perinatal torch' infections identified by serology: correlation with abnormalities in the children...

8/8/2019 Perinatal TORCH' Infections Identified by Serology: Correlation with Abnormalities in the Children through 7 Years of

1/8

International Journal of Epidemiology International Epidemiological Association 1992 Vol. 21 , No. 2Printed in Great Britain

Perinatal TORCH' InfectionsIdentified by Serology: Correlationwith Abnormalities in the Childrenthrough 7 Years of AgeJOHN L SEVER, JONAS H ELLENBERG. ANITA C LEY. DAVID L MADDEN. DAVID A FUCCILLO,NANCY R TZAN AND DOROTHY M EDMO NDSSever J L (National Institute of Neurological Disorders and Stroke, National Institute of Health, Bethesda, Maryland20892, US A), Ellenberg J H, Ley A C, Madden D L, Fuccillo D A , Tzan N R and Edmonds D M. Perinatal TOR CH 'infections identified by serology: correlation with abnormalities in the children through 7 years of age. InternationalJournal of Epidemiology1992; 21 : 285-292.A matched case-control methodology was used to assess the risk for a wide range of abnormalities in childrenassociated with serological evidence for 'TORCH' infections in the mothers. Specimens were selected from the largebank of sera from the approximately 54000 pregnant women who participated in the Collaborative Perinatal Project.There was no clear association between any of the antigens studied and any specific damage to the child. These'nega tive' findings are consistent w ith the absence of frequen t significant effects due to these agents in the second andthird trimesters of pregnancy.

The association between maternal infections withToxoplasma gondii, rubella, cytomegalovirus, andHerpes simplex, the 'TORCH' infections, during preg-nancy and certain types and frequencies of damageto the children of these pregnancies has been wellestablished.1 Most studies have concentrated on oneinfection at a time and the length of paediatric obser-vation has been variable.2 A few studies have used pro -spective serological testing to identify the maternalinfections.3

The present investigation employed a matched case-control methodology to assess the risk for a w ide rangeof abnormalities in children associated with serologicalevidence of 'TORCH' infections in the mothers. Wetook advantage of the large bank of serial serumspecimens from approximately 54000 pregnant w omenwho participated in the Collaborative Perinatal Pro-ject of the National Institute of Neurological and Com-municative Disorders and Stroke (NCPP) and the datafrom the 7-year follow up of the children. In theNCPP the pregnant women were studied prospectivelyfrom their first prenatal visit through delivery to 6National Institute of Neurological Disorders and Stroke NationalInstitutes of Health, Belhesda, Maryland 20892. USAReprint requests to : John L Sever, Children's National Medical Center111 Michigan Ave., NW Suite 2108 Washington. DC 20010, USA

weeks postpartum and serial blood samples were takenthroughout their pregnancy and postpartum. Womenin the NCPP whose children had certain abnormalities(e.g. fetal death, congenital heart disease, etc.) werematched with control mothers who did not have theseabnormal outcomes and the sera were tested undercode for antibodies to the 'TORCH' agents. The fre-quencies of seroconversions and significant increasesin antibodies for the abnormals and controls werecompared.METHODSAbout 54000 pregnant women entered the NCPP bet-ween 1959 and 1966, when they registered for prenatalcare at one of the 12 co-operating university hospitals.The general methodology and sampling frame for thisstudy has been described elsewhere.4 Of this popula-tion 46% was white, 46% was black, and most ofthe remainder were of Hispanic origin. Overall, thepopulation was slightly lower in socioeconomic statusthan the US population.5 The pregnant women werescheduled for serial serum specimens at the time ofregistration, every 2 months throughout pregnancy, atdelivery, and 6 weeks postpartum. Serum specimenswere aliquoted, frozen, shipped to the National In-stitutes of Health and held at the Serum Center of theInfectious Diseases Branch, NINDS for later study.

