- Email: [email protected]
The laboratory investigation of acute paralysis
Pttl,I. ttlth, I.,,ml. <1¢}71)85, 107-110
The Laboratory lnvest;=jation of Acute Paralysis DR T. H. FL.EWETT
,XHlu~V¢,~ in some tropical countries poliomyelitis is still common, it is not now in Great Britain the commonest cause of" acute paralysis by communicable disease. It has almost cumplctel~ been eliminated by vaccination of the population. But in every case of acute paralysis poliomyelitis virus must be looked for, if only to exclude it. If poliovirus is not found, investigation is still useful to establish the trim diagnosis: to be able for example to ~av n,q only "'potiovirus not isolated" but also "C.S.F. changes suggestive o f the GuiilainBarrc syndrome": or "'Coxsackie 13 virus isolated". The iirst question the physician will ask is: Is it polio or n o t ? The most useful specimens are: Cerebr,~-spinal fluid (l\)r a rapid differential diagnosis); two samples of faeces from ,,~vo successive stools, or rectal swabs if faeces are not available; and a throat swab; a n d clotted blood (for serum) taken as early as possible and again 10-14 days later. C..~.t.-. e.vammation. In the ea,'liest stages o f meningitis and myelitis due to enterovirus inl'cction polymorphonuclear cells may predominate; but after the first few days the cells are predominantly lymphocytes. Cell counts of 10--500/mm 3 are usual, but 2/mm3 does not reliably exclude. Protein concentration is usually raised, figures of 50-150 mg/100ml being usual: but ~ery high iigures (up to 500 mg/ml) are occasionally seen. The C.S.F. glucose concentration is not usually reduced. Chloride levels may or may not be reduced depending on whether the patient has been vomiting much. N o ~ a d a y s the most dilticul( differential diagnosis is acute polyneuritis--the GuillainBarrc syndrome no~' more c o m m o n than paralytic poliomyelitis. One might think that the clinical differential diagnosis should be easy; but the sensory changes in polyneuritis are not always ~:ery great and in very young children are almost impossible to detect. The C.S.F. picture of very high protein and normal cell count is helpful but not diagnostic; l haxc seen (liftecn years ago) bilateral flaccid paralysis in a ct|ild a few months old with C.S. I::. protein 250 rag/100 ml, cells 2/ram3; type 1 polio virus was isolated from the stools; the course of the disease was that of poliomyelitis. A n d in acute polyneuritis, despite Guillain's postulate, the C.S.F. protein m a y be within normal limits. Sera from 50-60]°o of patients with the Guillain-Barre syndrome at some stage of their illness contain complement-fixing antibodies for CNS extracts--especially spinal cord. But this reaction, though interesting, is not diagnostic and a negative test certainly does not exclude. Positives are, however, unusual in acute paralytic poliomyelitis. Although most useful aids, C.S.F. changes are not infallible guides to correct diagnosis. Isolation of poliovirus from the stools is by inoculation of tissue cultures o f cells o f primate origin, hun;an embryo kidney cells if available are best; m o n k e y kidney Cells are very useful. HeLa cells are useful for isolating p61ioviruses but not E C H O viruses. If the faeces contain m u c h virus, cytopathic effects (CPE) may be visible the next day, but if virus is scanty CPE may take up to 10 days to appear. Isolation from a throat swab is easit:st in the few days before paralysis sets in, and virus is often undetectable irt the throat by the time the paralysed patient has been admitted to hospital. Isolation from faeqes is not always possible: I have occasionally isolated poliovirus from the spinal cord after death when cultures from faeces yielded no ~irus. It is exceedingly rare to isolate poliovirus from the CSF of a paraiysed patient; isolates almost invariably turn out to be some other virus, identilication of an isolate is by showing that it is neutralized by a particmlar
]08
PUHI.I( ttt{AI.Ttl V()l.+ S5 N O . . l
