The present status of the IgM fluorescent antibody technique in the diagnosis of congenital toxoplasmosis

1116

December, 1969 T h e J o u r n a l o[ P E D I A T R I C S

The present status of the IgM fluorescent antibody teclmique in the diagnosis of congenital toxoplasmosis The I g M fluorescent toxoplasma antibody test was positive in 11 cases o[ suspect congenital toxoplasmosis. The diagnosis was established by findings at autopsy or by demonstration o[ persistence o[ toxoplasma antibodies. IgM antibodies were also demonstrable in in[ants with asymptomatic congenital toxoplasmosis in whom this injection would not ordinarily be suspected. The problem o[ positive I g M fluorescent antibody titers in in[ants who in later li[e do not have serologic evidence o[ congenital toxoplasmosis is discussed, as is the problem o[ obtaining sufficient volumes o[ a fluorescein-tagged antiserum to human IgM to allow [or ready availability of this method. Automation o[ the fluorescent antibody test has been suceess[ul and should allow [or the development of a screening program to detect congenital injections in the newborn.

Jack S. Remington, M.D. PALO ALTO,

CALIF.

I N 1966 we reported that the presence of IgM toxoplasma antibodies in a newborn infant, in the absence of evidence of a placental leak, was diagnostic of congenital toxoplasmosis? In this study gel filtration and sucrose density gradient ultracentrifugation were employed initially to isolate IgM From the Division of Allergy, Immunology, and In[ectious Diseases, Palo Alto Medical Research Foundation, and the Department o[ Medicine, Stan[ord University School o[ Medicine. Supported by Grant No. AI04717 [rom the National Institutes o[ Health and a grant [rom The John A. Hart[ord Foundation. Reprint address: Division o[ Allergy, Immunology, and In[ectlous Diseases, Palo Alto Research Foundation, 860 Bryant St., Palo Alto, Cali]ornia 94301. "

Vol. 75, No. 6, part 2, pp. 1116-1124

antibodies from the infants' sera. These techniques, however, were far too cumbersome to allow for easy availability of this method of diagnosis for general laboratory use. Further studies 2-4 performed in an attempt to develop more simplified methods for demonstration of these antibodies included reductive cleavage of the infants' sera with 2mercaptoethanol prior to testing in the hemagglutination and Sabin-Feldman dye tests. This method did not prove useful for demonstrating IgM antibodies in the early newborn period, since the effect of 2-mercaptoethanol on the infants' IgM antibodies was sometimes masked by the presence of high levels of maternal IgG antibodies. The infants' IgM

Volume 75 Number 6, part 2

IgM fluorescent toxoplasma antibody test

toxoplasma antibodies were, however, demonstrable by an indirect fluorescent antibody test. This procedure proved simple, highly specific, and rapid to perform and, except for the fluorescence microscope, does not require expensive equipment. Our suggestion that this method might be applicable to other congenital infections which are difficult to diagnose in the newborn period has now been substantiated by other reports. This technique has now been used successfully for the diagnosis of congenital infection with cytomegalovirus by Hanshaw and associates, ~ with rubella virus by Baublis and Brown, 6 and with Treponema pallldum by Scotti and Logan ~ and Alford and associates. 8 We have now had more than 3 years' experience with the IgM toxoplasma fluorescent antibody technique. The organization of this meeting prompted us to review the data which have accumulated subsequent to the studies which appeared in our initial reports. 2-4 It is the purpose of this communication to describe results with the IgM fluorescent toxoplasma antibody test in a "prospective"* study in infants with suspect congenital toxoplasmosis whose sera were sent to us for examination by physicians from various areas of the United States. Since a question has been raised (Dr. Georges Desmonts, personal communication, 1968) as to whether the IgM fluorescent antibody test could identify congenital toxoplasmosis in the asymptomat~c infant, a study to clarify this point is also reported here. MATERIALS

AND METHODS

Dr. Georges Desmonts supplied the sera from the cases of asymptomatie congenital toxoplasmosis. The Sabin-Feldman dye test was performed as described by Frenkel and Jacobs2 The IgM f/uorescent toxoplasma antibody test was performed as previously described, 2-~ except that Evans blue dye was added to the diluted (1:150) tagged antiserum in a concentration of 0.2 per cent. *The term " p r o s p e c t l v e '~ is used here to signify that we were unaware of the diagnosis at the time the results of the test in the first serum sample studied were reported.