285

byguestonJanuary6,2011

ije.oxfordjournals.org

Downloadedfrom
http://ije.oxfordjournals.org/http://ije.oxfordjournals.org/http://ije.oxfordjournals.org/http://ije.oxfordjournals.org/http://ije.oxfordjournals.org/http://ije.oxfordjournals.org/http://ije.oxfordjournals.org/http://ije.oxfordjournals.org/http://ije.oxfordjournals.org/http://ije.oxfordjournals.org/http://ije.oxfordjournals.org/http://ije.oxfordjournals.org/http://ije.oxfordjournals.org/http://ije.oxfordjournals.org/http://ije.oxfordjournals.org/http://ije.oxfordjournals.org/http://ije.oxfordjournals.org/http://ije.oxfordjournals.org/http://ije.oxfordjournals.org/http://ije.oxfordjournals.org/http://ije.oxfordjournals.org/http://ije.oxfordjournals.org/http://ije.oxfordjournals.org/http://ije.oxfordjournals.org/http://ije.oxfordjournals.org/


2/8

286 I N T E R N A T I O N A L J O U R N A L O F E P I D E M I O L O G YChildren born into the project underwent a seriesof examinations, among which were standardizedphysical and neurological examinations at newborn,1 year, and 7 years, a psychological examination at 4years of age, and a speech, language and hearingexamination at 3 years. Standardized follow-up of thechildren continued through 1973, when the last childcompleted the 7-year follow-up examination.Seventeen abnormalities were selected for study, andare listed in Table 1. A case/control methodology wasemployed to examine the association of infection witheach abnormality. This approach required serologicaltesting of only a fraction of the entire cohort of over54000 women, for the five antigens selected forstudy. Matching was introduced to control for con-founding factors and thereby increase the validity ofthe inferences.

TABLE 1 Abnorm al outcomes in case-control studies

Abnormalout comesFetal deathBirthweight ^2 00 0 gNeonatal seizuresNeonatal deathCongenital hearl diseaseCongeni tal malformat ionsNewborn bi li rubin > I 7 mg^oMicrocephalyMacrocephalyHypoton i aDelayed motor developmentFine or gross motor developmentAbn orm ality of liver at 1 yearHearing deficitI Q < 7 0Cerebral palsyEpilepsy

Number of caseswith adequateblood samples

580577122 .296258

276956959937310037140 224 958582597170

Phase of studyin which sero-logical testing

was performed111

II1II1

IIII111111

IIII

Mothers who gave birth to singleton children witheach of the abnormalities were matched with womenwhose singleton children had none of the specified ab-norm alities. The control mothers were selected by mat-ching each case mother with a control from the samehospital, of the same race, with age 5 years, withlast menstrual period (LMP) 4 weeks, and early andlate serum specimens taken at comparable times duringpregnancy ( 6 weeks).

Single controls were picked for each of the casemothers from those within the limits above using analgorithm which ranked the 'closeness' of each poten-tial control to its case mother. For each case mother

the unweighted sum of the squares of the differencesbetween each of the factors, LMP (in weeks), age (inyears), date of earliest serum specimen and date oflatest serum specimen (in weeks) was computed. Threesuch controls were selected, ranked according to theircloseness to the cases. The closest control was chosenunless insufficient serum was available for testing, inwhich case the next closest subject was chosen. Thisprocedure gave least importance to the age differencecriterion. Controls were used only once.Before selection of matched controls, both cases andcontrols were screened for the adequacy of their bloodsamples. All cases and controls were required to haveboth an early (


3/8

PERINATAL TORCH INFECTIONS AND CHILDHOOD ABNORMALITIES 2 8 7TABLE 2 Distribution of time of early an d late blood samples in relation to last menstrual period (LMP) and termination of pregnancy