antiserum. In practice an enterovirus isolate is ptlt tip agai(l~,t a pool el' antiser;i to each ~,!' the 3 polio types, a p{~ol against the 6 C'oxsackie B types, and a series of pin)b, against group, of the E C H O serotypes. When a ',~ool' is lotitld which neutralizes the \.itus+ the ~iru~ is put up against each individual a,ltiserun't to litld out which antiserum in the r+t~ol is doing the neutralizing. If a virus is isolated from a paticl~t with acute paralysis one v, nuld put it up immediately to each of the 3 serotypes of poliovirus separatel3. Occasionally two viruses may be presen! togelher in the faeces tit" a paralysed paliem. one a polio'eirus and the other an echovirus or Coxsackie A. The ech,)virus m:ty g r c ~ t'astcr than the poliovirus and so mask the latter. If the st,.ml is again inocutat~.'d int,~ tissue ctdttllc in the presence of the appropriate echovirus antiserum, the p~lio',irus ~ill {ht.'n Ive dctucted. This is always worth doing i[the expected virus ix not isolated from a badly paralyzed patmnt. Double infections o f this kind are not uncomNton in sonic tropical :ountrics. at+d l+a~c been encountered in Great Britain. Isolation o f a poliovirus used to be almost diagnostic. Hut m+waday:- isolation of xacuit~c strains is frequent, and presents a considerable diagnostic problem. I~xa',]lple: ~t box .'.l~,cd 2, was in hospital with tuberculosis of his spine. He .,,uddcnty deveh~pcd bilateral [taccid paralysis of his legs. Sensory loss could not be detected '~ith contidence. Lumbar puncture was excluded lk~r lear of provoking a T.B. meningitis. Poliovirus type I1t t~as i_,;otated from his faeces. He had never been given poliovaccine, nor had his family or ~isitors. Smgicat exploration revea]ed a tuberculoma pressing on the cord. and the viru.,, ',',as timttl~ identitied as a vaccine strain. The source of the vaccine ~,irtls was probably a ]~ur.,,e immunized not long previously at another ~lospital where she had been working. It i.', still import,tnt tu characterize such isolates, not only to elucidate the aetiology o l the paral.v.,,is, but especially to make sure that vaccine strains are not reverting to virulence and causing paralysis. We are now much more confident about their stability, but surveillance is still neces~,ar.v. Th.c dilferentiatJon between "wild" strains and vaccine strains is on the basis o1' genetic markers. These are : The 'V' (virulence) marker; wild strains are virulent for monkey CNS. The +rct 40' (temperature) marker; the titre of vaccine strains in tissue culture at 40 is much lower than at 37"; wild strains titre the same in both. The *dextran' marker: vaccine strains grow poorly in a medium containing dextran. The 'serological' marker; there is a serological dilference between wild and vaccine strains. The 'A' (aluminium chloride) marker; vaccine strains are more stable at 50 in presence o f aluminium chloride. The +M.S,+ marker; vaccine strains grow poorly in M.S. cells. Theo~d" marker; vaccine strains give a low plaque c o u r t in a medium ol'a low bicarbonate content. All these markers may be of value: the most useful are the rct 40 and serological markers. and for type 1 isolates the dextran marker also. When strains are clearly 'wild' ".~r clearly "vaccine' there is no great difficulty; but sometimes strains of intermediate belmviour are encountered and these may be dil~cutt to place with confidence. The neurovirulence test in monkeys is the final arbiter, but itS, ~.ost ensures that it is only done as a last resort. These tests are time-consuming and tedious and require a practised hand. In England they are only done for diagnosis by the Central Public Health Laboratory, though firms producing vaccines o.1"course have their own facilities for controlling their products. Other enleroviruses may als() cause paralysis. Coxsackie A7, first detected as causiw+g clinical poliomyelitis in the southern I;.S.S.R. appeared again nearer home i,t Glasgow in 1959. The virus is i.,,olated ~ml\ in suckling mice, in which it causes a widespread myositis. Extracts of the rnou~c carcas~,es agglutinate "vaccinia--positive' avian erythrocytes, i.e.
