laboratory diagnosis of virus infections …jcp.bmj.com/content/jclinpath/1/1/2.full.pdfthe...

1. clin. Path. (1947), 1, 2.

THE LABORATORY DIAGNOSIS OF VIRUSINFECTIONS OF MAN: A REVEW

BY

S. P. BEDSONLondon Hospital and Medical College

(RECEIVED FOR PUBLICATION, JULY 25, 1947)

The last fifteen years have seen a considerableadvance in our knowledge of viruses. Admittedlyit is still impossible to say whether or not virusesare living micro-organisms: there is even somedoubt as to whether all those agents which we grouptogether as viruses are of the same nature. But,interestixqg though these particular problems are,they have no direct bearing on the practical questionof the diagnosis and control of virus disease, sincefor these purposes viruses can be regarded asmicro-organisms. And the inability to answer thesequestions unequivocally does not mean that in otherrespects our knowledge of viruses has not madegreat strides. Many of the larger viruses have beenstained and seen with the ordinary microscope, andthe greater power of microscopic resolution obtain-able by the use of ultra-violet light and a quartzoptical system comprising a dark-ground condenser-an advantage which we owe largely to the workof J. E. Barnard in this country-has made possiblethe photography ofthese large viruses in an unstainedand unaltered state, and has also brought some ofthe smaller viruses within the range of visibility.Furthermore, the introduction of the electronmicroscope, with its still greater powers ofresolution,enables us to see even the smallest virus.We are no longer confined to the use of animals

for the cultivation of viruses. The adaptation of thetechnique of tissue culture to this purpose, and theuse of the developing hen's or duck's egg-whichwe owe to the ingenuity of the American pathologistGoodpasture-have provided us with simple meansfor growing many of the animal viruses in thelaboratory. And when we turn to the question ofthose serological reactions, the precipitin, agglutina-tion, and complement fixation tests which have beenof such invaluable service in the study of bacteriaand bacterial disease, we find that, contrary to whatwas af one time thought, these methods are applic-able to virus work. It is the purpose of this article

to consider some of this recent work and its applica-tion to the diagnosis of diseases of man of virusaetiology.

MicroscopyDemonstration of viruses by means of the

microscope.-Sixty-one years ago Buist announcedthat he had succeeded in demonstrating particleswith the appearance of minute cocci in smears ma.defrom vaccinial lesions (see Gordon, 1937); heconcluded that these were the virus. As a recentAmerican writer has remarked: "Buist was a manfar ahead of his time; his contemporaries were notmerely sceptical, they were not even interested."In 1904 Borrel published a paper on the virus offowlpox, in which he showed that, by the use ofLoeffler's flagella stain, minute coccal bodies couldbe demonstrated in smears of virulent material, andhe considered these to be the virus. Two years laterPaschen of Hamburg, using a very similar techniqueto Borrel, rediscovered the virus of vaccinia.Although this work did not pass unnoticed it didnot receive general acceptance, in fact most bac-teriologists adopted a sceptical attitude which ittook nearly twenty years to dispel. The reason forthis scepticism was due largely to the fact that thestaining methods then employed, although producingadmirable preparations in the hands of the expert,gave rise to artefacts bearing a remarkably closeresemblance to stained virus particles, and thetendency to dismiss all these appearances as artefactswas all too readily adopted.

Since then, improved methods of staining and thedevising of technical procedures for the separationof virus from the non-specific material in the crudesuspensions of virulent material have made itpossible to show that the particles one can stain andsee in smear preparations are, in fact, virus. In thiswork, fractional centrifugation has played animportant part. Saline suspensions of virulent

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LABORATORY DIAGNOSIS OF VIRUS INFECTIONS

material are first centrifuged at a relatively lowspeed to throw down gross particles of tissue, andthe resulting supernatant is then spun at a speedsufficient to deposit the virus. After removing thesupernatant fluid, the deposit is resuspended insaline and centrifugation, first at a low speed andthen at a high speed, is repeated. A deposit isfinally obtained which consists very largely ofparticles resembling in size and staining propertiesthose seen in the smears made from the originalvirulent material and thought to be virus, and thedemonstration that the virulence of the originalsuspension has moved with the particles and thatthey react specifically with a known antiserumcompletes the proof that virus and particles are oneand the same. There is, therefore, no doubt thatthe larger viruses can be seen with the microscope.They look like minute micro-organisms, and thisconception of their nature finds support in thedark-ground photomicrographs that Barnard hasproduced of unfixed and unstained viruses.

