Journal of Medical Virology 82:1255–1265 (2010)

Immunovirological Correlates in Human RabiesTreated With Therapeutic Coma

M. Hunter,1 N. Johnson,2 S. Hedderwick,1 C. McCaughey,3 K. Lowry,4 J. McConville,5 B. Herron,6

S. McQuaid,7{ D. Marston,2 T. Goddard,2 G. Harkess,2 H. Goharriz,2 K. Voller,2 T. Solomon,8

R.E. Willoughby,9 and A.R. Fooks2*1Department of Infectious Diseases, Royal Victoria Hospital, Belfast, United Kingdom2Rabies and Wildlife Zoonoses Group, Veterinary Laboratories Agency—Weybridge, Woodham Lane, Addlestone,Surrey, United Kingdom3Department of Regional Virology, Royal Victoria Hospital, Belfast, United Kingdom4Regional Intensive Care Unit, Royal Victoria Hospital, Belfast, United Kingdom5Department of Neurology, Royal Victoria Hospital, Belfast, United Kingdom6Department of Pathology, Royal Victoria Hospital, Belfast, United Kingdom7Royal Victoria Hospital, Belfast, United Kingdom8Brain Infections Group, Divisions of Neurological Science and Medical Microbiology, University of Liverpool,Walton Centre for Neurology and Neurosurgery, Liverpool, United Kingdom9Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin

A 37-year-old woman was admitted to hospitaland over the next 5 days developed a progressiveencephalitis. Nuchal skin biopsy, analyzed usinga Rabies TaqMan� PCR, demonstrated rabiesvirus RNA. She had a history in keeping withexposure to rabies whilst in South Africa, but hadnot received pre- or post-exposure prophylaxis.She was treated with a therapeutic coma accord-ing to the ‘‘Milwaukee protocol,’’ which failed toprevent the death of the patient. Rabies viruswas isolated from CSF and saliva, and rabiesantibody was demonstrated in serum (from day11 onwards) and cerebrospinal fluid (day 13onwards). She died on day-35 of hospitalization.Autopsy specimens demonstrated the presenceof rabies antigen, viral RNA, and viable rabiesvirus in the central nervous system. J. Med.Virol. 82:1255–1265, 2010.� 2010 Wiley-Liss, Inc.

KEY WORDS: rabies; Milwaukee protocol;therapeutic coma; humancase; virus

INTRODUCTION

Rabies is endemic in most regions of the world andcauses a fatal encephalomyelitis following infection ofthe central nervous system. The causative agent, rabiesvirus, is from the genus lyssavirus, within the familyRhabdoviridae [Tordo et al., 2004]. The virus can bedivided into terrestrial lineages, transmitted by dogs,and bat-borne lineages. The lyssavirus genus containstwelve species, seven of which have caused fatal disease

in humans. Some regions of the world have activelyexcluded the disease, including the United Kingdom, ortaken action to control and eliminate rabies in dogs(North America) and terrestrial wildlife (westernEurope). The burden of rabies however, continues tofall particularly heavily in Asia and Africa where dograbies remains a notable public health problem. It isestimated by the World Health Organisation that over50,000 rabies deaths occur annually as a result of dogbites [World Health Organisation, 1996], although thisfigure is considered to be a conservative estimate [Fooks,2005; Mallawa et al., 2007]. The UK has been free of dograbies since 1902, with a brief incursion occurring afterthe end of the First World War. As a result, humandeaths from the disease have been restricted to thosewho have been infected abroad and subsequentlyentered the country (Table I). The majority of caseshave occurred in individuals who were infected in India,Pakistan or Bangladesh [Johnson et al., 2005]. Indeedthe most recent human rabies case was in a woman whowas bitten by a dog in Goa in 2005 whilst on holiday

The authors have declared that no competing interests exist.{Honorary Senior Lecturer QUB; Adjunct Professor; NUI

Galway.

Grant sponsor: Department for Environment, Food and RuralAffairs (Defra) (to Rabies diagnosis in the UK); Grant number:SV3500.

*Correspondence to: A.R. Fooks, Weybridge, New Haw, Addle-stone, Surrey KT15 3NB, United Kingdom.E-mail: t.fooks@vla.defra.gsi.gov.uk

Accepted 3 February 2010

DOI 10.1002/jmv.21785

Published online in Wiley InterScience(www.interscience.wiley.com)

� 2010 WILEY-LISS, INC.

[Solomon et al., 2005]. The dog in question wasdomestically owned, appeared normal and was leashedwhen the encounter occurred. In suspect cases incountries where rabies in not endemic, suspicion usuallybegins with an accurate case history that includesforeign travel and a dog or other mammal bite or contactwith a bat. This should be subsequently confirmedthrough laboratory diagnostic testing. An accuratediagnosis should include: a history of foreign traveland a dog, bat or bite from a mammal. In the UK afurther challenge to accurate diagnosis is caused by thepresence of European Bat Lyssavirus type-2 (EBLV-2)in one species of British bat. This virus has caused thedeath of a Scottish bat worker in 2002 [Fooks et al.,2003a] and should also be included in a possiblediagnosis of human rabies, in the absence of a travelhistory.