TttI! I . A B ( ) R A T O R Y INVI:!SI"IGATION O F A C U T E P A R A L Y S I S
I09
crvlhroc,,te,, !,'~m~ these hens whose erythrocytes are also agglutinated by vaccinia virus. The inlMctivitv I~r suckling mic~ and the haemagglutination are both specilically neutralized by the a)~propriate antiserum. Fortunately outbreaks due to this virus appear to be rare: m~ xacdm: is av,~ilable as ",el. (',~xsackic Ag. (.'oxsackic B strains, especially B5, and some E C H O serotypes, especially (, and 9. m'.~v ai~,~ cause lo~er rotator neurone paralysis, but this is usually very mild and 1r~t tt %i,,211t.
These ~iruscs c.'i~ all frequently be isolated from tile C.S.F. Coxsackie A strains other than .\ 7 ~omctimcs .:re is~Hated together uith poliovirus: according to D a t l d o r f t h e double i~Hcc~ion prcdiyposes t~., a lm~re severe paralysis than infection by poliovirus alone. [)iagm,si:, ~1 infection by these viruses can be established retrospectively by tinding antib~.~dics ill lht: con\alc>c.21~l s e r u m ~v)lich w e r e n o t present in the acute phase serum. Tile complement I}\ation test. easy and quick, is useful for mumps, but of very doubtful value ~n p~diomyclit~s dIld t,sc]ess for the other enteroviruses. The neutralization test is precise and ~pctitic. i~tlt (unless the cntcr(}virus has a good haemagglutinin)is tedious, time-con~unllng 'dmi e~pcnsixc. I~ i-, ~H'vatue t'~w special epidemiological investigations, e.g. for assessi~+g herd immunity but of limited diagnostic application. ()thor ~iruscb ,)cc:~,ionaltv causing acute paralysis are varicella-zoster and tile m o n k e y W ~iru, (herpc~',iru~ simiae). In zoster, virus particles may be detectable by electron m i c r ~ ' ~ p > it, the centrifuged deposit of the C.S.V.. and complement fixing antibodies are u,,u~)l ~, lt~und in high ~itre. Virus can be isolated from vesicle fluid in tissue culture. The B ,ru~ uaH bc is,>latcd from (,'S.I., or brain and other organs at autopsy, in tissue cultures of m~mke5 and rabbit kidne5 cells. .~¢IIH(" (H/t~'g [¢ll'Yll.~ ~P['(t('gll( ~ D(lt'a]!'.~'iS
,\nothcr "()td Ptaguc" nol on the programme and regrettably still with us, namely )c!~ro~.~. can also cause acute parab:sis, perhaps more commonly than is generally recognized. (.arc!u) clinical examination, provided the diagnosis has been thought of. will lead to ~crapings from misal scptum or nicked ear lobe, and perhaps a biopsy reaching the ]aboralor}. Acute paralysis is usually associated with an acute lepromatous exacerbation of the "intermediate" form of leprosy, and c,rganisms in smears or sections stained by Fire's method can be found by a persistent pathologist. In a properly taken skin biopsy extending ~ell dox~'n into tilt: dermis, cpithelioid infiltration of nerve fibres is diagnostic. Myasthenia grav;s can sometimes present in the guise o f acute bulbar palsy. Clinical suspicion leads to a therapeutic test with prostigmine, the )aboratory can confirm by finding, by immunofluorescence, antibodies reacting with striated muscle. In one-thirct of all cases o f MuIfiple sclerosis the first s y m p t o m is.weakness of one or both lower limbs. Tile C.S.F. may contain raised protein and show pleocytosis. There is no spediic laboratory test. The commonest cause of acute paralysis is of ccoarse a stroke of some kind. Acute hemiplegia can occur in children especially in association with whooping cough. But in these and the many forms of acute paralysis not mentioned above the clinical findings are o f more value than the virological
References LkNNISTIE, E. K. & SCttMIi)I, N . . l , (!969). Diagnostic Procedures for I/iral and Victecltsial Diseases.