Time sinceLM P (weeks)0-12

13-2021-28>2 9

Total

Early blood sample

N o.942

25592261655827

%16.243.938.8I.I

100.0

Late blood

Time oflate blood

At delivery (37-43 weeks)Post delivery (4-12 weeks)OtherToial

samplea

N o.16523865

3065827

7o28.466.4

5.3100.1

aWomen whose pregnancy terminated in an abortion, stillbirth or a very low birthweight baby are not included.

earlier times, and are not included in this Table. Thecohort is such that we are able to assess the impactof infections essentially on the second and thirdtrimesters of pregnancy.Materials and Methods of Laboratory TestingThe 'TORCH' agents were selected for this initialstudy because of their established role as perinatalinfections which cause morbidity and mortality inchildren. The Indirect Haemagglutinat ion (IHA) andComplement Fixat ion (CF) with Microtechnique wereused for this study. Previous studies utilizing thesesystems for serological diagnosis 'en masse' demon-strated the useful applicat ions of these techniques. 6 7Methods of Preparation of AntigensThe antigens used, their host-cell source and the strainare shown in Table 3. Antigens were stored at -70C.A control antigen consisting of uninfected tissue andfluids maintained in the same way as the antigen wasprovided for each infectious agent.TABLE 3 Antigens used in NCPP studyAntigen

CytomegalovirusHerpes 1Herpes IIToxoplasmaRubella (Phase 1)Rubella (Phase II)

Host-cell sourceIndirect haemagglulinaiion test

MA-184MA-196MA-196Mouse peritoneal fluidComplement fixationBHK 21Vero

Strain

AD-169MaclntyreM SBeverlyGilchristTherien

Indirect haemagglutination antigensCytomegalovirus and Herpes I and II antigens were pre-pared according to the methods reported previously.8-9

Briefly, they consisted of infected tissue cultures show-ing 3 + to 4 + CP E and processed as cell pack antigensusing centrifugation and freeze/ thawing proce dures.Toxoplasma antigen was prepared from Toxo-

plasma gondii organisms obtained from peri tonealexudates of infected mice. The organisms were rup-tured by freeze-thawing once, followed by sonicationfor 20 seconds at 4C. The cell debris was removed bycentrifugation at 10000 g for 30 minutes. The superna-tant fluid was stored as antigen.Complement fixing antigensRubella antigen for Phase I of this study was preparedwith BHK-21 infected tissue cultures in a 5% suspen-sion according to the methods reported previously. l0Antigen for Phase 2 was prepared in Vero Tissue usinga 10% suspension of tissue culture material and fur-ther purification with glycine hydroxide, freezing andthawing three times, sonication for 3 minutes andalkaline-extraction at 37C for 6 hours.Other ReagentsAll control positive and negative sera were obtainedfrom personnel and remained the same throughout thestudy.The commercial ly prepared complement was anti-body free of a screen of viruses including rubella. Eachlot was pretested before purchase to maintain a con-stant titre.Sheep red blood cells were from one of two sheepchosen for their similarity in reaction and maintainedat the NIH farm.Consistency of Procedures and ReagentsThroughout the test ing programmes many techniqueswere applied to assure consistent results. All person-nel, biologists, microbiologists, biology laboratory

bygu

estonJanuary6,2011


Downloadedfrom


4/8

288 INTERNATIONAL JOURNAL OF EPIDEMIOLOGYtechnicians and medical technicians, were trained inMicrotechnique by the same person. The Micro-technique materials, i.e. plates, loops, etc. remainedconstant throughout these tests.The paired serum specimens were coded andscrambled for testing. These specimens which wereshipped and stored at -20C were thawed rapidly.Initial dilutions of 1:4 for CF, 1:8 for IHA viruses and1:32 for toxoplasmosis were held at 4C during testingand -20C for storage, even if only overnight. Thisminimized the anticomplementary effect in CF andsome nonspecific reactions in IHA. Repeat tests usedthis initial dilution unless anticomplementary ornonspecific reactions were noted. In that case a newdilution was made from the stock vial.