1117

RESULTS

The resu]ts of the dye test and IgM fluorescent antibody test in 11 cases of suspect congenital toxoplasmosis are shown in Table I. Clinical findings in each infant led their respective physicians to consider congenital toxoplasrnosis in the differential diagnosis. The IgM fluorescent antibody test was positive in each case and the diagnosis was later established by findings at autopsy or by demonstration of persistence of toxoplasma antibodies. Patient T. M. is included to demonstrate the persistence of IgM antibodies and high dye test titers. To determine if IgM antibodies are demonstrable in infants with asymptomatic congenital toxoplasmosis in whom this infection would not ordinarily be suspected, sera from 5 such infants were run in the IgM fluorescent antibody test. The results, shown in Table II, demonstrate that such infants do form IgM toxoplasma antibodies and in significant enough concentration to be measured by this technique. Each of these infants was born of a mother who had acquired toxoplasmosis during pregnancy. Each was followed for at least one year and the diagnosis established by persistence of toxoplasma antibodies? ~ In Table I I I are the serologic test results in an asymptomatic infant whose dye test titer decreased over a one-year period to 1:16. At age one year she was in perfect health. The results in this case are presented because the positive IgM fluorescent antibody test may in this instance represent a "false positive" result. DISCUSSION The results described above, along with those previously reported from our laboratory2, 4 and those of Dr. Charles Alford presented here today, ~ suggest that the IgM fluorescent antibody test is of definite value in the diagnosis of congenital toxoplasmosis in the early newborn period. This is true not only for infants suspected of having the infection, but for infected infants who are asymptomatic as well. The validity of our interpretation of positive results in the initial

1 1 18

Remington

The Journal of Pediatrics December 1969

Table I. Results of IgM fluorescent toxoplasma antibody test (FAT) in infants with suspect congenital toxoplasmosis Dye test ~

IgMFAT*

too. too. mo. too.

8,192 8,192 8,192 4,096

80 40 40 40

At 7 mo., pyrimethamine and sulfadiazine for 1 wk., discontinued because of bone marrow toxicity

Jaundice at birth. Random eye movement first 6 too. Chorioretinitis noted at 7 too. At 8 too. had partial clearing of retinae and improved vision

M.B.

1 mo. 2 too. 4 mo.

8,192 8,192 8,192

80 80 80

Two exchange transfusions shortly after birth. At 6 and 9 too., pyrimethamine and sulfadiazine for I mo.

Premature (1,700 Gm.), jaundice, microcephaly, bilateral chorioretinitis. Complete loss of vision O.S., partial loss O.D. Retarded growth but developing

T.C.

2 mo.

16,384

10

2 wk. of apneic spells; spastic, hydrocephalus. Died at 2 mo. Autopsy revealed toxoplasma in cerebral cortex

W.O.

9 too.

16,384

80

Chorioretinitis, esotropia, poor fixation O.D., autonomic seizures

B.J.

6 too. 15 too.

4,096 16,384

40 10

Intracranial calcifications, ehorioretinitis. At 6 mo., slight hepatomegaly, microphthalmia. Concurrent C M V infection

T.M.

7 yr. 8 yr.

4,096 4,096

40 80

At 7 yr., pyrimethamine and sulfadiazine

Bilateral chorioretinitis, psychomotor retardation, petit real seizures

K.M.

1.5 mo. 2.5 mo.

16,384 16,384

160 160

At 1.5 mo., pyrimethamine and sulfadiazine for 7 wk.

Hydrocephalus, chorioretinitis, blind, deaf, spastic, intracranial calcifications, abnormal cerebrospinal fluid

B.N.

Birth 0.5 mo. 4 mo. 10 mo.