Critically, in virtually all the reported cases in the UK,post-exposure prophylaxis in the form of rabies vaccineand rabies immune globulin was not applied. Thisprophylaxis, when administered before the onset ofclinical symptoms following regimes recommendedby the World Health Organisation (WHO), is highlyeffective at preventing the development of disease. It iswidely accepted that there is no proven effective treat-ment once clinical symptoms of rabies are observed.These non-specific symptoms include pain or tingling atthe bite site, progressing to behavioral changes, hydro-phobia and paralysis. However, a recent report hasdescribed a form of induced-coma that is believed to havecontributed to the survival of a teenager infected withrabies in the United States [Willoughby et al., 2005].This was in a child with bat rabies, who was also unusualin having anti-rabies antibodies at the time of presenta-tion. Although subsequent attempts to repeat thistherapy have failed for a number of human presenta-tions with rabies [Hemachudha et al., 2006; US Centresfor Diseases Control and Prevention, 2007; Schmiedelet al., 2007; McDermid et al., 2008; Rubin et al., 2009]the absence of suitable animal models to develop thisapproach has limited investigation into its mode ofaction or its refinement. This case report describes ahuman case of imported rabies in Northern Ireland, adescription of its diagnosis and subsequent monitoring,and the clinical interventions applied.

PATIENT

A 37-year-old woman was admitted to a districtgeneral hospital with a 4-day history of sweating, legweakness, and paraesthesia in her fingers (day 0). Oninitial examination her temperature was 37.58C, pulse78 per minute, and blood pressure was 136/94 mm Hg.She was anxious, but fully orientated in time, space, andperson. She had reduced power in her legs; left leg powerwas 3/5 (MRC grading) and right leg 4/5. There was nosensory or cranial nerve deficit. On admission, herhemoglobin was 13.6 g/dl, white blood cells 8.6�109/L,platelets 281� 109/L, sodium 139 mmol/L, potassium4.1 mmol/L, urea 3.6 mmol/L, creatinine 66 mmol/L,

J. Med. Virol. DOI 10.1002/jmv

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1256 Hunter et al.

C-reactive protein less than 0.2 mg/L, and she hadnormal liver function tests. On day 3 of her hospitaladmission, she developed a fever of 39.08C, confusion,verbal aggression, difficulty swallowing, and progres-sive leg weakness. Neurological examination demon-strated areflexia and marked weakness (power rangingfrom 0/5 in her legs to 2/5 in her arms). She wascommenced on aciclovir, 600 mg, three times per day,and ceftriaxone, 2 g, twice daily. On day 5 of hospital-ization, a neurological examination suggested menin-goencephalitis with a polyradiculoneuritis. Later thatday, her neurological deficit progressed to a flaccidquadraparesis with associated hypercapnic respiratoryfailure due to neuromuscular weakness (arterial bloodgas [FiO2 28%]: pH 7.38; pO2 15.6 kPa; pCO2 6.42 kPa;HCO3 28.1 mmol/L). She required intubation formechanical ventilation. She had not displayed anyhydrophobia or aerophobia.

At this time, further questioning of friends andrelatives revealed she had a notable animal exposurehistory. This included a visit to a monkey sanctuary inLimpopo province, South Africa, 24 months prior toadmission, when she separated two fighting dogs. One ofthese dogs allegedly died of rabies. During a further visitto the South African monkey sanctuary, 9 monthspreviously, she was bitten by a monkey. She had beenworking in a Northern Irish cat sanctuary for a yearprior to presentation, but had not traveled outside of theUK subsequently. There was no history or serologicalevidence of pre- or post-exposure rabies vaccination.

On day 7, rabies was considered in the differentialdiagnosis. She was transferred to the regional intensivecare unit with appropriate infection control precautions.Saliva taken on day 8 was negative for rabies (Table II).On day 9, a rabies nuchal biopsy was obtained; RabiesTaqMan� PCR was strongly positive in this specimen(Table III). Further nested PCR analysis on this samplegenerated a 606 base pair amplicon that was sequencedand used for virus characterization.

In view of the diagnosis and absence of vaccinationhistory, a decision, in conjunction with her family,was made to manage her aggressively according to amodified Milwaukee protocol. Figure 1 showsa timeline of the principal medications administeredfollowing her rabies diagnosis. We administered ket-amine (2 mg/kg/hr) and midazolam (0.5–0.67 mg/kg/hr)to induce a ‘‘burst suppression’’ pattern on the electro-encephalogram. The ketamine and midazolam dosage

was titrated to suppress background electroence-phalogram activity to 1–2 sec of interspersed cerebralactivity. Antiviral therapy was commenced with aman-tadine (100 mg twice daily) and ribavirin (3 g IV loading,then 1.2 g four times per day for 2 days). Metabolicsupplementation with co-enzyme Q (100 mg four timesper day), tetrahydrobiopterin (‘‘BH4’’) (50 mg four timesper day) and vitamin C (500 mg daily) was given. In anattempt to prevent rabies-associated vasospasm, shewas given nimodipine (60 mg six times per day), and herserum sodium was maintained between 145 and150 mmol/L. Neither rabies vaccine nor rabies immuneglobulin was administered.