New York: American Public 14ealth Association, inc. Gt~isr, N. R. (1962). Type A7 coxsackie (type 4 poliomyelitis) virus infection in Scotland. Hyg., Camb. 60, 323-332.
l l(J
PLJI+I.I(" I l I I A I + ' I I I V. +
(;1{1~,1, ]'~. J'~. ,~ IIJ-iZ. Ii. J. (197()),
I:lllel~)~.il;.~i ~,:tlt)J,.)~)' t)l+ Ih¢ I'~;.tl':tl)'ti': I)<)li(:',l].".cilti'-. ~)'t]/irt:,lll,.'
( ' l i t I i I ~ N I , JL,. ( i . ++~ |')Ax, L~, 'l'. I . (l<)~+4). l.cl+r+~.+,'( I'H 17+'<'++r~' ++++d l+'r~:+~lJt', ", 111+ 2-]+~, ~7(>. l'hi,~tt+I: J~tl++l+ W l l ~ h t ,~ S~}I+': l.l~l. PVll.i ml+ I':., ~. (". ( J¶)f>+~J..-++N +~.t'+,+', t)l + tit+ (;tlilJ[zli+-l:~+li're '+).Itt.ht:+it]+: ~,~] il+~1111Ll11~,It~+i~+':ll ~ftld)'. lip ~+I+,,/. ,I. i .~++,~. NAI+.~N, l~+. ('. (l-d.)~']Y+FI+))t I//l~.++ll'.+l('L'Io+f l*l¢++l+t'l'lt ,~Jll',ll'+~lt,/l,~+, .+J'+.J cdil+(]i'l, l)J'~. 2NN ~). li(+~+h~++h ;t:+d l..tJl+dl, H1'. J . t~. ~+ I.i~,'HI~,L~HIC+
The Laboratory lnvest;=jation of Acute Paralysis DR T. H. FL.EWETT
,XHlu~V¢,~ in some tropical countries poliomyelitis is still common, it is not now in Great Britain the commonest cause of" acute paralysis by communicable disease. It has almost cumplctel~ been eliminated by vaccination of the population. But in every case of acute paralysis poliomyelitis virus must be looked for, if only to exclude it. If poliovirus is not found, investigation is still useful to establish the trim diagnosis: to be able for example to ~av n,q only "'potiovirus not isolated" but also "C.S.F. changes suggestive o f the GuiilainBarrc syndrome": or "'Coxsackie 13 virus isolated". The iirst question the physician will ask is: Is it polio or n o t ? The most useful specimens are: Cerebr,~-spinal fluid (l\)r a rapid differential diagnosis); two samples of faeces from ,,~vo successive stools, or rectal swabs if faeces are not available; and a throat swab; a n d clotted blood (for serum) taken as early as possible and again 10-14 days later. C..~.t.-. e.vammation. In the ea,'liest stages o f meningitis and myelitis due to enterovirus inl'cction polymorphonuclear cells may predominate; but after the first few days the cells are predominantly lymphocytes. Cell counts of 10--500/mm 3 are usual, but 2/mm3 does not reliably exclude. Protein concentration is usually raised, figures of 50-150 mg/100ml being usual: but ~ery high iigures (up to 500 mg/ml) are occasionally seen. The C.S.F. glucose concentration is not usually reduced. Chloride levels may or may not be reduced depending on whether the patient has been vomiting much. N o ~ a d a y s the most dilticul( differential diagnosis is acute polyneuritis--the GuillainBarrc syndrome no~' more c o m m o n than paralytic poliomyelitis. One might think that the clinical differential diagnosis should be easy; but the sensory changes in polyneuritis are not always ~:ery great and in very young children are almost impossible to detect. The C.S.F. picture of very high protein and normal cell count is helpful but not diagnostic; l haxc seen (liftecn years ago) bilateral flaccid paralysis in a ct|ild a few months old with C.S. I::. protein 250 rag/100 ml, cells 2/ram3; type 1 polio virus was isolated from the stools; the course of