Turning now to the practical application of thiswork to the diagnosis of virus disease, it is obviousthat direct microscopy can be of use only in thecase of the large viruses. Even here the limitationsof the method are greater than in bacterial infections.The largest viruses are so much smaller than bacteriathat they can be detected only if they are present inconsiderable numbers, and the occasions when theiridentity can be assumed because they have been seenin smear preparation are very few. However, themethod is not without value and it finds its useparticularly in the diagnosis of infections due tothat group of large viruses which are distinguishedfrom the others by staining by Castaneda's orMacchiavello's methods. For that matter, thisgroup possesses other characteristics that mark itoff from the rest: the viruses are larger, and whenthey multiply they present a regular sequence ofmorphological change, recalling those changes insize and shape which a bacterium often undergoeswhen multiplying. This group contains amongstothers the agents of trachoma, inclusion conjunc-tivitis or inclusion blenorrhoea, lymphogranulomavenereum, and psittacosis. The use of microscopyin confirming the diagnosis of trachoma, particularlyin its early stages, is well known. Halberstaedterand von Prowazek (1907) were first to demonstratethe characteristic cytoplasmic inclusions in theconjunctival epithelium in this condition, and themore recent work of Thygeson and Proctor (1935)has shown that the particles which occur both freeand inside the cells and which are readily stained byCastaneda or Giemsa are, in fact, the virus.

Inclusion conjunctivitis.-Infection with the closelyrelated virus of inclusion conjunctivitis is of greaterinterest to us, since it is of commoner occurrencethan trachoma in this country, This virus ismorphologically indistinguishable from that oftrachoma; the exact relationship of the two, thoughobviously close, is yet to be determined. Like thetrachoma virus, it is a parasite of man and is foundproducing infection of the conjunctiva and themucous membrane of the cervix and the maleurethra. Cells infected with this virus presentinclusions which in situation, structure, and stainingproperties bear an extremely close resemblance tothose of trachoma. They consist of masses of virusparticles held together by inclusion material whichis basophilic and scanty in amount. These inclusionsdiffer, therefore, from what one might call the"typical" virus inclusion, in which the inclusionmaterial is acidophil and present in such amount ascompletely to mask the virus, and it is this scantinessof inclusion material in the inclusions produced bythe group of Castaneda-positive viruses which hasmade it so much easier to study and recognize theirtrue structure. Like the other members of thisgroup, the virus of inclusion conjunctivitis showsthe sequence of developmental forms to whichreference has already been made. The large forms,which are almost the size of staphylococci, have beencalled by Lindner (1910) "initial bodies," since inhis opinion, they represent the initial stage in thedevelopment of the virus, a view which is fullysupported by recent work. As the virus multiplies-and all appearances suggest that it does so by simpledivision-smaller and smaller forms are produceduntil, when the colony is fully grown and the cell isready to break down and discharge its virus content,all the virus is in the form of elementary bodies.

Inclusion conjunctivitis occurs either as a neonatalinfection, when it is acquired in precisely the sameway as gonococcal ophthalmia, or in older childrenor adults, when it is often called swimming-bathconjunctivitis. Both forms are associated withinflammation of the conjunctiva and some dis-charge; scattered follicles may be present. Diagnosisis made by the microscopy of conjunctival scrapingssuitably stained, and some idea of the incidence ofthe neonatal form can be gathered from a recentpaper by Sorsby and others (1944), who foundtwenty-seven cases in 269 of all forms of conjunc-tivitis in infants.

Cervicitis and urethritis.-Infection of the mucosaof the female genital tract is usually silent, but it canbe revealed by the examination of stained smears ofcervical scrapings. In the male, infection of the

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urethra takes a subacute form which may becomechronic and persist for months; this infection of thegenital tract is transmitted by sexual intercourse and,although it undoubtedly occurs in this country, itappears to be uncommon.