Antigens, except toxoplasma were prepared undercontract by Microbiological Associates, (MBA) Inc.Strains, host sources and preparation methods re-mained constant. Specificity and antigenicity werepretested by MBA and our laboratories for agreementbefore acceptance. A common source of complement,haemolysin, red blood cells and positive and negativeantisera was maintained from a minimum of differentlots.Reproducibility within twofold dilution was re-quired for each specimen tested by CF and fourfoldby IHA. All sera were titrated in duplicate on the dayof testing. Any questionable data and all fourfold(CF) and eightfold (IHA) or greater differences wereretested for explanation and verification.

Anticomplementary effect or non-specific reactionsnoted with control antigen were interpreted as shownin Table 4.All CF results were read by the same person. TheIHA tests had three different readers.DefinitionsTwo definitions of antibody responses were appliedto the serological data. The first or seroconversion oc-curred if the early titre was negligible and the later titresatisfied the values indicated in Table 5. Significant in-crease in antibody occurred if the difference betweentitres for early and late bloods indicated a specific risefrom a low titre. The observed frequency of these in-fections among the control cases according to type ofantibody response is given in Table 5. The high rate ofrubella reflects the occurrence of the rubella epidemicin 1963.

The paediatric outcomes were defined as follows:Fetal death: abortion or stillbirth.Neonatal seizures: seizures occurring within the first28 days of life.Congenital heart disease: gross structural abnorma-lity of the heart or intrathoracic great vessels that wasactually or potentially of functional significance ."Congenital malformations: major central nervoussystem malformations and related skeletal conditions,eye, ear, respiratory tract, thoracic, alimentary tractmalformations, abnormalities of liver, bile duct andspleen, genitourinary conditions, tumours and skin

T A B L E 4 Interpretation of anticomplem entary effect or nonspecific re actions noted w ith control antigenAnticomplementery or nonspecificreactionScrum dilution

Test Reactions'1Serum Dilution

1:41- 41-41- 41- 41-44321Ir

si ir

1:83-421tr

si tr000000

1:4XXXXXXXXX

4 b Accept3 b XAccept

1:8XXX

4 b Accept3 b XAccept

-

1:16XX

Accept

1:32XXAccept*

1:64XAccept

*

1:128XAccept**

"Test results rejected (X) wiih various levels of anticomplementary effect or nonspecific reaction.bRead ing of 4

byguestonJanuary6,2011


Downlo

adedfrom


5/8

PERINATAL TORCH INFECTIONS AND CHILDHOOD ABNORMALITIESTABLE 5 Criteria for seroconversion and significant increase in antibody

28 9

Antigen

To.xoplasmosisRubellaCytomegalovirusHerpes 1Herpes 11

Seroconversion

Early

0 or 32a04 a

0 o r 8 a"

Late

1283=43=8

5*32

Observedfrequency

among controlsRate/10 000

18.988 .4 b35.710.86.3

Significant increase

Early3=323:45=8

in antibodyLate

3:2563:163=16

(8-fold)(4-fold)(4-fold)

Observedfrequencyamong controls

Rate/10 0007.3

55.4185.020.021.9

indicates t race act ive at indicated dilution.Study period covers lime in which 1963 rubella epidemic occurred

malformat ions . All syndromes, including mongolismand Herler ' s Syndrome, and Pompe's diseases, wereexcluded from this classification if they were the onlycondit ion present in a child. Congenital heart condi-tions were treated separately and were not included inthe congenital malformation category. Minor malfor-mations, other than polydactyly, syndactyly andenlargement of thigh and leg, were also excluded.