2,048 16,384 8,192 16,384

20 80 80 80

At 1.5 mo., pyrimethamine and sulfadiazine for 3 wk.

Premature, growth retardation, abnormal cerebrospinal fluid, microcephaly, chorioretinitis, hepatosplenomegaly, intracranial calcifications

B. We.

Birth

16,384

160

B. Wo.

2 mo. 4 mo.

2,048 2,048

80 10

Pyrimethamine and triple sulfa

Bilateral chorioretinitis, intracranial calcifications

2 days 1 mo. 1.5 too. 3 mo. 6 too. 9 mo. 22 mo.

16,384 16,384 65,536 32,768 65,536 32,768 8,192

80 80 40 80 80 80 20

At 1 mo., pyrimethamine and sulfadiazine for 2 wk.

Hydrocephalus, chorioretinitis O.D., hepatomegaly, splenomegaly, abnormal cerebrospinal fluid. At 6 too., developmentally and neurologically normal; at 22 too., appeared normal

Patient T.B.

T.T.

Age 7 8 10 15

*Reciprocal of titer.

Treatment

Remarks

Hydrocephalus, hepatomegaly, splenomegaly. Died at 14 hr. Autopsy revealed generalized toxoplasmosis

Volume 75 Number 6, part 2

IgM fluorescent toxoplasma antibody test

11 19

T a b l e I I . Results of I g M fluorescent t o x o p l a s m a a n t i b o d y test ( F A T ) in infants w i t h a s y m p t o m a t i c congenital toxoplasmosis*

Po.e.

[

Age

I Dye ,estt t

r

B.C.

1 mo. 10 mo.

256 4,096

80 20

Premature birth

B.L.

Birth 2 mo. 10.5 too.

16,384 4,096 256

20 20 10

Toxoplasma isolated from placenta

B.R.

1 too. 8 too.

1,024 4,096

160 40

B.M.

Birth 2.5 mo. 6 mo.

1,024 4,096 4,096

10 20 20

Toxoplasma isolated from placenta; child with very mild "nearly asymptomatic" infection (a peripheral small scar in fundus)

B.S.

3 mo.

4,096

40

Premature birth; twin dead few days after birth

Premature

birth

*Sera supplied by Dr. Georges Desmonts, Hopital Saint-Vincent-de-Paul, 74, Avenue Denfert-Rochereau, Paris (XIVe), France. Each of these infants was studied because acquired toxoplasmosls had been diagnosed in their mothers during pregnancy?0 tReciprocal of titer.

T a b l e I I I . Positive I g M - F A T in " n o r m a l " i n f a n t ( P a t i e n t O. S.)

Age Birth 2.5 mo. 5 too. 7.5 too. 1 yr.

] Dye test* 8,192 4,096 1,024 64 16

[

IgM-FAT* 80 40 Neg. +10t Not done

*Reciprocal of titer. "tNot completely negative at 1:10 compmed with COlltrol; less than 50 per cent of organisms had clear fluorescence.

sera tested was substantiated by the persistence of dye test antibody in the infant or by identification of toxoplasma infection at autopsy. I n a recent collaborative study with Drs. M i c h a e l Miller a n d Philip Sunshine, 5,006 consecutive cord sera were e x a m i n e d to determine the significance of elevated levels of I g M a n d I g A in the newborn. T h e sera were also tested in the I g G fluorescent t o x o p l a s m a a n t i b o d y test a n d a p p r o x i m a t e l y 20 p e r cent were positive. ( T h e results of this m e t h o d are the same as those o b t a i n e d in the dye test w h e n the sera are screened for positivity or negativity at a dilution of 1:10.) Sera positive in the I g G fluorescent a n t i b o d y test were studied by the I g M fluorescent a n t i b o d y m e t h o d to a t t e m p t to identify unsuspected