By day 11 the patient had a dense quadraparesis withno respiratory effort. She did not respond to pain andpupils were fixed and unresponsive to light stimulus.On day 13, she developed small bowel ileus. Low-doseinotropes were commenced on day 15 of hospitaladmission and inotrope requirements increasedfrom day 22 onwards. Autonomic instability (manifestas labile blood pressure) was noted on day 21. On days11, 14, 15, 17, and 24 she had cranial diabetes insipidis.This was managed with desmopressin boluses. On day27 she developed abnormal liver function tests. This wasattributed to an adverse drug reaction to metronidazole.

Daily electroencephalogram was consistent with anon-specific encephalitis, until the trace became ‘‘flat,’’even after the adjustment of sedation, on day 24.Previous case reports have reported cerebral artery

J. Med. Virol. DOI 10.1002/jmv

TABLE II. Detection of Virus in Saliva DuringHospitalization

Day ofhospitalization

Taqman PCR(N gene)

Virus isolation(RTCIT)

8 � þ9 � þ10 þ �11 þ �12 þ �15–28 � �

TABLE III. Conclusive Tests for Diagnosis of Rabies

Day ofhospitalization

TaqmanPCR

Virusisolation FAT

CSF 8 þ þ Not doneSaliva 8 � þ Not doneSaliva 10 þ � Not doneSaliva 15 � � Not doneSkin biopsy 9 þþ Not done Not doneBrain Post mortem þþþ þ þþþ

þ, weak positive; þþ, positive; þþþ, strong positive; �, negative.

Fig. 1. Medication administered.

Immunovirological Correlates in Human Rabies 1257

vasospasm or cerebral oedema. Therefore appropriateneuroimaging was undertaken. This comprisedmagnetic resonance imaging on days 6, 15, 21, compu-terized tomography on days 3, 23, and 26, and single-photon emission computed tomography on day 20. Therewas no evidence of vasospasm or oedema.

On day 26 sedation with ketamine and midazolamwas tapered to zero over 24 hr. After the removal ofsedation, she had no response to pain, no respiratoryeffort, no deep tendon reflexes, and no pupillaryresponses. From day 28 she required full neuroendo-crine support to maintain vital organ function. This waswith noradenaline, corticosteroids, thyroxine, and vaso-pressin. In view of the belief that rabies encephalitismay mimic brain death, the patient received supportivecare between days 28 and 35. However, on day 35,magnetic resonance angiography revealed no cerebralblood flow and loss of definition between cerebral greyand white matter. In conjunction with her family,cardiorespiratory support was withdrawn and she died.Her family consented to a limited post mortem exami-nation. Specimens were taken from left and rightoccipital lobes, left and right cerebellar hemispheres,and dura mater (Table IV).

MATERIALS AND METHODS

Rabies virus was detected in both ante-mortem andpost-mortem samples. Virus isolation was achievedusing the rabies tissue culture infection test employingneuroblastoma (N2a) cells and using standard protocols[Webster and Casey, 1996]. Rabies antigen was detectedin brain tissue using the fluorescent antibody test aspreviously described [Dean et al., 1996]. Rabies virusgenomic RNA was detected using a lyssavirus differ-ential TaqMan� RT-PCR [Wakeley et al., 2005] and ahemi-nested RT-PCR [Heaton et al., 1997] after extrac-tion of RNA with TRIzol (Invitrogen, Paisley, Scotland)using the manufacturers protocol. The amplicon pro-duced by this method was purified using a spin column(Qiagen, Crawley, UK) and sequenced using the flank-ing primers Jw12 and Jw6dpl. The sequence derivedwas compared to a range of RABV sequences publishedpreviously [Kissi et al., 1995] and available fromGenBank. The phylogenetic tree presented (Fig. 2) wasgenerated using the MegaAlign program. Bootstrapvalues greater than 70 were considered significant.Rabies neutralizing antibodies were detected using thefluorescent antibody virus neutralization test usingpublished protocols [Cliquet et al., 1998].

The limited post mortem was performed 18 hr afterdeath. Tissue was taken from the dura and brain via

an occipital approach. Each sample was taken usingseparate disposable forceps and scalpel with no crosscontamination. Samples of dura and brain were placedin sterile containers and taken directly to the virologylaboratory. Additional samples were taken for histology.These were fixed in 10% buffered formalin for 14 daysbefore processing and double-embedding in low viscositynitrocellulose paraffin wax.

Formalin-fixed 7-mm paraffin embedded tissue sec-tions were deparaffinized, and antigen retrieval wasundertaken in a pressure cooker at full power for 2 minin 0.01 M TRIS-EDTA buffer (pH 6.0). Viral antigen wasdetected with anti-rabies nucleocapsid protein mono-clonal antibody (1:80; mAb HAM 5DF123BO, SwissRabies Centre). Sections were incubated in primaryantibody overnight at 48C, and specific antibody-antigenbinding sites were detected using an Envision-Perox-idase system with DAB (Dako UK Ltd., Ely, UK) assubstrate (light microscopy).

