856

Inhibiting effect of resorcinol on uptake of 1181 by rats’ thyroids,compared with that of methyl thiouracil and sodium thiocyanate.

Each rat is represented by a horizontal bar whose length gives the countsper min, recorded from its digested thyroid. The rats were killed 2 hr.after injection of 10 fix 113’, except all rats in experiment 3, which werekilled 1’/. hr. afterwards, and those shown as open bars in experiment 4,which were killed t hr. afterwards.

to 1/5-1/9 of that found in controls-an extent notexceeded with higher doses and comparable to the maxi-mal reduction obtained with methyl thiouracil. Further

experiments will be needed to establish the minimumdose required to produce the maximal antithyroid effect.A number of rats were given the resorcinol in two doses-the second, 1 hr. 10 min. before they were killed.As is shown in the figure, the results did not differ greatlyfrom those produced by single doses. When the resorcinolwas administered orally instead of parenterally, therewas an indication in some animals of a smaller, butdefinite, antithyroid effect, which will be further studied.

Comparison of the antithyroid action of resorcinolwith that of other known drugs throws some light on itsmode of action on the thyroid. The thyroid is knownto concentrate iodide from the serum in at least twosteps-first by concentrating the iodide in the gland,and then by organic binding of this iodide into protein,in which form most of the thyroid’s store is made. Thefirst step is inhibited by thiocyanate and the second stepby the thiouracils ; in each case this is the only knownantithyroid effect of the drug. Autoradiographs of alcohol-fixed paraffin-embedded thyroids show the presence oforganically bound iodine only. Autoradiographs madeafter pretreatment with resorcinol, as after methylthiouracil, failed to show any organically bound 1.The prevention of organic binding was further confirmedby finding that the antithyroid action of resorcinolcould be further enhanced by injecting sodium thio-

- cyanate 1 hr. before killing ; and the antithyroid actionof methyl thiouracil could be enhanced in exactly thesame way (see figure, experiments 2 and 3). This additionof thiocyanate further reduced the concentration ofp31 from 1/5 to 1/10 of that obtained with resorcinol ormethyl thiouracil alone. In this connection, it shouldbe noted from experiment 4 that the thyroid uptake inthe presence of resorcinol at the end of 1 hr. is not

significantly different from that at the end of 2 hr. ;thus the addition of thiocyanate at 1 hr. before killinghad evidently discharged some of the iodide ion whichhad been concentrated there despite the presence ofresorcinol. Vanderlaan and Vanderlaan (1947) havereported similar results with propyl thiouracil andthiocyanate. From the figure (experiments 2 and 3) itwill also be noted that when thiouracil is given in additionto resorcinol antithyroid action was not enhanced.With regard to the toxicity of resorcinol, 50 mg. doses

never proved fatal, but the injected animals all showeda severe tremor for the first half-hour. Smaller doseshad no overt toxic results.

CONCLUSIONS

Preliminary assessment of the antithyroid action ofresorcinol on rats, using Il3i to measure thyroid uptake,shows it to be similar to that of methyl thiouracil in allthe respects tested-namely, the maximal depressionof thyroid uptake of iodine which can be induced, theabolition of organic binding as evidenced by autoradio-graphy, and the enhancement of its antithyroid effectobtainable by the additional injection of thiocyanatebut not of methyl thiouracil.We are indebted to Mr. D. G. Arnott, B.sc., for the isotope

measurements and help in the experiments, and to Mr. S. R.Pelc, PH.D., for the autoradiographs.

REFERENCES

Bull, G. M., Fraser, R. (1950) Lancet, May 6, p. 851.Vanderlaan, J. E., Vanderlaan, W. P. (1947) Endocrinology, 40, 403.Veall, N. (1948) Brit. J. Radiol. 21, 347.

SEROLOGICAL DIAGNOSIS OF

HERPES SIMPLEX INFECTIONS

MARGARET E. HAYWARD *

B.Sc. Melb.

