Gut 1993; 34: 299-302

PAPERS

Varicella-zoster virus DNA in the oesophagealmyenteric plexus in achalasia

C S Robertson, B A B Martin, M Atkinson

AbstractIn a search for past or present infection withherpes viruses, serum antibody titres to herpessimplex type 1 virus, cytomegalovirus, andvaricella-zoster virus were measured by com-plement fixation test in 58 patients withachalasia. Serum was also taken from 40 ageand sex matched patients without oesophagealsymptoms who formed a control group. Alltitres were low, and those for herpes simplextype 1 virus and cytomegalovirus did not differin the achalasia patients and the controls.However, the incidence of varicelia-zostervirus antibodies was significantly greater inthe achalasia than in the control group(p<005). Using oesophageal tissue contain-ing myenteric plexus removed at the timeof cardiomyotomy in nine patients withachalasia, in situ DNA hybridisation showedevidence of varicella-zoster virus in three, butall were negative for the other two viruses.No positive results were obtained for herpessimplex type 1 virus, cytomegalovirus, orvaricella-zoster virus in oesophageal tissuefrom 20 patients undergoing oesophagealresection for diseases other than achalasia.The incidence of positivity for varicella-zostervirus was significantly increased in theachalasia group compared with the controls(p<002). The findings indicate that varicella-zoster virus DNA may persist in the oeso-phageal myenteric plexus in some patients withachalasia and raise the possibility that thisvirus is of aetiological importance in achalasia.(Gut 1993; 34: 299-302)

Department of Surgery,University of NottinghamC S RobertsonM Atkinson

Department of CancerStudies, University ofBirminghamB A B MartinCorrespondence to:Professor M Atkinson,Department of Surgery,E Floor, University Hospital,Queen's Medical Centre,Nottingham NG7 2UH.

Accepted for publication20 August 1992

Although the cause of achalasia of the cardiaremains obscure, there is convincing evidencethat the oesophageal motor disorder results fromdamage to the oesophageal myenteric plexuswith ganglion cell depletion.'2 An intriguingfeature of the disease is its localisation to theoesophagus without affecting the rest of the gut.The oesophagus is unique in being the only partof the alimentary tract lined by squamousepithelium and the known predilection of theherpes class of viruses for squamous ratherthan columnar epithelium is consonant with theoesophageal mucosal involvement reported invaricella,34 herpes simplex,56 and cytomegalo-virus infections.78 These viruses are neurotropicand the varicella-zoster virus has been shown topersist in posterior nerve root ganglia9 where itmay remain dormant for years until reactivated

in herpes zoster. This led us to the hypothesisthat the ganglion cell loss in achalasia may be theresult of persistent herpes virus infection in theoesophageal myenteric plexus.

This study aimed to test this hypothesis, firstlyby examining the serum for antibodies to herpessimplex type 1 virus, varicella-zoster virus, andcytomegalovirus and secondly by in situ hybrid-isation studies on resected oesophageal tissueusing DNA probes to these viruses.

Methods

SEROLOGICAL STUDIESSera were obtained from 58 patients (26 men;median age 48 years; range 19-90 years) withestablished achalasia of the cardia, and 40 ageand sex matched controls without oesophagealdisorders (23 men; median age 57 years; range15-76 years) recruited from the gastroenterologyclinic. Control subjects were matched to within5 years of the patients' age.Serum antibody titres. Serum antibody titres to

herpes simplex type 1 virus, cytomegalovirus,and varicella-zoster virus were determined by amodification of the complement fixation testdescribed by Bradstreet and Taylor.'0

IN SITU HYBRIDISATION STUDIESSpecimens of oesophageal muscle were obtainedfrom nine patients with proved achalasia (fivemen; median age 32 years; range 14-45 years)and from 20 control patients (10 men; medianage 59 years; range 5-75 years), 17 of whomhad undergone oesophagectomy for malignantdisease and three of whom were cadaveric renaltransplant donors.

Fresh tissueAll specimens from the achalasia patients andfive from the control subjects were collectedfresh at the time of operation. The tissue wasorientated transversely under a dissecting micro-scope by a consultant neuropathologist beforesnap freezing in liquid nitrogen and isopentane.The tissue blocks were stored at - 196°C.Cryostat sections (5 [tm) were subsequently cutand the slides were stored at - 70°C until use.Every fourth slide was stained with haema-toxylin and eosin to confirm the correctorientation of the tissue.