For immunofluoresence and confocal microscopy viralantigen binding sites were detected with goat anti-mouse-Alexa 488 (Invitrogen). Sections were counter-stained with propidium iodide (Sigma) and mounted incitifluor for examination under a 40� oil-immersionobjective on a Leica TCS/NT SP2 confocal microscopeequipped with a krypton-argon laser.

RESULTS

A summary of rabies diagnostic tests undertaken onpatient samples is shown in Tables II–IV. The firstand second saliva samples submitted (days 8 and 9)tested negative by rabies TaqMan� (Tables II and III). ACSF sample taken on day 8 was initially considerednegative, although a marginal increase in fluorescencewas observed, and a very weak band observed whenthe reaction sample was separated by gel electropho-resis. Both CSF and saliva samples taken on day 8 weresubsequently shown to be rabies virus positive by virusisolation after 5 days culture in neuroblastoma cells.The CSF cell count, biochemistry, and serology resultsare summarized in Table V. The first confirmationof rabies was made on a skin biopsy sample takenon day 9. Rabies TaqMan� PCR was strongly positive inthis sample. Further nested PCR analysis on thissample generated a 606 base pair amplicon that wassequenced and used for virus characterization (Fig. 2).Saliva samples taken on days 10, 11, and 12 testedpositive by TaqMan� PCR but negative by virusisolation. All subsequent saliva and CSF samplestaken were negative by PCR and virus isolation(Table II).

J. Med. Virol. DOI 10.1002/jmv

TABLE IV. Post Mortem Virology Tests

PM specimen Antigen detection Taqman (ct value) Virus Isolation

Dura Negative Positive (21.74) NegativeLeft occipital Strongly positive Positive (24.57) Weak positiveRight occipital Strongly positive Positive (22.48) Weak positiveLeft cerebellum Strongly positive Positive (19.45) Weak positiveRight cerebellum Strongly positive Positive (10.89) Weak positive

1258 Hunter et al.

The skin biopsy sample taken on day 9 produced aweak product after first-round of amplification and astronger band after nested amplification. The bandwas sequenced using flanking primers (Jw12 and Jw6dpl) to produce a 400 base pair (bp) fragment from therabies virus nucleoprotein between genome positions 71and 470. An initial blast search demonstrated thatthis sequence showed 100% homology with Africanrabies viruses that are circulating in dogs. Additionalphylogenetic analysis confirmed that the sequence

was closely related to a South African canine strain(Fig. 2).

Detection of rabies neutralizing antibody was con-ducted on both cerebrospinal fluid (CSF) and serum(Fig. 3). All results from this test are presented asInternational Units per milliliter (IU/ml). The first bloodsample was taken on day 7 and generated a rabies virusneutralizing antibody value of 0.06 IU/ml; interpretedas negative. The first CSF sample taken on day 8 gave avalue of 0.17 IU/ml and was also considered negative.

J. Med. Virol. DOI 10.1002/jmv

Fig. 2. Phylogenetic tree of virus sequence amplified from the patient (skin biopsy sample). Themegalign-derived tree compares a 400 bp of nucleoprotein gene from the patient (isolate RV2481N) with anumber of African RABV isolates. The initial AFS (South Africa), MOZ (Mozambique) and NAM (Namibia)have been used to describe sequences from southern Africa.

Immunovirological Correlates in Human Rabies 1259

A second serum sample taken 4 days later demonstratedthe presence of neutralizing antibodies with a value of3.42 IU/ml, well above the recognized level of sero-conversion 0.5 IU/ml (Fig. 3). A second CSF sampletaken on day 13 gave a value of 1.5 IU/ml. Serumneutralizing antibody increased to a peak of 40.5 IU/ml(sample taken on day 16) and fluctuated around thislevel during the period of hospitalization (Fig. 3A). TheCSF neutralizing antibody peaked at 2.6 IU/ml on day

16 and a further sample taken on day 21 also gave thesame value (Fig. 3B).

A total of six brain samples and a section of the duramater were submitted for testing. With the exception ofthe dura mater sample, all tested positive for rabiesantigen and positive for rabies genome by TaqMan RT-PCR. There was a wide variation in TaqMan PCRreactivity between different areas of brain sampled(Table IV) as indicated by ct value. A difference of 13 ctvalues between the highest and lowest values implies agreater than 4 log difference in amount of virus genomebetween these samples. Immunostaining of brain tissuesampled at post-mortem reveals the presence of exten-sive rabies virus nucleoprotein (Fig. 4). Histologicalexamination showed meningoencephalitis. Meningeallymphocytic inflammation was present and the braintissue showed perivascular inflammation in the cortexand white matter of the occipital lobes and cerebellum.There was extensive tissue destruction especially incortical areas with very few recognizable neuronesapart from those in the cerebellar granular layer. Norecognizable purkinje cells remained. Negri bodies werenot observed. Immunohistochemical detection showedpositive staining in the neuropil, in axonal processes andfocally in nerve cell body cytoplasm (Table VI).