cases of congenital toxoplasmosis. O n l y a brief s u m m a r y of the d a t a is presented here, as they will be published in a separate communication. O n e h u n d r e d five of t h e m were positive in the I g M fluorescent a n t i b o d y test at a titer of 1:10 or greater. Eighty-two h a d at least a titer of 1:20 and 6 h a d a titer in excess of 1:80. I n only 2 of these infants d i d q u a n t i t a t i o n of I g M a n d I g A reveal a " p l a c e n t a l leak" h a d occurred, e F o l l o w - u p sera o b t a i n e d between 1 m o n t h a n d 12 months from 104 of the infants were negative in the I g M fluorescent antibody test. T h e infant in w h o m no follow-up serum was obtained died several hours after birth, a n d at autopsy there was no evidence of toxoplasmosis. These results suggest t h a t "false positives" in this g r o u p were a b o u t 10 p e r cent. W e are not certain, however, t h a t the t e r m "false positive" is applicable since there are insufficient d a t a on the i m m u n e response of the fetus in utero to infection at different stages of gestation with virulent a n d avirulent strains of toxoplasma. F o r t u n a t e l y , no instance of a false negative case has been identified to date. Patient O. S. in T a b l e I I I was included to demonstrate serial serologic test results in *If the level of IgA was found to be higher than that of IgM, a placental leak was considered to have occurred.

1 1 20

Remington

an infant, normal at birth, who had a positive I g M fluorescent antibody test. T h e infant's mother had previously given birth at Stanford Hospital to an infant with congenital toxoplasmosis. 12 T h e infant's dye test titer decreased progressively from 1:8,192 at birth to 1:16 at one year of age, and the I g M fluorescent antibody titer also decreased progressively. According to the data of Stiehm and associates, 13 if the I g M were passively transferred maternal antibody, the I g M titer should have disappeared after the first few weeks. In an attempt to resolve the question of "false positive" in this case, Dr. Hugh Fudenberg kindly typed the maternal I g M and that of the infant. Unfortunately, they were found to be the same so that we could not prove that the IgM was manufactured by the infant. T h a t the high IgG titer of maternal antibody in the infant did not cause a "false positive" result has been proved by results of previous studies in which IgG isolated from sera with dye test titers of 1:32,000 or greater was negative when tested in the I g M fluorescent antibody test2 Low molecular weight g a m m a globulins with I g M specificity ( L M W - I g M ) and antibody activity have been described a,*, 1,, 15 and we have postulated that they may play a role in the observed positivity--but to explain the observed persistence of such I g M antibodies the half-life of L M W - I g M would have to differ from that of the maternally transmitted I g M described by Stiehm and associates. 16 Since there are marked antigenic differences between certain of these L M W I g M and other known classes of immunoglobulins, further studies are needed to define their physicochemical and immunochemieal nature and half-life, as well as that of other I g M species formed by the fetus and in the newborn in response to infection to determine if they are similar to I g M found in the adult. The presence of I g M toxoplasma antibodies in such infants may indeed reflect true infection. T h e "antigenic load" may have been slight and eradicated by the infant, but not in time to prevent stimulation of antibody formation. In the absence of a state of

The Journal o[ Pediatrics December 1969

immune tolerance, it is difficult to postulate that antibody is no longer formed after several weeks because parasites have become encysted or are in a latent phase. Infants and older children who have asymptomatic toxoplasmosis, and in whom the parasite has encysted, retain positive serologic test titers for years and perhaps for their entire life span. This does not, however, rule out the possibility that foci of infection with small numbers of relatively avirulent strains might persist and yet be "insufficient" as a continuing antigenic stimulus. As is often the case for any new serologic method, the problem of sensitivity in relation to specificity has arisen. It is noteworthy that with the exception of Infant T. C., the I g M fluorescent antibody test was greater than 1 : 10 in each of the infants in the present study and in 17 of the 18 infants reported previously. 4 We feel, however, that there is insufficient experience with this method at this time to allow us to suggest using a titer of greater than 1:10 as a minim u m for diagnosis. We have attempted for the past year to obtain a commercial antiserum to I g M which can be recommended, in order to make the IgM-toxoplasma fluorescent antibody method available to individuals desiring to use it. The antisera have been tested as previously described a to assure the absence of toxoplasma antibodies as measured in the dye test and to document their unispecificity to IgM. We tested each tagged antiserum at various dilutions from 1:10 to 1:200 with at least 5 sera from cases of established congenital toxoplasmosis. Despite the specificity of such antisera and their ability to reveal I g M antibodies in control sera, certain of them gave negative results when tested against sera demonstrated to contain I g M antibodies not only with our original antiserum, 3 but by serologic testing of fractions from gel filtration and sucrose gradients. The reason(s) underlying this lack of reactivity despite proved specificity of the antisera and apparently adequate sensitivity of the method is unclear. The question must be raised as to whether the I g M produced by the fetus in-