Psittacosis.-The virus of psittacosis is anotherthat can be recognized microscopically; and,although it is not suggested that direct microscopy isof value as a diagnostic procedure in human infec-tions with this virus, it is undoubtedly of use in thecase of infection of birds. It is often possible tofind the virus in smears made from the spleen, whichis commonly enlarged, or with material from the airsacs, where infection often resides, and so to makean early diagnosis.Lymphogranuloma venereum.-Lymphogranuloma

venereum virus, another member of the Castaneda-positive group, can also be detected in suitablystained smears of the pus from the inguinal buboeswhich occur in the male and when the primary lesionis penile, or when it is situated in the anterior partof the genital tract of the female. Though possiblyworth making, this investigation has not the samevalue in the diagnosis oflymphogranuloma venereumas isolation of the virus, the Frei test, or the com-plement fixation reaction.

Smallpox. -As a further example of the diagnosticvalue of the microscopical demonstration of virus,mention might be made of its use in smallpox. Thisvirus is ofa size readily visible under the microscope,and a number of staining methods are available forits demonstration, sbme of which, such as the alka-line methyl-violet method of Gutstein (1937), arequite simple. The virus is present in quantity in theskin lesions and, as van Rooyen and Illingworth(1944) have shown, microscopic examination ofsuitably stained smears made from scrapingsobtained from the base of papules or vesicles can beof considerable diagnostic value. In the cutaneouslesions due to herpes febrilis, zoster, or varicella, thevirus particles, though present and staining by themethods applicable to variola, are neither sonumerous nor so large as variola virus. VanRooyen and Illingworth used Paschen's stainingmethod. Commenting on this diagnostic procedure,Downie (1946) states a preference for Gutstein'sstain and points out that smears made from pustularlesions or crusts are unreliable, that a negative resultdoes not exclude smallpox, and that the test wouldnot differentiate between the lesions of smallpoxand vaccinia.Dem ra oof specic histological

Quite apart from the possibility of demonstrating

the virus itself, there are, in many virus diseases,demonstrable histological changes in the infectedcells which are sufficiently distinctive to be of diag-nostic value. These are the inclusion bodies foundin the cytoplasm or the nucleus of the affected cells,bodies of varying size, usually homogeneous inappearance and acidophil in staining reaction, whichmay come to occupy the major part of the nucleusor a considerable part of the cell's cytoplasm.Doubt still exists as to the exact nature of thenuclear inclusions, but it has been shown that manycytoplasmic inclusions are virus colonies, masses ofvirus embedded in a matrix of homogeneousmaterial. It is this latter material which gives tomany of these inclusions their appearance ofhomogeneity and affinity for acid dyes.- In the caseof the cytoplasmic inclusions produced by theCastaneda-positive viruses, inclusion material is ininsufficient quantity to mask the virus particles, andthese can usually be readily seen either in Castaneda-or Giemsa-stained preparations. Even where theinclusions appear homogeneous when fixed andstained, it is still possible, as Barnard has shown inthe case of ectromelia, to demonstrate the truenature of the inclusion by using dark-groundillumination. The difference in refractility ofvirus andinclusion material shows up the former as bright dots.At one time it was thought that only infection

with a virus could produce inclusion bosdies, but itis now known that very similar appearances can beproduced experimentally by other means. Thisapplies particularly to nuclear inclusions. None theless, the finding of inclusion bodies can be taken aspresumptive evidence of a virus infection, and insome instances their demonstration has diagnosticvalue. The finding of Negri bodies, the acidophilcytoplasmic inclusions produced by rabies virus innerve cells, confirms the diagnosis of suspectedrabies in the dog. And mention has already beenmade of the demonstration of the Halberstaedter-Prowazek bodies in trachoma and the -similarinclusions in inclusion conjunctivitis, cervicitis, andurethritis as diagnostic procedures. Paul's test forsmallpox, which depended on the production oflesions in the rabbit's cornea characterized bycytoplasmic inclusions, has been largely supersededby the demonstration of the infective agent in theskin lesions either by direct microscopy or by thecomplement fixation test.