DISCUSSION

Once clinical symptoms have occurred, human rabiesis accepted to have a case fatality rate approaching100%. Throughout the world, treatment is directed atpalliation and symptom control until death occurs[Jackson et al., 2003]. A small number of patientspresenting with clinical rabies have survived, some withsevere neurological damage. All but one had evidence ofeither pre-exposure or post-exposure prophylaxis [Hatt-wick et al., 1972; Porras et al., 1976; Tillotson et al.,1977; Alvarez et al., 1994; Madhusudana et al., 2002].However, in 2004, Willoughby et al. [2005] described thecase of a 15-year-old girl who developed clinical rabiesfollowing a bat exposure, and survived in the absenceof rabies vaccination, though she was unusual inhaving antibodies at the time of presentation. Theunderlying hypothesis is that death in rabies resultsfrom ‘‘neurotransmitter imbalance’’ and autonomicfailure [Willoughby et al., 2009] with evidence ofdeficiencies in BH4, dopamine and serotonin. However,the effect of these deficiencies on the clinical manifes-

J. Med. Virol. DOI 10.1002/jmv

TABLE V. Serial Cerebrospinal Fluid Results

Day ofhospitalization

White blood cells(cells/mm3)

Red blood cells(cells/mm3) Protein (mg/L) Glucose (mg/L)

Neutralizingantibodies (IU/ml) Rabies PCR

3 70 0 0.59 4.8 — —8 4 4 3.8 3.5 0.17 Positive13 — — 4.16 4.8 1.5 Negative16 — — — — 2.6 Negative21 48 <1 1.79 3.3 2.6 Negative

—, specimen not analyzed.

A

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2120191817161514131170.01

0.1

1

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100

Serum antibody

(IU/ml)

21161380.01

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CSF antibody

(IU/ml)

Fig. 3. Detection of rabies neutralizing antibodies in serum (A) andCSF (B) Horizontal axis—day from hospitalization. Note that alogarithmic scale has been used to display the relative antibodyresponse. Results are expressed as international units per milliliter ofblood or CSF (IU/ml).

1260 Hunter et al.

tations of rabies remains to be discovered. Death afterprolonged rabies survival has been reported to beassociated with post-mortem findings of clearance ofrabies virus suggestive that normal immune response issufficient to clear virus if survival is sufficiently long[Rubin et al., 1970; Emmons et al., 1973]. Therefore, ifsupportive therapy can protect the nervous system untilimmune response clears virus, then the patientmay survive with acceptable neurological function.Willoughby et al. developed a protocol comprisingdrug-induced coma (using ketamine, a N-methyl-D-aspartate receptor antagonist and midazolam), anti-viral agents (ribavirin, amantadine, and ketamine), andneurotransmitter substrate replenishment (using met-abolic supplementation). Subsequently, 20 cases ofrabies have been treated with the Milwaukee protocol,or its modification [Rabies Registry Children’s Hospitalof Wisconsin, 2009; Table VI]. To date, two further cases,a Brazilian and a Colombian patient with rabiesacquired from a bat (unpublished), are considered to besurvivors. The full treatment regimens used in each casehave yet to be published. Non-immune patients, treatedusing the same protocol, in Thailand [Hemachudhaet al., 2006], USA [US Centres for Disease Control andPrevention, 2007], Germany [Schmiedel et al., 2007],Canada [McDermid et al., 2008], and Equatorial Guinea[Rubin et al., 2009] have died. The actual number offailed treatments may be higher due to failure toreport unsuccessful treatment attempts. Our case alsodied despite being treated with the modified protocol.However, the close monitoring of this case provides

useful information on the development of the immuneresponse to rabies virus infection in association withtherapy.

In terms of exposure to rabies, the patient traveled toSouth Africa on two occasions. Further investigationwould be required to ascertain whether transmissionoccurred 2-years following an encounter with dog or9 months after the bite of a monkey. Phylogeneticanalysis supports the information provided in the casehistory. There is also documented evidence that anepizootic of rabies occurred in Limpopo province in 2006during the time when the patient was resident in thatarea and had exposure to a dog [Cohen et al., 2007;Sabeta et al., 2007]. However, available sequencesmay not provide sufficient variation to connect this toa specific year.

Diagnosis remains problematic in non-endemic coun-tries, such as Northern Ireland. This was principallybecause rabies is rarely considered as a cause ofundiagnosed encephalitis unless a direct exposurehistory is available. In recent years, there has beenincreasing emphasis on the clinical utility of salivabased diagnosis using sensitive molecular approaches[Nagaraj et al., 2006]. In clinical practice, saliva ispreferred because specimen collection is convenient andnon-invasive. However, this case highlights the limi-tation of only sampling saliva. The saliva was onlypositive by PCR from days 10 to 15, at the point whereneutralizing antibody was detected. Salivary specimenscollected before and after this diagnostic window werenegative for rabies RNA, although virus isolation was

J. Med. Virol. DOI 10.1002/jmv

Fig. 4. Upper: Light microscopy of paraffin sections of brain tissue. Lower images: Confocal imaging ofdeparaffinized tissue sections with anti-rabies antibodies.

