decarboxylase inhibitors (carbidopa, benserazide, selegiline) may be indicated for selected patients in whom the bioavailability of BH₄ to the brain can vary.

This work must be carefully replicated in future patients and extended in animal models. Unfortunately, BH₄ is not analytically stable in fluids and tissues and so is not amenable to retrospective investigations and, to date, there is no ideal animal model for rabies therapy. Given the lack of an effective alternative to rabies management, application of the Milwaukee protocol and BH₄-related diagnostics to humans will necessarily proceed in parallel with development of a survival model, and before formal clinical trials. For such ad hoc attempts, and while we develop an animal model, we offer an updated protocol and simple, web-resident case-registry at www.mcw.edu/rabies.