Isolation of the VirusIf the application of microscopy to the diagnosis

of virus infections has been dealt with at some

length this has been done deliberately in order to

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emphasize that, contrary to common belief, manyviruses are sufficiently large to be seen and recog-nized by means of the microscope. The limitationsof this procedure have not, however, beeni lost tosight, and, in virus infections as in any other infec-tive process, the aim of the laboratory should bethe isolation and identification of the infective agent.Unfortunately this is less easy to achieve than inbacterial disease. Not only is the isolation andidentification of a virus a lengthy and often difficultprocedure, involving the infection of animals orembryonated eggs, the study of the histologicallesions produced by this infection, and the applica-tion of serological tests usually in the form of theneutralization reaction, but there are quite a numberof viruses infecting man for which a suitableexperimental animal has not been found. Theviruses of zoster, varicella, and infective hepatitisexemplify this: to them, man alone would seem tobe susceptible. Or it may be that the only suscep-tible species of animal is so expensive as to precludeits routine use; it is this factor, probably more thanany other, which has delayed our understanding ofpoliomyelitis and its epidemiology. Even where avirus can be isolated by the use of the incubated eggor an inexpensive animal, the time taken to establishand identify it is too long to make the procedure ofimmediate diagnostic importance. That does notmean that every attempt should not be made toisolate the agent of a suspected virus infection,because this should, of course, always be done wherethe diagnosis is in doubt. The following examplesillustrate some of the commoner uses of this formof investigation.Egg inoculation.-It is at times important to

know whether an outbreak of clinical influenza isdue to on¢ of the known influenza viruses and, ifso, which one. This might be necessary, for instance,at the commencement of an epidemic when prophy-lactic immunization is contemplated; and, thoughat one time the ferret would have been chosen as themost suitable animal for the purpose, probably theincubated hen's egg would now be the choice.Hirst (1942 and 1945) has found the injection ofnaso-pharyngeal washings into the amniotic sac ofthirteen-day eggs slightly superior to ferret inocula-tion for the isolation of influenza A virus, anobservation confirmed by Burnet and others (1942).And Beveridge and others (1944) employed the samemethod successfully for the isolation of influenza Bvirus. The combination of penicillin and sulpha-diazine with the crude washings does away with thenecessity for filtration. The eirbryonic membranesof the developing hen's or duck's egg-the amniotic,

allantoic, and yolk sacs and the chorio-allantois-provide admirable means for the cultivation of anumber of viruses to which man is susceptible; inaddition to influenza A and B, the viruses of herpessimplex, psittacosis, lymphogranuloma venereum,mumps, measles, lymphocytic meningitis, andsmallpox can all be grown on one or other of thesemembranes. Incidentally, North and others (1944)utilized inoculation of the chorio-allantois withsuspensions of skin crusts in the diagnosis ofsmallpox, a procedure the value of which has beenfully confirmed by Downie (1946) and Downie andDumbell (1947).

Value of animal inoculation.-Invaluable thoughthe egg technique has proved in virus work, it has byno means superseded the use of experimentalanimals, for there are occasions when only thelatter are suitable to the work in hand. This is true,for instance, of poliomyelitis, where inoculation ofthe monkey is our only means of demonstrating thepresence of the virus. Even in those cases wherethe virus sought will infect both eggs and animalsit is advantageous to use both; it increases thechance of success and it may well be that the diseaseproduced in the animal is more distinctive than thechanges resulting from infection of the egg. Thelatter may, in fact, be so inconspicuous as to maketheir detection difficult and uncertain, as in infectionof the chorio-allantois with the virus of lymphocyticchorio-meningitis. Or, as recent work on rinderpesthas shown (Shope and others, 1946), a virus maymultiply within the developing egg withoutproducingany detectable naked-eye or histological changes oreven death of the embryo.

Meningitis.-So where both egg and animal areavailable for our purpose we use both. This is true,for instance, in the investigation of acute asepticmeningitis, a syndrome due to a number of viruses,some of which have been identified. Of these, thevirus of lymphocytic choriomeningitis of Armstrongand Lillie is responsible for about one-third of cases.At any rate, that would appear to be so in NorthAmerica, though one has the impression that it is aless frequent cause of benign meningitis in thiscountry. Other viruses which have been foundproducing this form of meningitis are the pseudolymphocytic choriomeningitis virus of MacCallum,Findlay, and Scott (1939) and the virus oflymphogranuloma venereum. Infection with thelast-named virus may rarely present as a meningitis(Sabin and Aring, 1942), and the virus of MacCallumand Findlay has been isolated on only two occasions;more than half the cases of this condition are due toa virus or viruses yet to be identified. The three