Immunovirological Correlates in Human Rabies 1261

J. Med. Virol. DOI 10.1002/jmv

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pre

sen

tati

on

No

vir

us

isol

ate

dN

ovir

al

an

tigen

sd

etec

ted

(2sk

inbio

psi

esan

d9

sali

va

sam

ple

s)

Su

rviv

al

Not

ap

pli

cable

US

Cen

ters

for

Dis

ease

Con

trol

an

dP

reven

tion

[2007]

Ind

ian

a,

US

Gen

otyp

e1/b

at

Ab

pre

sen

tat

clin

ical

pre

sen

tati

on

RT

-PC

Rp

osit

ive

for

rabie

svir

us

(sk

inan

dsa

liva)

Sk

inan

dsa

liva

pos

itiv

evia

dir

ect

flu

ores

cen

tan

tibod

y(D

FA

)

Dec

ease

d,

34

days

aft

ersy

mp

tom

onse

t

Rabie

svir

us

an

tigen

det

ecte

din

bra

inti

ssu

eat

pos

t-m

orte

mU

SC

ente

rsfo

rD

isea

seC

ontr

olan

dP

reven

tion

[2007]

Cali

forn

ia,

US

Gen

otyp

e1/b

at

Neg

ati

ve

at

pre

sen

tati

on.

Bec

am

ep

osit

ive

ond

ay

12

RT

-PC

Rp

osit

ive

for

rabie

svir

us

(sali

va)

Rabie

svir

us

an

tigen

sd

etec

ted

by

corn

eal

imp

ress

ion

s(D

FA

)

Dec

ease

d,

29

days

aft

ersy

mp

tom

onse

t

Rabie

svir

us

an

tigen

det

ecte

din

bra

inti

ssu

eat

pos

t-m

orte

mH

emach

ud

ha

etal.

[2006]

Th

ail

an

dG

enot

yp

e1/d

ogN

oan

tibod

yd

etec

ted

du

rin

gcl

inic

al

cou

rse

NA

SB

Ad

etec

ted

rabie

sR

NA

(hair

foll

icle

)

No

com

men

tD

ecea

sed

,8

days

aft

ersy

mp

tom

onse

t

Rabie

svir

us

was

isol

ate

dfr

ombra

inan

dsp

inal

cord

tiss

ues

at

nec

rosc

opy

Un

pu

bli

shed

[2006]

Tex

as,

US

Gen

otyp

e1/b

at

Not

kn

own

Not

kn

own

Not

kn

own

Dec

ease

d�

7d

ays

pos

t-h

osp

itali

zati

onN

otk

now

n

Sch

mie

del

etal.

[2007]

Ger

man

yG

enot

yp

e1/d

ogA

bn

otd

etec

ted

at

pre

sen

tati

on.

Ser

um

Ab

resp

onse

from

day

11

RT

-PC

Rp

osit

ive

for

rabie

sR

NA

(sali

va

an

dco

rnea

lsw

ab)

No

com

men

tD

ecea

sed

,31

days

aft

ersy

mp

tom

onse

tA

uto

psy

per

form

ed‘‘f

urt

her

vir

olog

ical

inves

tigati

ons

ongoi

ng’’

McD

erm

idet

al.

[2008]

Can

ad

aG

enot

yp

e1/b

at

‘‘Over

tim

era

bie

sIg

Gan

dIg

Mro

se’’

RT

-PC

Rp

osit

ive

for

rabie

sR

NA

(sk

inbio

psy

an

dsa

liva)

Rabie

sA

gd

etec

ted

by

flu

ores

cen

tan

tibod

yte

st

Dec

ease

d,

65

days

aft

ersy

mp

tom

onse

tR

abie

svir

us

an

tigen

det

ecte

dat

au

top

sy.

Neg

ribod

ies

pre

sen

ton

path

olog

yU

np

ubli

shed

[2008]

Bra

zil

Gen

otyp

e1/b

at

An

tira

bie

san

tibod

yin

blo

odan

dC

SF

.In

crea

sed

over

clin

ical

cou

rse

RT

-PC

Rid

enti

fied

vir

us

RN

A(s

kin

foll

icle

)

No

com

men

tS

urv

ival

Not

ap

pli

cable

Un

pu

bli

shed

[2008]

Ger

man

y(l

un

gtr

an

spla

nt

reci

pie

nt)

Gen

otyp

e1/d

ogN

on

eutr

ali

zin

gan

tibod

yre

spon

seR

T-P

CR

det

ecte

dra

bie

svir

us

(CS

F,

spu

tum

,B

AL

)

Vir

us

isol

ate

dby

cell

cult

ure

Dec

ease

d,

49

days

pos

t-tr

an

spla

nt

Rabie

sR

NA

isol

ate

dfr

omC

NS

tiss

ue

Un

pu

bli

shed

[2008]