Volume 75 Number 6, part 2

I g M fluorescent toxoplasma antibody test

fected with toxoplasma in utero is antigenically the same as the I g M of normal adults. In some cases, it may be that the L M W - I g M antibodies discussed above play a role by acting as blocking antibodies by binding antigenic sites on the surface of the toxoplasma organisms, but are not themselves able to bind enough fluorescein-tagged antibody to give a positive test result. We are continuing to work in collaboration with cbmmercial suppliers of fluorescein-tagged antibody in an effort to make available a large supply of an adequate antiserum. When such antisera become available it is hoped that the I g M fluorescent antibody test may be useful as a screening device for detecting infection early in the newborn period. Before it can be used in this manner, however, it is necessary to develop means whereby more serum samples can be examined per unit time. In recent studies conducted by the United States Public Health Service Venereal Disease Research Laboratory, Stout and her colleagues have demonstrated the feasibility of automation of the fluorescent antibody test. They made use of the SeroMatic System developed by the Space Division of AerojetGeneral Corporation, El Monte, California, and described their results at the 1968 annual meeting of the American Public Health Association. Such instrumentation to automate the fluorescent antibody test should allow for the development of a screening program to detect congenital infections in newborns. Such a screening program would detect infected infants who are asymptomatic at birth. At present we are identifying only the clinically apparent cases in the newborn per i o d - t h e tip of the iceberg. T h a t it is indeed only the tip has been shown in a carefully designed prospective study in Paris by Desmonts and Couvreur, 1~ who attempted to define the consequences on the offspring of maternal toxoplasmosis acquired during pregnancy. The risk of toxoplasmosis in the childbearing population in Paris is high, being 0.55 per cent per month. 1~ Thus the chances of infection in this group are approximately

112 1

5 per cent for a pregnancy of 9 months. The authors have taken into account the proportion of these women who have no demonstrable toxoplasma antibodies as measured by the dye test, and they have concluded that there may be as many as 80 cases of acquired toxoplasmosis among a total of 10,000 pregnancies reaching term. This figure is, however, far greater than the actual number of recognized cases of congenital toxoplasmosis in Paris. 1~ The French workers have studied 64 cases of toxoplasmosis acquired during pregnancy and 60 of the infants born of these mothers were examined; 22 (36 per cent) had congenital toxoplasmosis. Most noteworthy is their observation that 16 (73 per cent) of the infected infants presented no sign of congenital toxoplasmosis. Careful serologic follow-up in each of them established the diagnosis of congenital infection. Certain of the children received no treatment in their first year of life and revealed no untoward effect of the infection. A number, however, were treated and the authors recognize that the asymptomatic character of their infection may have been due at least in part to this treatment. Although we, 17 as well as others, is, 19 have observed and attempted to bring to the attention of physicians the wide clinical spectrum of congenital toxoplasmosis, this study by Desmonts and Couvreur finally defines the statistical significance of the asymptomatic congenital infection. It must be clearly borne in mind that such infants may later develop untoward sequelae such as chorioretinitis, blindness, epilepsy, etc. For this reason, we were interested in determining whether the I g M fluorescent antibody method would be of diagnostic value in such infants. The test was positive in sera of each of the 5 infants with asymptomatic infection in the present study. These infants were all from the study by Desmonts and Couvreur. And what is the role of specific therapy in congenital toxoplasmosis? In a recent conference on the subject of prevention of mental retardation through control of infectious diseases, Frenkel 2~ stated, "more rapid diagnosis and more effective chemotherapy