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Robertson, Martin, Atkinson

Fixed TissueOesophageal specimens from 15 patients whohad undergone subtotal oesophagectomy forcancer were also used as controls. After beingfixed in buffered formalin and embedded inparaffin wax in the usual way, 5 urm sectionswere cut on to poly-l-lysine coated slides toimprove tissue adhesion. Tissues known to beinfected by herpes viruses were used as positivecontrols (sections of a chickenpox vesicle, renaltissue infected with cytomegalovirus, and braininfected with herpes simplex virus).

DNA HYBRIDISATION

Preparation ofDNA probesThe following DNAs were used: the HindIII BLfragment, cloned in pTR262, of the genome ofa varicella-zoster virus strain obtained from achickenpox patient; genome DNA of herpessimplex type 1 virus strain Troisbel; the EcoRIEK fragment of the genome of herpes simplextype 1 virus strain Troisbel; the HindIlI Ffragment of the genome of cytomegalovirus(Towne strain); and pAT153, a plasmid probeused to detect bacterial contamination. TheDNAs were labelled with biotin using biotin-dUTP or biotin dLTP by nick translation (BRL,Paisley) and used for hybridisation of slides atconcentrations of about 2-3 ,tg/ml. Probes weretested for efficacy by hybridisation to controlDNA dot blots prepared by loading sampleson to a nitrocellulose filter using a Bio-Dotapparatus (Bio-Rad Instruments, Watford); andalso by hybridisation to Cytospin preparations ofinfected and uninfected cells on glass slidescoated with aminopropyltriethoxysilane (APES)by immersing in 2% Apes in acetone, rinsing,and allowing to dry.

Hybridisation ofdot blotsThe filter was wetted in 0-2% bovine serumalbumin, 0-2% Ficoll, 0-2% polyvinylpyrroli-done (lOx Denhardt's solution) in 0-6M NaCl,0-06M sodium citrate (4x SSC), and shaken atroom temperature for 2 hours. It was then sealedin a polythene bag with 45% formamide,5 x SSC, I x Denhardt's solution, 0-1% sodiumdodecylsulphate (SDS), 1mM EDTA, and 100,ug/ml denatured salmon sperm DNA contain-ing probe and was incubated at 42-450C over-night. Probe consisted of about 0-1 [ig/mlbiotinylated DNA. The probe and the filter wereremoved from the bag and the filter was washedsuccessively in 2 x SSC, 0-1% SDS and0 2xSSC, 0 1% SDS twice each for 5 minutes atroom temperature, then in 0-16xSSC, 0-1%SDS twice for 15 minutes at 50°C. Finally filterswere rinsed in 2 x SSC. The filter was incubatedwith a streptavidin-alkaline phosphatase con-jugate (using the BluGENE kit from BethesdaResearch Laboratories, Paisley).

Colour development was achieved by incubat-ing the filters in a solution of buffer (0 IMTris-HCl; pH 7-5), 0-1M NaCl, 2mM Mg C12,3% bovine serum albumin) containing 75 mg/mlof nitro blue tetrazolium in 70% dimethyl-

formamide, and 50 mg/ml of 5-bromo-4-chloro-5indolyl phosphate in dimethylformamide.

Pretreatment and dehybridisation offresh tissueCryostat sections were thawed at room tempera-ture and processed according to a standardmethod" except that they were processed onuntreated glass slides, and paraformaldehydewas prepared in Dulbecco A solution.'2

After pretreatment the cryostat sections wereimmersed in 2xSSC 50% formamide for 10minutes at room temperature and prehybridisedat room temperature with hybridisation buffernot containing probes for 20 minutes.

Pretreatment offixed tissueSections were dewaxed and were then hydratedby sequential washes of twice for 1 minute ingraded ethanol (95%, 80%, 60%, and 30%)followed by a brief wash in distilled water and1 minute in Dulbecco A solution. The specimenswere then processed according to a methodmodified from'3 that considerably shortened thepreparation time. No prehybridisation wasnecessary for paraffin wax embedded specimens.Before the hybridisation mixture was applied,the slides were dipped briefly into 2 x SSC andthe excess liquid was wiped away.