From the Department of Bacteriology, University of LiverpoolPRIMARY infection with herpes simplex virus occurs

frequently in young children (Dodd et al. 1938, Burnetand Williams 1939). The primary infection may be

symptomless (Anderson and Hamilton 1949) or there

may be clinical manifestations varying from inconspicuousvesicular lesions about the mouth, with or withoutstomatitis, to more extensive eruptions on the skin ofother parts of the body, especially in children or adultssuffering from chronic eczema. The acute stomatitis(aphthous stomatitis) may be accompanied by constitu-tional upset (McNair Scott et al. 1941). Cases of herpeticmeningo-encephalitis may also be commoner than theliterature suggests. Infected individuals apparentlycarry the virus throughout life and, their sera containantibodies to the virus.The diagnosis of herpetic infection may be confirmed

or established by isolation of virus or by demonstratingan increase in antibody in the serum during the illness.Estimation of serum antibody has generally been madeby testing the power of the serum to inhibit the actionof the virus in susceptible animals or to prevent thedevelopment of lesions on the chorioallantois of develop-ing chick embryos (neutralisation test). Such methodsrequire several days for their performance and a sensitivein-vitro serological test would offer obvious advantages.Sera containing antibodies to herpes virus were shownby Bedson and Bland (1929) to fix complement in thepresence of a herpetic antigen ; their technique whenused in the examination of human sera (Brain 1932)did not give as high a proportion of positive reactionsin adults as did the neutralisation test in the hands ofother workers (Andrewes and Carmichael 1930, Burnetand Williams 1939).* Holding the John W. Garrett International Fellowship in

Bacteriology.

857

TABLE I—COMTItSMENT-BTXATION AND NEUTRALISATION WITH IMMUNE GUINEAPIG SERA AGAINST FOUR STRAINSOF VIRUS

* Complement-fixation titres are expressed as reciprocal of highest dilution of serum giving < 50 % haemolysis.

Observations on a complement-fixation test for thedetection of antibody to the virus of herpes simplexwere reported in a previous paper (Hayward 1949).The antigen for these tests was prepared from herpes-infected extra-embryonic fluids from developing chickembryos several days after inoculation of virus into theyolk-sac, into the amniotic cavity, or into the allantoiccavity. The activity of the antigen was found to bedue chiefly to a " soluble " specific substance separablefrom the virus by high-speed centrifugation ; the antigenwas stable on storage at 4&deg;C but its activity was con-siderably weakened by heating at 560C for 1 hour.

Serological differences were not detected among fourrecently isolated strains of herpes simplex virus by thecomplement-fixation technique using immune sera pre-pared in guineapigs and sera from individuals whoprovided three of the strains. Infected chick-embryofluids were more sensitive antigens than extracts oftissue from infected animals, and a preliminary examina-tion of human sera by this complement-fixation testgave a high proportion of positive reactions.

Before accepting this technique as a sensitive methodfor the detection of herpetic antibody in human sera,it seemed advisable to find out whether it gave resultscomparable to those obtained by neutralisation testswhich are commonly used by other workers for thispurpose. Immune guineapig sera prepared againstfour strains of virus were therefore tested against eachof the strains by both complement-fixation and neutralisa-tion techniques. A similar comparison was made of 95specimens of sera from healthy adults. The results ofthese tests are reported in this paper.

METHODS

The virus strains studied, the preparation of the antigenand the technique of the complement-fixation tests have

already been described (Hayward 1949). To obtain potentantigen, the fluids from individual eggs were tested separatelyin doubling dilutions against an immune human serum diluted1 in 8. Only those fluids which showed fixation in highdilution (1 in 64 or 1 in 128) were pooled and used for routinetests.