Ger

man

y(k

idn

eytr

an

spla

nt

reci

pie

nt)

Gen

otyp

e1/d

ogN

on

eutr

ali

zin

gan

tibod

yre

spon

seR

T-P

CR

det

ecte

dra

bie

svir

us

(CS

F,

sali

va)

No

vir

us

cult

ure

dD

ecea

sed

,52

days

pos

t-tr

an

spla

nt

Rabie

sR

NA

isol

ate

dfr

omC

NS

tiss

ue

Un

pu

bli

shed

[2008]

Ger

man

y(k

idn

eyan

dp

an

crea

str

an

spla

nt

reci

pie

nt)

Gen

otyp

e1/d

ogR

T-P

CR

det

ecte

dra

bie

svir

us

(CS

F,

spu

tum

,co

rnea

lsw

ab)

Vir

us

isol

ate

dby

cell

cult

ure

Dec

ease

d,

95

days

pos

t-tr

an

spla

nt

Rabie

sR

NA

isol

ate

dfr

omC

NS

tiss

ue

van

Th

iel

etal.

[2009]

Ken

ya

Gen

otyp

e4

(Du

ven

hage)

/bat

Ab

not

det

ecte

d(p

ati

ent

giv

enH

DC

Vd

1,

4,

8,

an

d15)

RT

-PC

Rd

etec

ted

DU

VV

(Nu

chal

skin

bio

psy

an

dsa

liva)

No

vir

us

cult

ure

dD

ecea

sed

,45

days

pos

t-in

cid

ent

wit

hbat

Rabie

svir

us

an

tigen

infr

onta

lan

dte

mp

oral

cort

ex,

hip

poc

am

pu

san

den

torh

inal

cort

exR

ubin

etal.

[2009]

Equ

ato

rial

Gu

inea

Gen

otyp

e1/d

ogA

bd

etec

ted

inse

rum

d18

an

dC

SF

d20

RT

-PC

Rd

etec

ted

rabie

svir

us

inS

ali

va

Rabie

svir

us

isol

ate

dfr

omsk

inbio

psy

wh

enin

ocu

late

din

tom

ice

Dec

ease

d.

(NB

dea

thatt

ribu

ted

tom

aln

utr

itio

nan

dre

nal

fail

ure

)

No

com

men

t

Th

isca

se,

2008

Bel

fast

,U

KG

enot

yp

e1/d

ogA

bn

otd

etec

ted

at

pre

sen

tati

on.

Ser

um

an

dC

SF

Ab

resp

onse

from

day

8

RT

-PC

Rd

etec

ted

rabie

svir

us

(CS

F,

spu

tum

,sk

in)

Vir

us

isol

ate

dby

cell

cult

ure

Dec

ease

d,

35

days

aft

ersy

mp

tom

onse

tR

abie

sR

NA

isol

ate

dfr

omC

NS

tiss

ue.

Via

ble

rabie

svir

us

cult

ure

d.

An

tigen

pre

sen

t.

1262 Hunter et al.

possible in some early samples where RT-PCR failed.This may be due to a limitation in the RNA isolation insamples where the nucleic acid concentration is parti-cularly low. Therefore, nuchal skin biopsy should beregarded as the key diagnostic sample for ante-mortemdiagnosis where rabies is suspected [Dacheux et al.,2008].

Molecular methods of diagnosis demonstrated utilityin terms of speed of diagnosis in this case. RabiesTaqMan� provided a rapid, reliable method for detect-ing rabies in a range of samples. This re-affirms the useof RT-PCR for ante-mortem diagnosis of rabies [Crepinet al., 1998]. However, conventional virus isolation diddetect virus in some samples that were PCR negative.Analysis of the samples that were positive in this patientsuggests that the ability to detect virus in ante-mortemsamples is highly dependent on the immune status of thepatient. Once peripheral seroconversion had occurred(day 11) only one further saliva sample (day 12) waspositive by rabies TaqMan� PCR. This suggests thatthere is a narrow window for ante-mortem diagnosis ofdisease based on detection of virus in accessible ante-mortem samples such as saliva, CSF, and skin biopsy. Inpractice this places the burden of diagnostic testing onserology. This may account for the failure to detect virusin the Wisconsin case, where the patient presented withhigh anti-rabies antibody titers in CSF [Willoughbyet al., 2005].

Serology provided a useful tool to monitor serocon-version and thus possible clearance of the rabies virus bythe immune response in this case. In the ‘‘MilwaukeeProtocol,’’ prediction of the development of the immuneresponse is believed to have utility in order to determinethe time to withdraw drug-induced coma [MedicalCollege of Wisconsin, 2009]. However, in this case,results were often delayed due to transport timebetween Belfast and the Veterinary LaboratoriesAgency based in southern England, and to the 2-daylaboratory turnaround time for serology tests. If thedevelopment of the immune response can accuratelypredict neurologic virus clearance then this delay maygenerate an unacceptable increase in the time thepatient spends in a drug induced coma, with thepotential for additional complications. Rapid serologytesting for antibody is needed to support futureintensive interventions in cases of human rabies.