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known causal agents are to be found in the cerebro-spinal fluid, and all these are pathogenic for themouse and guinea-pig. Wherever the diagnosis ofacute aseptic meniingitis is in question, cerebro-spinal fluid collected as early in the disease aspossible should be injected intracerebrally in miceand subcutaneously in the thigh of guinea-pigs.Should any of the three viruses be present, encephali-tis may develop in one or more of the mice, and thevirus so isolated can be established and studied.The virus of Armstrong and Lillie (1934) will ofteninfect the guinea-pig, producing a generalizedinfection with pyrexia, wasting, and salivation, theanimal dying usually in from nine to sixteen days.Should the case ofmeningitis prove to be tuberculous,and in the early stages of this condition the differen-tial diagnosis from benign lymphocytic meningitis ofvirus origin may well arise, the mice will remainwell whereas the guinea-pigs will develop tuber-culosis.Lymphogranuloma venereum.-In addition to pro-

ducing an encephalitis when inoculated intracereb-rally in mice, lymphogranuloma virus also producesa local lesion when injected sub-cutaneously inthe guinea-pig's thigh, and an enlargementof the inguinal lymph nodes occurs; the lesionscan be examined histologically; and, by furtherpassage in animals and eggs, the virus canbe established and identified. The use of monkeysin such an investigation has not been mentionedbecause they are expensive and not essential,though, if available, one or possibly two might beinoculated intracerebrally. And it has beenassumed that eggs, if available, will be used inparallel with the animals.

Psittacosis group.-Another example ofthe use ofanimal inoculation, in addition to eggs, in the diag-nosis ofvirus infection is provided by diseases causedby viruses ofthe psittacosis group. Human infectionwith psittacosis virus is often associated with somedegree of pneumonia, and either sputum or lungpuncture materia4tan beused for animal inoculation,though in psittacosis pneumonia sputum is oftenscanty. The mouse is the animal usually employed;and, if sputum is the material chosen, the presence ofbacteria, particularly the pneumococcus, may-introduce a complication. Though the bacteria canbe got rid of by filtration, this also reduces theamount of virus, and a better procedure is toprotect the mouse against the bacteria by means ofsulphonamides, to which psittacosis virus is insen-sitive, or relatively so. This procedure has theadditional advantage, if the mice fiave been inocu-lated by the nasal route, of preventing a lighting up

of latent infection of the mice with the virus of Nigg(Nigg and Eaton, 1944), present in many mousestocks. This virus belongs to the Castaneda-positivegroup and is fortunately susceptible to sulphon-amides. And, in connexion with the use of mice forthe isolation of psittacosis virus, it is important thatboth the intranasal and intraperitoneal routes ofinoculation should be employed, because certainstrains of psittacosis virus produce only an inappar-ent infection when introduced into the peritonealcavity. This is true of pigeon strains, and here itmight be pointed out that the host range of thisvirus is much more extensive than was at one timethought. In addition to birds of the parrot tribe,certain species of finch, fulmar petrels, pigeons, thedomestic fowl, and ducks have been found to sufferfrom natural infection with psittacosis virus and tobe responsible for human infections.

Serological InvestigationIt is still far from being widely appreciated that

those serological tests which are in daily use in theidentification of bacteria and the diagnosis ofbacterial disease are equally applicable to viruses.Virus workers themselves have been to some extentresponsible for the tardy recognition of this fact.There is no need here to consider the reasons forthis; it is sufficient to note that it is now well estab-lished that the precipitin, agglutination, and comple-ment fixation tests are all available for the investi-gation of viruses and the diseases they cause.And, because it is more difficult to obtain a suspen-sion rich in virus than is the case with a bacterium,the complement fixation test is the serological,reaction which has been found most suitable forvirus work, since, as Merril has shown (1936), thistest has a lower antigen threshold than the precipitinor agglutination reactions. A serological test couldbe used in the diagnosis of a virus infection eitherfor the purpose of demonstrating the presence ofvirus in the infective material or for the detectionof antibodies formed in response to the infection.

Demonstrating virus in infective material-Asan example of the former we have the use of thecomplement fixation test in the diagnosis of small-pox. Craigie and Wishart (1936) have shown thatthis is a reliable test for the presence of variolaantigen in the vesicle fluid or in an extract madefrom the crusts of older skin lesions; the serumemployed is one made against vaccinia virus in therabbit. Tulloch (cited by Downie, 1946) has hadwide experience of this test in the outbreaks ofsmallpox which occurred in Scotland in the war

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of 1939-45; he considers the test to have. a highdiagnostic value. This opinion is shared by Downie(1946), who found that vesicle fluid or crusts fromtwenty-two cases suspected of being smallpox onclinical or epidemiological grounds gave a positiveresult; his material came from twelve differentoutbreaks. Similar material from other conditions,such as varicella, zoster, or septic skin rashes, gavea negative reaction. He concludes that this is themost valuable single laboratory test available for thediagnosis of smallpox, an opinion which seems fullyjustified by the evidence.