the disease was that of poliomyelitis. A n d in acute polyneuritis, despite Guillain's postulate, the C.S.F. protein m a y be within normal limits. Sera from 50-60]°o of patients with the Guillain-Barre syndrome at some stage of their illness contain complement-fixing antibodies for CNS extracts--especially spinal cord. But this reaction, though interesting, is not diagnostic and a negative test certainly does not exclude. Positives are, however, unusual in acute paralytic poliomyelitis. Although most useful aids, C.S.F. changes are not infallible guides to correct diagnosis. Isolation of poliovirus from the stools is by inoculation of tissue cultures o f cells o f primate origin, hun;an embryo kidney cells if available are best; m o n k e y kidney Cells are very useful. HeLa cells are useful for isolating p61ioviruses but not E C H O viruses. If the faeces contain m u c h virus, cytopathic effects (CPE) may be visible the next day, but if virus is scanty CPE may take up to 10 days to appear. Isolation from a throat swab is easit:st in the few days before paralysis sets in, and virus is often undetectable irt the throat by the time the paralysed patient has been admitted to hospital. Isolation from faeqes is not always possible: I have occasionally isolated poliovirus from the spinal cord after death when cultures from faeces yielded no ~irus. It is exceedingly rare to isolate poliovirus from the CSF of a paraiysed patient; isolates almost invariably turn out to be some other virus, identilication of an isolate is by showing that it is neutralized by a particmlar
]08
PUHI.I( ttt{AI.Ttl V()l.+ S5 N O . . l
antiserum. In practice an enterovirus isolate is ptlt tip agai(l~,t a pool el' antiser;i to each ~,!' the 3 polio types, a p{~ol against the 6 C'oxsackie B types, and a series of pin)b, against group, of the E C H O serotypes. When a ',~ool' is lotitld which neutralizes the \.itus+ the ~iru~ is put up against each individual a,ltiserun't to litld out which antiserum in the r+t~ol is doing the neutralizing. If a virus is isolated from a paticl~t with acute paralysis one v, nuld put it up immediately to each of the 3 serotypes of poliovirus separatel3. Occasionally two viruses may be presen! togelher in the faeces tit" a paralysed paliem. one a polio'eirus and the other an echovirus or Coxsackie A. The ech,)virus m:ty g r c ~ t'astcr than the poliovirus and so mask the latter. If the st,.ml is again inocutat~.'d int,~ tissue ctdttllc in the presence of the appropriate echovirus antiserum, the p~lio',irus ~ill {ht.'n Ive dctucted. This is always worth doing i[the expected virus ix not isolated from a badly paralyzed patmnt. Double infections o f this kind are not uncomNton in sonic tropical :ountrics. at+d l+a~c been encountered in Great Britain. Isolation o f a poliovirus used to be almost diagnostic. Hut m+waday:- isolation of xacuit~c strains is frequent, and presents a considerable diagnostic problem. I~xa',]lple: ~t box .'