In situ hybridisation procedureSlides were air dried before in situ hybridisationwith 15-20 ,ul of probe solution, consisting ofbiotinylated DNA in 40% formamide 4x SCC,lx Denhardt's solution, 0 2 mg/ml denaturedsalmon sperm DNA. After pipetting the hybrid-isation mixture onto the tissue sections, theedges of the siliconised glass coverslips weresealed with rubber solution and allowed to airdry. Simultaneous denaturation of cellular DNAin the tissue sections and probes was achieved byheating the slides at 80-85°C for 10 minutes andthey were then incubated for 16-18 hours at38 5°C in a humidified box. The slides were thenimmersed in 2 x SSC and the rubber seal wascarefully peeled off. The slides were washedsequentially in SSC and buffers I and II beforedetection of hybridisation signals.

Detection ofhybridisation signalsBiotinylated DNA was detected using a strep-tavidin-alkaline phosphatase system (BluGENE)in a humidified box and incubation for 30minutes to 4 hours in the dark, monitoringcolour development at frequent intervals. Afterdevelopment the colour reaction was stoppedand counterstaining with 2% methyl green andmounted in glycerol (20% in Dulbecco Asolution) was used.

STATISTICAL ANALYSISAnalysis was by Fisher's exact test with a value ofp less than 0 05 taken to denote statisticalsignificance.

Informed consent was obtained for operativetissue samples to be obtained and the study was

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Varicella-zoster virusDNA in the oesophageal myenteric plexus in achalasia

Positivity of complement fixation tests for herpes simplex type I virus, cytomegalovirus, andvaricella-zoster virus in achalasia patients and controls

Achalasia Controls(n=58) (n=40)

Titres 16 32 64 16 32 64

Herpes simplex type 1 virus 20 3 - 12 1Cytomegalovirus 17 - 2 10 1Varicella-zoster virus 14 2 - - - 1

approved by the Nottingham Hospital EthicalCommittee.

Results

SEROLOGICAL STUDIESViral serology was positive in 37 of the 58patients (63-8%) with achalasia (23 herpessimplex type 1 virus; 19 cytomegalovirus; 16varicella-zoster virus and in 19 of the 40 (47 5%)control sera (13 herpes simplex type 1 virus; 11cytomegalovirus; one varicella-zoster virus). Allthe titres were low, however, and are shown inthe Table. When patient and control groups

were compared for each type of virus, a statistic-ally significant difference was seen only forvaricella-zoster virus (X2=8 7148; p less than0 05; Fisher's exact test and Yates's correction),for which a positive titre was obtained in serafrom 16 of 58 patients with achalasia comparedwith one of 40 controls.

DETECTION OF DNA IN TISSUE SECTIONSOesophageal tissue sections from nine patientswith achalasia and from 20 controls were hybrid-ised with varicella-zoster virus, herpes simplextype 1 virus, cytomegalovirus, and pAT153probes. After staining, a clearly positive hybrid-isation signal was detected with varicella-zostervirus in tissue from three patients' with achalasia(Fig 1) but in none of the 20 control samples.This difference was clearly significant, with a pvalue of0-02 by Fisher's exact test. Positivity didnot correlate with the age of the patient or theduration of disease. No clearly positivehybridisation signal was detected in any of thespecimens (patients or controls), with cytomega-lovirus, herpes simplex type 1 virus, or pAT 153probes (Fig 2).

Figure 1: Transverse section through the myenteric plexus in a tissue samplefrom a patient withachalasia showing positive stainingfor varicella-zoster virus.

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Figure 2: Transverse section through the myenteric plexus in a tissue samplefrom a patient withachalasia showing negative staining for herpes simplex type I virus. This sample had shownpositive stainingfor varicella-zoster virus.