The guineapig immune sera, as previously described, wereobtained from animals which had received several injectionsof active virus from herpes-infected guineapig pads. The95 specimens of human serum were kindly supplied byDr. D. Lehane, director of the Liverpool Regional BloodTransfusion Service. When testing these sera, known nega-tive and positive control sera were included with each batch.The neutralisation tests were carried out by the chorio-

allantois titration technique, 4 membranes being inoculatedwith each serum-virus mixture. All the sera were inactivatedat 560C for 20 minutes before the test. The guineapig serawere tested in dilutions of 1 in 10, 1 in 100, and 1 in 1000,the human sera in a dilution of 1 in 10 only. Serum froma normal uninoculated guineapig and sera from individualswith no history of herpetic infection were used as controlnegative sera. Herpes-infected allantoic and amniotic fluids- collected as for complement-fixation tests served as a sourceof virus for the neutralisation tests. After clarification by

low-speed centrifugation the virus content of the fluids wasestimated by titration on the chorioallantois of 12-day chickembryos. For tests with sera a dilution of the virus wasused which gave a count of over 1000 lesions on controlmembranes. The serum dilutions were mixed with equalvolumes of virus suspension and the mixtures were allowedto stand for 1-2 hours at room-temperature before inoculationof 0-2 ml. volumes on the chorioallantois of 12-day chickembryos. After two day’s incubation, the membranes werecut out and placed in 2 % formol saline and the number oflesions counted. The results were expressed as the percentagereduction in the number of lesions compared with those onmembranes which had received the control mixtures of virusand diluted negative serum.

RESULTS

Titration of antibody in immune guineapig sera bycomplement-fixation and neutralisation tests.-Table i

shows the results when the immune guineapig sera weretested against each of the four viruses both by comple-ment-fixation and neutralisation techniques. With thecomplement-fixation technique the sera are arranged infixing potency in order P<H<McC<N. This order ismaintained in tests with antigens from all four virusesas mentioned in the previous paper. i

Similarly when the sera were tested by the neutralisa-tion technique, the relative titre of antibody was of thesame order except that serum P gave better neutralisationof virus H than did the homologous serum H.These results support the opinion based on complement-

fixation tests only (Hayward 1949) that there are nomarked antigenic differences between the four virusstrains. The titre of antibody measured by the comple-ment-fixation technique parallels that demonstrated byneutralisation tests ; there is no evidence that differentantibodies are measured by the two techniques.

Comparison of complement-fixation and neutralisationwith human sera.-The results of the complement-fixation and neutralisation tests with the 95 humansera are shown in table 11.

In neutralisation tests each serum was tested in a

single dilution of 1 in 10. Those sera which contained

antibody showed 95-100 % neutralisation, and sera withweaker neutralising capacity were not observed, thoughthe high lesion counts on the control membranes did not

TABLE II-HERPETIC ANTIBODY IN 95 HUMAN SERA DETER-MINED BY COMPLElYfENT-FIXATION AND NEUTRALISATION

858

facilitate the detection of such sera. - The all-or-nothingcharacter of herpes-neutralising antibody in adult humansera is, however, generally recognised (Burnet 1946).The testing of a single serum dilution did not enable aquantitative estimate of the amount of antibody presentin individual sera to be made.

It can be seen that 75 sera (80%) gave a positive testand 19 (20%) gave a negative test for herpetic antibodyby both techniques. One serum gave a positive resultby neutralisation and was negative by complement-fixation. Unfortunately this specimen was too smallto allow the test to be repeated.These tests with human sera, like those with the

guineapig sera, show close agreement by the two tech-niques. The proportion of sera showing herpetic anti-body (80%) is similar to that found by others (Burnetand Lush 1939).

SUMMARY

Immune guineapig sera for four strains of herpessimplex virus were tested for antibody against -the fourstrains by complement-fixation and neutralisation tech-niques. The results by the two techniques showed closeagreement and did not disclose antigenic differences

among the four virus strains.Tests on 95 adult human sera by the two methods gave

similar findings with the two techniques. 80% of thesesera contained herpetic antibody.The complement-fixation technique, using as antigen

extra embryonic fluids from herpes-infected chickembryos, provides a simple and reliable test for thedetection and titration of herpetic antibody in humansera.