Microcephaly and macrocephaly: A child was con-sidered micro or macrocephalic if his fronto-occipitalhead circumference normed for gestational age, sexand race deviated more than 2 standard deviat ions forthe mean on two or more examinations between new-born and 7 years (newborn, 4, 8, 12, 48 mont hs and 7years).Hypot on i a at 1 year: hypotonic with deep tendonreflexes.Delayed motor development: delayed milestones ofmotor development at 1 year.Fine or gross motor development: clinical impres-sion of abnormal fine or gross motor development at 4years.Enlarged liver at 1 Year: liver size greater than 2cm.below the costal margin in the right midelavular line orconsistency unusual .Hearing deficit at 3 years: failed to respond to oneor more of the frequencies 500, 1000 or 2000 at 20 db.in either ear.IQ


6/8

290 INTERNATIONAL JOURNAL OF EPIDEMIOLOGYinfection. Odds ratios greater than one indicate apositive association of the infection with the risk of theoutcome. The OR in a matched case-control study iscomputed as the ratio of the number of matched pairswhere the affected child was born to a mother with aspecified infection and the control child was not, to thenumber of matched pairs where the control child wasborn to a mother with a specified infection and theaffected child was not. The importance of the OR isa function not only of its magnitude, but also of theseverity and prevalence of the outcome. A doubling ofrisk (OR = 2) for fetal death would be considered moreimportant than the same OR for the outcome ofasthma. Similarly, a doubling of risk when the pre-valence of the outcome is of the order of magnitudeof one in a million is not equivalent to a doubling ofrisk when the prevalence is around one in a thousand.In addition, the observed OR must be assessed alongwith measures of its statistical significance and thewidth of the confidence interval with which it isestimated, both of which are affected by the samplesize of the study as well as the inherent variability ofthe observations. The confidence interval for anobserved OR gives the precision with which one makesan estimate. A wide interval indicates a large varia-bility in the estimate.

There were over 170 case-control studies involvingone of the five antigens and one of the 17 outcomes. Intesting whether differences in rates of seroconversionor significant increase in antibody associated with theabnormal cases were statistically significant (as in-dicated by a high OR), MacNemar's test for matchedpairs was utilized.12 Since there was such a largenum ber of tests involved, with some, but not all, of thetest comparisons specified prior to the onset of thestudy , som e of the tests could be significant by chancealone. We did not adjust significance levels usingmultiple comparisons techniques, which would lessenthe chance of observing some clinically important riskfactors.13 Rather, we chose to consider the significancevalues as indicators for risk factors that are candidatesfor confirmation in future studies. Nevertheless, toprovide some protection against this problem onlystatistical tests with a tabled P value of P


7/8

PERINATAL TORCH INFECTIONS AN D CHILDHO OD ABNORMALITIES 291

TABLE 6 Largest" and smallest odds ratios (OR) for ab normal outcome related to seroconversion by antigenAbnormalout come

Toxoplasma Rubella Cytome-galovirus Herpes I Herpes II

Fetal death (abortionsor stil lbirths)

Birthweight ^20 00 gNeonatal seizuresNeonatal deathCongenital heart diseaseCongeni tal malformat ionsBi l i rubin >17mg%Microcephaly 2SDMacrocephaly 2SDHypotonia 1 yearDelayed motor developmentFine/gross motor developmentEnlarge d liver 1 yearHearing deficit 3 yearsIQ 17mg%Microcephaly 2SDMacrocephaly 2SDHypotonia I yearDelayed motor developmentFine/gross motor developmentEnlarged liver I yearHearing deficit 3 yearsIQ