.l~,cd 2, was in hospital with tuberculosis of his spine. He .,,uddcnty deveh~pcd bilateral [taccid paralysis of his legs. Sensory loss could not be detected '~ith contidence. Lumbar puncture was excluded lk~r lear of provoking a T.B. meningitis. Poliovirus type I1t t~as i_,;otated from his faeces. He had never been given poliovaccine, nor had his family or ~isitors. Smgicat exploration revea]ed a tuberculoma pressing on the cord. and the viru.,, ',',as timttl~ identitied as a vaccine strain. The source of the vaccine ~,irtls was probably a ]~ur.,,e immunized not long previously at another ~lospital where she had been working. It i.', still import,tnt tu characterize such isolates, not only to elucidate the aetiology o l the paral.v.,,is, but especially to make sure that vaccine strains are not reverting to virulence and causing paralysis. We are now much more confident about their stability, but surveillance is still neces~,ar.v. Th.c dilferentiatJon between "wild" strains and vaccine strains is on the basis o1' genetic markers. These are : The 'V' (virulence) marker; wild strains are virulent for monkey CNS. The +rct 40' (temperature) marker; the titre of vaccine strains in tissue culture at 40 is much lower than at 37"; wild strains titre the same in both. The *dextran' marker: vaccine strains grow poorly in a medium containing dextran. The 'serological' marker; there is a serological dilference between wild and vaccine strains. The 'A' (aluminium chloride) marker; vaccine strains are more stable at 50 in presence o f aluminium chloride. The +M.S,+ marker; vaccine strains grow poorly in M.S. cells. Theo~d" marker; vaccine strains give a low plaque c o u r t in a medium ol'a low bicarbonate content. All these markers may be of value: the most useful are the rct 40 and serological markers. and for type 1 isolates the dextran marker also. When strains are clearly 'wild' ".~r clearly "vaccine' there is no great difficulty; but sometimes strains of intermediate belmviour are encountered and these may be dil~cutt to place with confidence. The neurovirulence test in monkeys is the final arbiter, but itS, ~.ost ensures that it is only done as a last resort. These tests are time-consuming and tedious and require a practised hand. In England they are only done for diagnosis by the Central Public Health Laboratory, though firms producing vaccines o.1"course have their own facilities for controlling their products. Other enleroviruses may als() cause paralysis. Coxsackie A7, first detected as causiw+g clinical poliomyelitis in the southern I;.S.S.R. appeared again nearer home i,t Glasgow in 1959. The virus is i.,,olated ~ml\ in suckling mice, in which it causes a widespread myositis. Extracts of the rnou~c carcas~,es agglutinate "vaccinia--positive' avian erythrocytes, i.e.
TttI! I . A B ( ) R A T O R Y INVI:!SI"IGATION O F A C U T E P A R A L Y S I S
I09
crvlhroc,,te,, !,'~m~ these hens whose erythrocytes are also agglutinated by vaccinia virus. The inlMctivitv I~r suckling mic~ and the haemagglutination are both specilically neutralized by the a)~propriate antiserum. Fortunately outbreaks due to this virus appear to be rare: m~ xacdm: is av,~ilable as ",el. (',~xsackic Ag. (.'oxsackic B strains, especially B5, and some E C H O serotypes, especially (, and 9. m'.~v ai~,~ cause lo~er rotator neurone paralysis, but this is usually very mild and 1r~t tt %i,,211t.