1122

Remington

a p p l i e d to infants with generalized toxoplasmosis should m a k e it possible to reduce the p r o p o r t i o n of those sustaining cerebral d a m a g e and blindness resulting in m e n t a l deficiency." A t this same conference Eichenwald 21 discussed his findings in 40 treated infants with congenital toxoplasmosis diagnosed within a few hours to days after birth. E a c h received p y r i m e t h a m i n e a n d sulfadiazinc for 28 days. N o n e of them h a d hydrocephalus or m i c r o c e p h a l y at the time thera p y was initiated. A comparison of these 40 cases with a similar group of infants with toxoplasmosis who were not t r e a t e d indicated t h a t the degree of central nervous syst e m d a m a g e observed on follow-up 3 years later was identical in both groups. Eichenwald concluded, therefore, that there is as yet no evidence that the use of p y r i m e t h a mine a n d sulfadiazine in neonatal toxoplasmosis offers a n y m e a s u r a b l e benefit to the infants. T h e statement was m a d e d u r i n g a discussion a n d unfortunately no d a t a were given as to the numbers of infants suffering sequelae or of the types of sequelae. W h e t h e r higher doses of p y r i m e t h a m i n e a n d sulfadiazine a n d / o r m o r e p r o l o n g e d t h e r a p y m i g h t have resulted in m o r e favorable resuhs remains to be d e t e r m i n e d in future studies. Since leucovorin m a y limit bone m a r r o w toxicity of p y r i m e t h a m i n e w i t h o u t interfering with the action of p y r i m e t h a m i n e on the protozoan, its administration in conjunction with p y r i m e t h a m i n e and sulfadiazinc m a y allow for use of higher d r u g doses. 2~ T h i s becomes even m o r e feasible with the i m p o r t a n t a d d i t i o n to t h e r a p y in the infant of the new intravenous form of p y r i m e t h a m i n e ( p y r i m e t h a m i n e isethionate) which has been developed by Burroughs Wellcome &

Co., I n c : ~Although this form of the drug has not been released for general use, it may be obtained from Dr. George Hitchings, Burroughs Wellcorne & Co., Tuckahoe, N. Y. 10707.

REFERENCES

1. Remington, J. S., and Miller, M. J.: 19S and 7S anti-toxoplasma antibodies in the diagnosis of acute congenital and acquired toxoplasmosis, Proe. Soc. Exper. Biol. & Med. 121: 357, 1966.