This is one of the few examples, however, of thisparticular use of serological reactions in the diag-nosis of human virus infections. It has, at times,been applied, in conjunction with animal inoculation,for the differentiation of zoster from herpes simplex,using a known positive convalescent zoster serumand the fluid from the vesicular eruption (Bedson,unpublished observations).

Detection of antibodies.-But, generally speaking,the concentration of antigen in infective materialfrom virus infections is too low even for its detectionby the complement fixation reaction, and it is forthis reason that the indirect use of such a test (thatis, for the demonstration of specific antibody in thepatient's serum) is of so much wider application as adiagnostic procedure. It would be impossible hereto give a comprehensive survey of this use of thecomplement fixation test; a few illustrative sampleswill have to suffice.

Lymphocytic choriomeningitis.-In the course ofinfection with the virus of lymphocytic chorio-meningitis, complement fixing antibodies are devel-oped (Smadel and others, 1939) for the solubleantigen of this virus. Although these antibodieshave probably nothing to do with immunity to thisvirus, they are specific and their presence diagnostic(Smadel and others, 1939; Lepine and Sautter, 1938).The soluble antigen is present in considerableamount in the tissues of the experimentally infectedanimals, and a suspension of the spleens of guinea-pigs succumbing to lymphocytic choriomeningitisprovides a satisfactory antigen which remains stablewhen stored in the refrigerator for months or evena year or more. It is important to note that afreshly made spleen suspension may give falsereactions, it should be allowed to age for two tothree weeks before use. Although the American andFrench work already referred to leaves no doubt asto the specificity and diagnostic value of this test,limited experience of its use in this country has beendisappointing (Bedson, unpublished findings). Dur-

ing the war a number of sera from cases of benignaseptic meningitis have been examined withoutobtaining a single positive result, and, although i

some of these cases this was probably due to theirnot being caused by the virus of Lillie and Arm-strong, there were two from which this virus wasisolated by mouse inoculation.

Influenza.-The complement fixation test can alsobe applied to the diagnosis of influenza (Smith,1936; Hoyle and Fairbrother, 1937; Fairbrotherand Hoyle, 1937; Francis and Magill, 1937; Tulloch,1939), but since antibodies to influenza virus are tobe found in detectable though low titre in the bloodof most adults and older children, diagnosis dependson demonstrating a significant increase in theseantibodies, and that makes the findings mainly ofretrospective interest. This being so the neutraliza-tion test, which takes so much longer to do, mightequally well be emaployed, and in the past it is theneutralization test which most workers have used.It may be, of course, that the Hirst phenomenon ofagglutination of the red cells of the domestic fowl byinfluenza virus (1941), which is specifically inhibitedby influenzal antibody and can be used as a methodof antibody titration, will eventually largely replaceboth complement fixation and neutralizationtests.

Virus agglutination of r.d cells.-The subject ofvirus agglutination of red cells, which, since Hirst'soriginal work, has been the object of much study,principally by Burnet and his colleagues in Australia,leads to mention of a recent observation fromBurnet's laboratory. This is not the place for anydetailed consideration of all the interesting workdone in this field; suffice it to say that the pheno-menon of red-cell agglutination is not confined tothe influenza virus and chick cells; other viruses willdo this, and the red corpuscles of certain species ofmammal, including man, are agglutinated as well aschicken red cells. The absorbed virus responsiblefor the agglutination is liberated after a few hours;this fact, incidentally, is utilized in the concentrationof the influenza viruses in the preparation ofinfluenza vaccine.The recent observation of Burnet and Anderson

(1946) to which it is desired to draw attention is thathuman red cells which have been agglutinated bythe virus of Newcastle disease of fowls and fromwhich the virus has been eluted have becomesusceptible to agglutination by the majority of serafrom recent cases of glandular fever. This work isstill in its initial stages. The evidence produced byBurnet and his colleague suggests that the change in

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the cells is due to the absorption ofantigenic material,other than virus, produced by Newcastle diseasevirus when growing in chick embryo cells. Burnet(1946) has also shown that human red cells treatedwith egg-grown mumps virus provide a sensitivetest for mumps antibody in human sera. Althoughmuch remains to be done, this work opens up suchexceedingly interesting possibilities that it seemedwell worth mentioning.