These ~iruscs c.'i~ all frequently be isolated from tile C.S.F. Coxsackie A strains other than .\ 7 ~omctimcs .:re is~Hated together uith poliovirus: according to D a t l d o r f t h e double i~Hcc~ion prcdiyposes t~., a lm~re severe paralysis than infection by poliovirus alone. [)iagm,si:, ~1 infection by these viruses can be established retrospectively by tinding antib~.~dics ill lht: con\alc>c.21~l s e r u m ~v)lich w e r e n o t present in the acute phase serum. Tile complement I}\ation test. easy and quick, is useful for mumps, but of very doubtful value ~n p~diomyclit~s dIld t,sc]ess for the other enteroviruses. The neutralization test is precise and ~pctitic. i~tlt (unless the cntcr(}virus has a good haemagglutinin)is tedious, time-con~unllng 'dmi e~pcnsixc. I~ i-, ~H'vatue t'~w special epidemiological investigations, e.g. for assessi~+g herd immunity but of limited diagnostic application. ()thor ~iruscb ,)cc:~,ionaltv causing acute paralysis are varicella-zoster and tile m o n k e y W ~iru, (herpc~',iru~ simiae). In zoster, virus particles may be detectable by electron m i c r ~ ' ~ p > it, the centrifuged deposit of the C.S.V.. and complement fixing antibodies are u,,u~)l ~, lt~und in high ~itre. Virus can be isolated from vesicle fluid in tissue culture. The B ,ru~ uaH bc is,>latcd from (,'S.I., or brain and other organs at autopsy, in tissue cultures of m~mke5 and rabbit kidne5 cells. .~¢IIH(" (H/t~'g [¢ll'Yll.~ ~P['(t('gll( ~ D(lt'a]!'.~'iS
,\nothcr "()td Ptaguc" nol on the programme and regrettably still with us, namely )c!~ro~.~. can also cause acute parab:sis, perhaps more commonly than is generally recognized. (.arc!u) clinical examination, provided the diagnosis has been thought of. will lead to ~crapings from misal scptum or nicked ear lobe, and perhaps a biopsy reaching the ]aboralor}. Acute paralysis is usually associated with an acute lepromatous exacerbation of the "intermediate" form of leprosy, and c,rganisms in smears or sections stained by Fire's method can be found by a persistent pathologist. In a properly taken skin biopsy extending ~ell dox~'n into tilt: dermis, cpithelioid infiltration of nerve fibres is diagnostic. Myasthenia grav;s can sometimes present in the guise o f acute bulbar palsy. Clinical suspicion leads to a therapeutic test with prostigmine, the )aboratory can confirm by finding, by immunofluorescence, antibodies reacting with striated muscle. In one-thirct of all cases o f MuIfiple sclerosis the first s y m p t o m is.weakness of one or both lower limbs. Tile C.S.F. may contain raised protein and show pleocytosis. There is no spediic laboratory test. The commonest cause of acute paralysis is of ccoarse a stroke of some kind. Acute hemiplegia can occur in children especially in association with whooping cough. But in these and the many forms of acute paralysis not mentioned above the clinical findings are o f more value than the virological
References LkNNISTIE, E. K. & SCttMIi)I, N . . l , (!969). Diagnostic Procedures for I/iral and Victecltsial Diseases.
New York: American Public 14ealth Association, inc. Gt~isr, N. R. (1962). Type A7 coxsackie (type 4 poliomyelitis) virus infection in Scotland. Hyg., Camb. 60, 323-332.
l l(J
PLJI+I.I(" I l I I A I + ' I I I V
(;1{1~,1, ]'~. J'~. ,~ IIJ-iZ. Ii. J. (197()),
I:lllel~)~.il;.~i ~,:tlt)J,.)~)' t)l+ Ih¢ I'~;.tl':tl)'ti': I)<)li(:',l].".cilti'-. ~)'t]/irt:,lll,.'
( ' l i t I i I ~ N I , JL,. ( i . ++~ |')Ax, L~, 'l'. I . (l<)~+4). l.cl+r+~.+,'( I'H 17+'<'++r~' ++++d l+'r~:+~lJt', ", 111+ 2-]+~, ~7(>. l'hi,~tt+I: J~tl++l+ W l l ~ h t ,~ S~}I+': l.l~l. PVll.i ml+ I':., ~. (". ( J¶)f>+~J..-++N +~.t'+,+', t)l + tit+ (;tlilJ[zli+-l:~+li're '+).Itt.ht:+it]+: ~,~] il+~1111Ll11~,It~+i~+':ll ~ftld)'. lip ~+I+,,/. ,I. i .~++,~. NAI+.~N, l~+. ('. (l-d.)~']Y+FI+))t I//l~.++ll'.+l('L'Io+f l*l¢++l+t'l'lt ,~Jll',ll'+~lt,/l,~+, .+J'+.J cdil+(]i'l, l)J'~. 2NN ~). li(+~+h~++h ;t:+d l..tJl+dl, H1'. J . t~. ~+ I.i~,'HI~,L~HIC+