The Journal o[ Pediatrics December 1969

2. Remington, J. S.: Characterization of antibodies to parasites, in Immunologic aspects of parasitic infections, Proceedings of the Special Session held during the Sixth Meeting of the PAHO Advisory Committee on Medical Research, June 13, 1967, Scientific Publication No. 150, Sept., 1967, pp. 50-57 and pp. 150-151 (Pan American Health Organization, Washington, D. C.). 3. Remington, J. S., Miller, M. J., and Brownlee, I.: IgM antibodies in acute toxoplasmosis. II. Prevalence and significance in acquired cases, J. Lab. & Clin. Med. 71: 855, 1968. 4. Remington, J. S., Miller, M. J., and Brownlee, I.: IgM antibodies in acute toxoplasmosis. I. Diagnostic significance in congenital cases and a method for their rapid demonstration, Pediatrics 41: 1082, 1968. 5. Hanshaw, J. B., Steiufeld, H. J'., and Cherie, J. W.: Fluorescent-antibody test for cytomegalovirus macroglobulin, New England J. Med, 279: 566, 1968. 6. Baublis, J. V., and Brown, G. C.: Specific response of the immunoglobulins to rubella infection, Proc. Soc. Exper. Biol. & Med. 128: 206, 1968. 7. Scottl, A. T., and Logan, L.: A specific IgM antibody test in neonatal syphilis, J. PEDIAT. 73: 242, 1968. 8. Alford, C. A., Polt, S. S., Cassady, G. E., Straumfjord, J. V., and Remington, J. S.: 3'M-fluorescent treponemal antibody in the diagnosis of congenital syphilis, New England J. Med. 280: 1086, 1969. 9. Frenkel, J. K., and Jaeobs, L.: Ocular toxoplasmosis, Arch. Ophth. 59: 260, 1958. 10. Desmonts, G., and Couvreur, J.: L'expression clinique de l'infection chez le nouveau-n6. 3. Toxoplasmose cong6nitale, In Congr~s des P4diatres de langue Francaise, 21st, Paris, 1967, Paris, L'Expansion Scientifique Francaise Rapports 3: 453, 1968. 11. Alford, C. A., Foft, J. W., Blankenship, W. J., Cassady, G., and Benton, J. W.: Subclinical CNS disease of neonates. A prospective study of infants who were born with increased levels of IgM, J. PEDIAT. 75: 1167, 1969. 12. Miller, M. J., Aronson, W. J., and Remington, J. S.: Late parasitemia in asymptomatic cases of acquired toxoplasmosis, Ann. Int. Med. In press. 13. Stiehm, E. R., Vaerman, J. P., and Fudenberg, H. H.: Plasma infusions in immunologic deficiency states: Metabolic and therapeutic studies, Blood 28: 918, 1966. 14. Perchalski, J'. E., Clem, L. W., and Small, P. A.: 7S gamma-M immunoglobulins in normal human cord serum, Am. Med. J. Sc. 256: 107, 1968. 15. Solomon, A.: Molecular heterogeneity of immunoglobulin-M (7M-globulin), J. Immunol. 102: 496, 1969. 16. Stiehm, E. R., Ammann, A. J., and Cherry, J. D.: Elevated cord macroglobulins in the diagnosis of intrauterine infections, New England J. Med. 275: 971, 1966.

Volume 75 Number 6, part 2

I g M fluorescent toxoplasma antibody test

17. Miller, M. J., Seaman, E., and Remington, J. S.: The clinical spectrum of congenital toxoplasmosis: Problems in recognition, J. P~.DIAT. 70: 714, 1967. 18. Eiehenwald, H. F.: A study of congenital toxoplasmosis with particular emphasis on clinical manifestations, sequelae and therapy in human toxoplasmosis, Internat. Conf. Pedlar. (Copenhagen, 1956), Copenhagen, 1960, Ejnar Munksgaards Forlag, p. 41. 19. Glasser, L., and Delta, B. G.: Congenital toxoplasmosis with placental infection in monozygotic twins, Pediatrics 35: 276, 1965. 20. Frenkel, J. K.: In Eichenwald, H. F., editor: Proceedings of a conference on prevention of mental retardation through control of infectious diseases, June 9-11, 1966, Cherry Hill, N. J., U. S. Public Health Service Publication No. 1692, p. 307. 21. Eichenwald, H. F.: In Eichenwald, H. F., editor: Proceedings of a conference on prevention of mental retardation through control of infectious diseases, June 9-11, 1966, Cherry Hill, N. J., U. S. Public Health Service Publication No. 1692, p. 307.

DISCUSSION DR. BELLANTX. I noticed in one of your slides there was a rise in the dye antibody to 246, and yet a fall in the IgM antibody from 86 to 20 in the same sera. Is this because one is reflective of an IgG response and the other IgM or a difference in sensitivity of detectability? DR. REMINGTON. This probably reflects the infant's IgG antibody response to antigen still present--the initial antibody titer being that of the mother, IgM titers in such infants may remain elevated for years. The reason for the decrease in IgM titer here is unclear. I have not seen this before. DR. BELLANTL A discrepancy like that? DR. REMINGTON. Yes. DR. BELLANTI. My second question is: Would it be of any value to use a fluorescent IgG test as well in terms of the late antibody responses? Since there may be a transition from IgM to IgG as the antigenic stimulation proceeds, would it be of any value in late specimens to use a fluorescent lgG as well as IgM test? DR. REMINOTON. We have not made it a point to study cases months or years after birth because the original purposes of our developing the technique was to pick up infection in newborn infants. After the first month of life I think it may be too late for definitive therapy. The damage is done. However, to make a definitive diagnosis of congenital toxoplasmosis in the later months of life one only needs to do the IgG fluorescent