I am indebted to Prof. A. W. Downie for encouragementand advice during the course of this work.

REFERENCES

Anderson, S. G., Hamilton, J. (1949) Med. J. Aust. i, 308.Andrewes, C. H., Carmichael, E. A. (1930) Lancet, i, 857.Bedson, S. P. Bland, J. O. W. (1929) Brit. J. exp. Path. 10, 393.Brain, R. T. (1932) Ibid, 13, 166.Burnet, F. M. (1946) Virus as Organism. Harvard University Press.

&mdash; Lush, D. (1939) Lancet, i, 629.&mdash; Williams, S. W. (1939) Med. J. Aust. i, 637.

Dodd, K., Johnston, L. M., Buddingh, G. J. (1938) J. Pediat. 12, 95.Hayward, M. E. (1949) Brit. J. exp. Path. 30, 520.Scott, T. F. McN., Steigman, A. J., Convey, J. H. (1941) J. Amer.

med. Ass. 117, 999.

TYPHOID FEVER TREATED WITH

CHLORAMPHENICOLDEVELOPMENT OF THE CARRIER STATE

A. D. M. DOUGLASM.B. St. And., D.P.H., D.P.M.

SENIOR PSYCHIATRIC REGISTRAR, SAXONDALE HOSPITAL,NOTTINGHAM

SEVERAL papers published in England in the last fewmonths have confirmed the original observation, madeby Prof. J. E. Smadel and his team in Malaya, of thevalue of chloramphenicol in typhoid fever. So far,however, the drug has not prevented the developmentof relapses or of the chronic carrier state. At a meetingof the epidemiological section of the Royal Society ofMedicine on March 17 the hope was expressed that’’ some cunning and at present unsuspected technique "would make it possible to prevent and cure the carrierstate with chloramphenicol. How much cunning is

likely to be required is indicated by the following 3 casestreated with chloramphenicol, of which 2 have becomechronic carriers.

CASE-RECORDS

Case l.-A female catatonic schizophrenic, aged 37, wastreated with chloramphenicol from the third day of her

1. See Lancet, March 25, p. 544.

Fig. I-Case J. Relapse in typhoid fever despite apparently goodresponse to chloramphenicol.

illness, when cultures of the blood and the stools grewS. typhi phage-type F 1. She was given 4 g. in the first hour, 0.25 g.two-hourly for the next four days, then 0-75 g. twice dailyfor nine days, and finally one dose of 0-75 g., making a totalof 31 g. Because there was clinical evidence of broncho-pneumonia she was also given 3,650,000 units of penicillinand 36 g. of Sulphatriad ’ in six days. On the third dayof chloramphenicol treatment her temperature fell sharply,and it swung at a lower level until four days later,when, to all intents, the patient’s illness seemed to haveresolved.

Relapse.-Sixteen days after the withdrawal of chloram-phenicol the patient relapsed. Blood-culture and stools wereagain positive for S. typhi. On the fourth day of this relapsechloramphenicol was started again, and she received a totalof 24-75 g. in thirteen days. Her temperature again settledafter three days (fig. 1). Her excreta became negative forS. typhi after four days and have remained so for the subse-quent five months. Clinically, the relapse was characterisedby what appeared to be neurological involvement. She hada well-marked " trombone " tremor of the tongue and acoarse tremor of the lips, arms, and legs, but no other

neurological abnormalities were detected. These symptomsappeared on the third day of her relapse and had disappearedforty-eight hours after the reintroduction of chloramphenicol.Thereafter the patient made an uneventful recovery.

Case 2.-A female schizophrenic, aged 34, was at first

diagnosed as having bronchopneumonia, so penicillin andsulphatriad were given at once. Three days later a blood-culture grew S. typhi Fl. Chloramphenicol was started next

Fig. 2-Case 2. Response in patient who became a chronic carrier.