Psittacosis-lymphogranuloma venereum group.-Itis perhaps in the diagnosis of iftfections with theviruses of the psittacosis-lymphogranuloma vene-reum group that the complement fixation test hasbeen most widely applied. This test has been usedin the diagnosis of psittacosis in man (Bedson, 1935and 1937) for eleven years or more, and the workof Meyer and his colleagues and others in Americahas confirmed its value. By means of this testsupport was given to the findings of Haagen andMauer (1939) that cases of an atypical pneumoniaoccurring in the Faroe Islands were due to psitta-cosis or some very closely related virus, and it wasused to show that a similar clinical condition inIceland had a like aetiology (Bedson, 1940). Infec-tion in these cases originated in fulmar petrels.Some involvement of the lung is of frequentoccurrence in human infections with psittacosisvirus, and it has long been recognized that theresulting pneumonia was unlike bacterial pneumoniain its clinical signs and histological picture. It wasnot, therefore, very surprising when Eaton andothers (1941) showed that some cases of the condi-tion known as primary atypical pneumonia, whichin the recent war assumed almost epidemic propor-tions in this country and elsewhere, were due topsittacosis virus. This observation was based onisolation of the -virus from sputum or lung materialand the demonstration of a positive complementfixation test with the patients' sera. Subsequentwork has shown that an appreciable proportion ofcases presenting this syndrome, which Reimann(1946) quite rightly prefers to name "virus pneu-monia," are due to viruses belonging to thepsittacosis group. Smadel (1943), for instance,found that some 15 per cent of forty-five cases ofso-called primary atypical pneumonia were probablydue to a psittacosis virus, and, in a serologicalsurvey mnade of this condition in England during thewar, nine cases out of 120 had a very high or risingtitre of antibody for psittacosis virus (Bedson;unpublished findings).The more extensive use of the psittacosis com-

plement fixation test resulting from these investiga-tions has shown that the mere presence of such an

antibody in the serum of an individual does notnecessarily mean active infection with a virus of thisgroup. As in the use of any serological test forantibody as a diagnostic procedure, the demonstra-tion of a rising titre of antibody is necessary topostulate active infection. Smadel (1943) rightlyinsists that only a fourfold rise in psittacosis antibodyshould be accepted as evidence of active infection,and with this the writer would fully concur. Therecent work in America showing that the virusesbelonging to the Castaneda-positive group areclosely similar in antigenic structure has introduceda further complication. Rake and others (1941)found that sera from patients infected with theviruses of lymphogranuloma venereum and thepsittacosis group all cross-reacted when tested bythe complement fixation test with these viruses.They further showed that cases of atypical pneu-monia due to a psittacosis virus might give a positiveFrei test. And the demonstration by Rake andothers a year later (1942) that the sera from casesof trachoma and inclusion conjunctivitis may fixcomplement with the antigens of lymphogranulomavirus suggests that these two viruses also shareantigens with the psittacosis-,lymphogranulomagroup. There is further evidence obtained fromcross-immunity experiments in animals confirmingthe close relationship between the viruses of thepsittacosis group-parrot psittacosis, pigeon psitta-cosis or ornithosis, and the virus isolated by Eatonand others from atypical pneumonia-and the virusof lymphogranuloma venereum. How close thisrelationship will prove to be remains to be seen, butthe immediate practical bearing of this work is thatthe serological tests which have been accepted in thepast as specific for psittacosis and lymphogranulomavenereum can no longer be so regarded. And theFrei test, as one might expect, would appear to beno more specific than the serological tests. Thoughthis does not mean that these tests are withoutvalue, it does emphasize the importance, which ofcourse always existed, of invariably interpretingthem in conjunction with the clinical findings.

General RemarksThis account of the part which the laboratory can

play in the diagnosis of virus infections is far fromcomplete. Only the more important examples ofthis have been mentioned, and they have been givenshorn of much detail and supporting evidence. Itis hoped, however,,that this paper will have shownthat the laboratory can be of help, not just occa-sionally by making some special investigation, butin an everyday routine way.

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