1 12 3

antibody test. I think, however, that this has been a problem all along. We have been satisfied with following serological tests such as the dye test for 4 to 6 months and observing that the dye test in the infant did not fall as it should have if infection is absent. By that time the infant may be damaged irreparably. DR. ALVORD. We have also been attempting to determine the causes for the technical difficulties associated with the IglV[ fluorescent toxoplasma test. Our preliminary results indicate that different variables may be at fault. Undoubtedly, the lack of standardization of the conjugated antiIgM antiserum from commercial sources is of major importance in this regard. However, early results indicate the antigen storage may influence the outcome. For example, using the same conjugated IgM antisera, higher titers of IgM toxoplasma antibody were detected with freshly prepared or recently frozen antigen when compared with antigen that had been stored in the frozen state for over 1 month. Methods of fixation of antigen prior to storage and the number of toxoplasma present per field examined may also influence the results. When attention is directed to these other variables, different batches of conjugated gaInma-M antisera which were originally nonreactive gave adequate results, although some lots remained nonreactive under any circumstances. DR. REMINOTON. Actually we have not found this to be a similar problem. I have brought some data from experiments in which we performed the IgM fluorescent antibody test in a single day on 2 adults and 4 infants that were known to have IgM antibody by other techniques. The adults were positive. The negative control was negative. The 4 infants were all positive by the Behringwerke antiserum we had originally used and all negative by another Behringwerke and Hyland antiserum. These were all run parallel. DR. ALFORD. We have had that. DR. BERENDES. IS there any relation betwe~-n the outcome in the baby and the time at ~hich the mother gets the infection during pregnancy? In other words, do we know if the worst cases occnr in a mother who had the infection in the first trimester versus the asymptomatics which may be late? Is there information on this? DR. REMINGTON. There is a good deal in the literature; many statements and nmch conjecture, but unfortunately little in the way of facts. I think the problem people don't take into regard

1 124

Remington

is the variation in virulence of the organism. An infection with an avirulent strain eariy in pregnancy may not be as damaging as one due to a virulent strain late in pregnancy, There are no data on this. I think it should have come from Dr. Desmont's study but it was not in his manuscript, DR. SEVER. Have you noticed any problem with cross-reactivity of the entire system. T h a t is, cord sera or newborn sera which has a high IgM and gives a high FA (fluorescent antibody) titer, and when tested for C M V (cytomegalovirus) or other indirect tests, also comes up positive? We have noticed this. DR. REminGTON. The only experience we have had is in a collaborative study with Dr. Hanshaw and he is perhaps better able to answer that question. I know we examined one baby with Dr. Hanshaw that had both C M V and toxoptasmosis but there were others with C M V infection who did not have a positive I g M fluorescent antibody test for toxoplasmosis. DR. H a ~ s H a w . We had a 6-month-old baby that we suspected had C I D (cytomegalic inclusion disease) because of chorioretinitis and cere-

The Journal of Pediatrics December 1969

bral calcifications. We cultured the urine and found C M V . Sometime later we sent some serum to Dr. Remington for a toxoplasma macroglobulin test. T h e titer was very high. Looking back on this infant I am sure the child really had congenital toxoplasmosis rather than CID. T h e calcifications were not periventricular and the C M V macroglobulin test was equivocal. In general, if a baby is symptomatic--and I will talk more about that later--there will be a positive specific macroglobulin test, with very few exceptions. DR. REI~INGTON. The data for that baby shows that at 15 months the dye test titer was still 16,000, so there is no question about the congenital infection with toxoplasma. DR. AL~'ORD. Were I g M levels determined in the cord sera collected from your 20 cases of symptomatic toxoplasmosls ? DR. RE~amGTON. We don't have that data. DR. HaNsI~,tw. But the 20 cases that you mentioned that were symptomatic were studied later? DR. REMINGTON. Only IglV[ antibody levels specific for toxoplasma but not for I g M levels of cord sera.