In this case, extensive virus was detected in brainsamples from this patient at post-mortem, 24 days afterseroconversion. This suggests that although the patientdid develop a significant immune response, this did notappear to restrict virus replication centrally. It isdifficult to explain the marked variance in amount ofvirus genome between different brain samples and thedifficulty in virus isolation. This could be interpreted aspossible initial evidence of viral clearance. However, thevery high levels of antigen and viral load argue againstthis. It is possible that the therapy applied to this casefailed because the patient seroconverted relatively lateafter the initiation of drug induced coma. Alternatively,the antibody levels in the CSF may not have increased

sufficiently to control the virus. In either case, theneurological damage could have been significant beforeinitiation and establishment of the therapy, such thatearlier treatment might have been successful. Thesurviving patient had neutralizing antibodies in bothCSF and serum at presentation, whereas the Belfastpatient seroconverted 11 days after presentation. Thesefacts may suggest that the speed of development of theimmune response relative to spread of the viruscentrally is crucial to eventual central nervous systemfunction.

Rabies is a highly neurotropic virus. The pathogenesisof human rabies is incompletely understood. The clinicalpresentation ranges from classical (encephalitic ‘‘furi-ous’’ or paralytic ‘‘dumb’’ forms) to non-classical forms[Hemachudha et al., 2002]. Commentators haveobserved that ‘‘bat’’ rabies (when compared with rabiesacquired via a dog bite) is associated with longerincubation, a more rapid antibody response and casereports of survivors [Hanna et al., 2000; Hemachudhaet al., 2002; Hemachudha and Wilde, 2005]. Isolatedsurvivor reports, animal models, and in vitro studieshave provided additional clues about disease mecha-nism, and have led to novel therapies. Furthermore,effective anti-rabies treatment must include the pro-duction of viral neutralizing antibody and immune-mediated viral clearance from the central nervoussystem [Hooper et al., 1998]. Animal models havedemonstrated that neuronal apoptosis is T-cell medi-ated rather than as a consequence of direct rabies-viruscytotoxicity [Galelli et al., 2000]. On the basis of thislimited evidence, doctors have undertaken trials ofnumerous medications which theoretically have anti-rabies activity. This has included accelerated rabiesvaccination, rabies-specific immunoglobulin, inter-feron-a, steroids, anti-thymocyte globulin, ketamine,and anti-viral drugs [Hattwick et al., 1972; Meriganet al., 1984; Warrell et al., 1989; Hemachudha, 1994].We believe that the ‘‘Milwaukee protocol’’ integratesclinical experience and research findings to date.Although the outcome in our case was unsuccessful,the ‘‘Milwaukee protocol’’ may still contain curativetreatments, especially if applied to patients with rabiesacquired from bats. Interestingly, the surviving patientdescribed by Hattwick et al. [1972] did not receive anyspecific treatment but only supportive care. An earlydetectable antibody response and absence of virus wereobserved, similar to the patient treated in 2004 using theMilwaukee Protocol suggesting that these are criticalfactors that may determine a positive response totreatment.

The Milwaukee Protocol case highlights the impor-tance of rabies vaccination. Vaccination against rabiesshould be considered for all persons who travel to arabies endemic region for either recreational or occupa-tional purposes bearing in mind that the majority of dog-bite exposures occur in urban and not rural regions. In astudy on UK travelers to rabies endemic countries, thereason provided for declining pre-exposure immuniza-tion against rabies was cost [Fooks et al., 2003b]. A cost/

J. Med. Virol. DOI 10.1002/jmv

Immunovirological Correlates in Human Rabies 1263

benefit analysis needs to be undertaken in order to offsetimmunization costs and increase vaccine uptake inrecipients who travel to rabies endemic countries. Indeveloping countries where resources are limited physi-cians are unlikely to adopt this protocol for routine useuntil the protocol has been clinically proven and wherethe cost of administration is prohibitive.

In conclusion, rabies diagnosis is dependent on a highlevel of suspicion in a patient with a relevant exposurehistory. Although saliva represents the most accessiblediagnostic sample, there is a diagnostic window whichmay be smaller than previously believed. Molecularmethods are rapid and reliable but serology is stilluseful, particularly to monitor the development of theimmune response.

The utility of the ‘‘Milwaukee Protocol’’ has not beenestablished partly due to the absence of appropriateanimal models. Its use must be considered carefully incases of rabies in consultation with patients’ families asit can intensify expectations of a positive outcome. Wewould suggest that detection of rabies antibodies inserum and CSF, early after presentation and in theabsence of a history of vaccination may be a positiveindicator for intervention. Conversely, seroconversionlate in the disease course is a negative prognosticindicator. Fundamental studies in animal models arerequired to provide support for continued use of the‘‘Milwaukee Protocol’’ for human use. Alternative smallanimal models should now be considered for the ‘‘proof-of-concept’’ assessment of different treatment regimensfor rabies leading to pre-clinical assessment in non-human primates. In our opinion, curative rabies treat-ment attempts should be only employed with theunderstanding that the regimen is used experimentally.

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