8/8

29 2 I N T E R N A T I O N A L J O U R N A L O F E P I D E M IO L O G Yprevalance of herpes in this population, the absence ofan association with fetal damage may be due to thesmall sample size.A similar low prevalence of infection was also seenfor toxoplasmosis. Our recent report on toxoplasmosisin 23000 pregnancies showed associations betweenmaternal infections and certain maternal andpaediatric findings.3 Several of the associations withpaediatric ou tcomes found were also examined bu t notconfirmed in the current study, and included hearingimpairment, microcephaly and 1Q. Several reasonsmay account for the lack of confirmation. The mor-bidity was defined differently for each of the outcomesin the two studies. For example, IQ was measured at 7years for the former study and 4 years here, and hear-ing impairment was defined as bilateral deafness in theformer study and hearing deficit in either ear in thisstudy. The former study also examined primarily highantibody to toxoplasma based on a single bloodsample at delivery or within 6-15 weeks postpartum,not a p redetermined high increase or seroconversion asin the present study. The most likely reason for theapparent inconsistency of the two studies is the moresensitive study design of the first study. The formercohort study involved the examination of some 23 000women and the association of the outcomes with anyincreases in titre to toxoplasmosis.ACKNOWLEDGMENTSThe Collaborative Study of Cerebral Palsy, MentalRetardation, and other Neurological and SensoryDisorders of Infancy and Childhood is supported bythe National Institute of Neurological Disorders andStroke. The following institutions participated: BostonLying-in Hospital; Brown University; CharityHospital, New Orleans; Children's Hospital of Buf-falo; Children's of Philadelphia; Children's MedicalCenter, Boston; Columbia University; The JohnsHopkins University; Medical College of Virginia; NewYork M edical College; Pennsylvania H ospital; Univer-sity of Minnesota; University of Oregon; University

of Tennessee; Infectious Diseases Branch, Biometryand Field Studies Branch, and the DevelopmentalNeurology Branch, National Institute of NeurologicalDisorders and Stroke.We thank Alan Talbert, who did the computer pro-gramming for this study.REFERENCES

' Sever J L, Larsen J W Jr. and Grossman J H. HI. Handbook ofPerinatal Infections. Boslon: Little, Brown and Company,1979.

2 Sever J L, Nelson K B and Gilke son J R. Rubella epid emic 1964:Effect on 6000 pregnancies. Am J Dis Child 1965; 100:395-406.

3 Sever J L, Ellenberg J H, Ley A C, el al. Toxoplasmosis: Maternaland Pediatric Findings in 23000 Pregnancies. Pediatrics 1988;82 : 181-92.

4 Niswander K, Gordon M. The Women and Their Pregnancies.National Institute of Health Publication, 73-379. US Depart-ment of Health, Education and Welfare, 1972.

5 Myrianthopo ulos N C, French K S. An A ppl ication of the USBureau of the Census Socioeconomic Index to a Large, Diver-sified Patieni Population. Soc Sci Med 1968; 2: 283-99.

6 Sever J L. Application of a Microtechnique to Viral SerologicalInvestigations. J Immunol 1962; 88 : 320.Sever J L, Huebner R J, Castellano G A, el al. Serologic Diagnosis

"en masse" with Multiple Antigens. Am Rev Resp Dis 1963;88: Part 2.

Fuccillo D A, Moder F L, Traub R, et al. Micro Indirect Hemag-glutination Test for Cytomegalovirus. Appl Microbiol 1971;21 : 104.

Fuccillo D A, Moder F L, Catalano L W Jr. el al. Herpes HominisTypes 1 and II: A Specific Micro Indirect Hema gglutinationTest . Proc Soc Exp Biol Med. 1970; 133: 735.10 Sever J L, Huebner R J, Castellano G A, el al. Rubella Comple-ment Fixation Test. Science 1965; 148: 385.

" Mitchell S C, Korones S B and B erendes H W. Conge nital HeartDisease in 56109 Births - Incidence and Natural History. Cir-culation 1971; 43 : 323.

12 Fleiss J L. Statistical Methods for Rates and Proportions. NewYork, John Wiley and Sons, 1973.13 Mantel N, Haenszel W. Statistical aspects of the analysis of datafrom retrospective studies of disease. JNCI 1959; 22: 719 -48.

14 Sever J L. Infections in pregnancy: Highlights from the col-laborative perinatal project. Teratology 1982; 25: 227-37.

(Revised version received June 1991)

byg

uestonJanuary6,2011


Downlo

adedfrom

perinatal torch' infections identified by serology: correlation with abnormalities in the children...

Documents