DiscussionThe three herpes viruses chosen for this studyare all recognised as causing oesophageal ulcera-tion but seldom affect the columnar epitheliumof the remainder of the gut. Herpes simplex type1 virus and varicella-zoster virus are known to beneurotropic, and cytomegalovirus infections ofthe central nervous system have been reported -hence all three seemed possible causes ofdamageto the oesophageal myenteric plexus.The serum antibody titres to all three viruses

were generally low and only in the case ofvaricella-zoster virus were they significantlygreater in patients with achalasia than in thecontrols. This suggests that these patients havehad greater exposure to varicella in the past. InBritain, varicella is prevalent in childhood, yetin only one of 40 control patients was antibodydetected that might point to past attacks ofgreater severity in the patients with achalasiathan in the control group. We know ofno clinicalevidence, however, to support this contention.Our studies did not encompass other viruses butan increased antibody titre against measles virushas been reported from Cardiff. 14The DNA probes used in this study are highly

specific for herpes simplex type 1 virus, varicella-zoster virus, and cytomegalovirus respectivelyand in this context the demonstration ofvaricella-zoster virus material in the oesophagealwall in three of nine patients with achalasia isclearly significant. It suggests that the virus maypersist in the oesophageal wall for many yearsafter an attack of varicella but only in some ofthose patients who have developed achalasia.Varicella-zoster virus positivity did not correlatewith either age or disease duration and unfor-tunately few subjects could recall if and whenthey had had varicella.

If varicella-zoster virus is of aetiologicalimportance in achalasia, an explanation must besought for why only three of nine patients with

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302 Robertson, Martin, Atkinson

this disorder were varicella-zoster virus positive.We do not know how much viral DNA is neededto give a positive signal and it could be thatthe six varicella-zoster virus negative achalasiapatients had this material present in amounts toosmall to be detectable by the method employed.Alternatively, the varicella-zoster virus may havedisappeared from these patients and, since wedid not obtain myenteric plexus ganglion cellcounts, we were unable to correlate varicella-zoster virus positivity with severity of myentericplexus damage. Positivity may be patchily distri-buted, as has been reported in the trigeminalganglia,9 and this would result in a samplingerror, particularly since the tissue samplesremoved at the time of cardiomyotomy wererelatively small in size. A third possibility is thatviruses other than varicella-zoster virus maycause oesophageal myenteric plexus damage, butour findings indicate that herpes simplex type 1virus and cytomegalovirus are unlikely to beinvolved.

Varicella-zoster virus is known to be neutro-tropic but may lie dormant in the tissues formany years before becoming reactivated as inherpes zoster. Whether this could be the case inthe oesophageal myenteric plexus is uncertainbut the much more gradual pattern of onset inachalasia indicates slowly progressive rather thanexplosive neural damage. Serial ganglion cellcounts in the myenteric plexus performed over aperiod oftime have not been reported but clinicalobservation suggests that the oesophageal motordisturbance is often steadily progressive,implying progressive neural damage.

In summary, both the serum antibody andDNA probe studies point to a relationshipbetween varicella-zoster virus infection andachalasia. Varicella lesions occur in the oeso-

phageal mucosa, which would give the virusready access to the oesophageal myentericplexus, but this route would seem more probableif the myenteric plexus elsewhere in the gut wereto be shown to be negative for varicella-zostervirus. Our findings suggest further studiesshould be undertaken to confirm and delineatethe extent of varicella-zoster virus involvementof neural tissue in achalasia of the cardia.

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2 Misiewicz JJ, Waller SL, Anthony PP, Gummer JWP.Achalasia of the cardia: pharmacology and histopathology ofisolated cardiac sphincteric muscle from patients with andwithout achalasia. QJ Med 1969; 38: 17-30.

3 Johnson HN. Visceral lesions associated with varicella. ArchPathol 1940; 30: 292-307.

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10 Bradstreet CMP, Taylor CED. Technique of complementfixation test applicable to the diagnosis of virus disease.Monthly Bulletin of the Ministry of Health and the PublicHealth Laboratory Service 1962; 21: 96-104.

11 Lewis FA, Griffiths S, Dunnicliff R, Wells M, Dudding N,Bird CC. Sensitive in-situ hybridisation technique usingbiotin-streptavidin polyalkaline phosphatase complex.J Clin Pathol 1987; 40: 163-6.

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14 Jones DB, Mayberry JF, Rhodes J, Munro J. Preliminaryreport ofan association between measles virus and achalasia.J Clin Pathol 1983; 36: 655-7.

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