000024929

8/8/2019 000024929

http://slidepdf.com/reader/full/000024929 1/9

Intervirology 1998;41:149–157

Cytomegalovirus Infection in

Pregnancy: A Still ComplicatedDiagnostic Problem

T. Lazzarotto

P. Spezzacatena

P. PradelliD.A. Abate

L. Gabrielli

S. Varani

M.P. Landini

Department of Clinical and ExperimentalMedicine, Division of Microbiology,University of Bologna, Italy

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Key Words

CytomegalovirusPregnancy

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Summary

The diagnostic problems linked to human cytomegalovirus (HCMV) in preg-nancy are many and not all have been fully defined. In long-term seropositivewomen there is a tacit agreement that no laboratory testing for HCMV shouldbe carried out. In seronegative women a test for HCMV-specific IgG should beperformed at least twice during the first 4 months of pregnancy, and if theseronegativity persists, further follow-up might be stopped. On the other hand,if a seropositivity appears the diagnosis of a primary HCMV infection is estab-lished and prenatal diagnosis should be offered to the mother. Finally, in thecase of a pregnant woman with unknown serological status, the diagnosis of

HCMV infection is a complex problem and several different questions need tobe addressed. In our opinion they should be screened with a reliable IgM test(confirmed by blot if necessary) followed, in the case of positivity, by an avidi-ty assay. Pregnant women undergoing a primary HCMV infection should beencouraged to seek prenatal diagnosis to be performed by PCR and virus isola-tion from amniotic fluid at the 21st to 23rd week of gestation.OOOOOOOOOOOOOOOOOOOOOO

Maria Paola Landini, MDDepartment of Clinical and Experimental Medicine

Division of Microbiology, Policlinico S. Orsola-MalpighiVia Massarenti 9, I–40138 Bologna (Italy)Tel. +39 051 346306, Fax +39 051 341 632, E-Mail [email protected]

ABC

Fax +41 61 306 12 34E-Mail [email protected]

© 1999 S. Karger AG, Basel0300–5526/98/0415–0149$17.50/0

Accessible online at:http://BioMedNet.com/karger

Introduction

Human cytomegalovirus (HCMV) is the leading cause

of congenital infection in developed countries accountingfor 0.5–2.2% of all live births. Recent data continue tosupport this epidemiological situation (table 1) [1–3]. Ap-proximately 10–15% of congenitally infected infants willhave major damage that may include asymmetricalgrowth retardation, hydrocephalus, chorioretinitis, opticatrophy, microphthalmia, cerebral calcifications anddeafness (approximately 50% of the HCMV-infected

newborns with symptoms at birth have severe auditorydefects). Furthermore, over half the patients with chorio-retinitis or optic atrophy have significant biocular vision

impairment [4–5]. Of the 85–90% infected newborns whoare asymptomatic at birth, 10–20% will have subtle,delayed impairment in the form of mental retardation (IQless than 70), hearing problems and visual impairment[6–8]. This means that approximately 3 out of 1,000 new-borns suffer from a congenital HCMV infection. Sinceapproximately 500,000 babies were born in Italy during1996, one can estimate that around 1,500 babies are

8/8/2019 000024929


Ivarsson et al. [1]

1977

150 Intervirology 1998;41:149–157 Lazzarotto/Spezzacatena/Pradelli/Abate/Gabrielli/Varani/Landini

Table 1. Frequency of congenital HCMV infection reported in1997

Authors Country Number positive/Number tested

Congenitalinfection, %

Sweden 44/9,806 0.5Natali et al. [2] Italy 6/1,045 0.6

Lazzarotto et al.1

Italy 6/564 1.1Fowler et al. [3] Alabama, USA 22/1,828 1.2

1 Unpublished data.

Table 2. Seropositivity for HCMV in Northern Italy amongblood donors

Year Samples tested Seropositivity, %

100 891985 300 801994 300 741997 300 71

The test was performed by complement fixation in 1977 and byELISA in 1985, 1994 and 1997.

experiencing or will experience clinical problems due toHCMV aquired in utero (approximately 40,000 per year

in the USA [7, 9]). Therefore, congenital HCMV infectionshould be considered a major medical and socioeconomicproblem. Furthermore as the percentage of HCMV-sero-positive adults is in general decreasing (table 2), the per-centage of HCMV-seronegative women of childbearingage is increasing. Therefore, it is likely that in the nearfuture an increase in the number of primary infections inadults will be observed, paralleled by an increase in con-genital HCMV infections.

Mother-fetus transmission can occur throughout gesta-tion and infant outcome does not seem to be related to thetrimester of maternal infection [10] although infection

during the first 16 weeks of pregnancy has been associatedwith a higher rate of damage [11]. HCMV can also betransmitted to the fetus when the initial maternal infec-tion occurred before conception but data are not availableon the consequences for the newborn in this event. Con-genital HCMV infection can be the result of both exoge-nous and endogenous maternal infection. While exoge-nous infection can be primary or secondary, endogenous

infections are the result of reactivation of a latent virus. Ingeneral over 90% of HCMV infections in pregnant wom-en, as in most otherwise healthy individuals, are asymp-tomatic and are likely to go undetected by a woman andher physician. Furthermore, symptomatic infections areusually accompanied by symptoms that are not specificfor HCMV, such as fever, fatigue, headache, myalgia and

sore throat. For these reasons, laboratory techniques re-present a decisive diagnostic approach. The diagnosticproblems linked to HCMV in pregnancy are many andnot all have been fully defined. A first distinction shouldbe made with respect to the prepregnancy serological sta-tus.

Diagnosis of HCMV Infection in Pregnant Women

with Known Serological Status

While primary maternal HCMV infection is linked to10–15% of symptomatic infections at birth and 10–15%of infections with delayed sequelae, nonprimary maternalHCMV infections rarely lead to fetal HCMV infection[12]. This implies that cellular and humoral immune areimportant for controlling detrimental effects of HCMVinfection. The role of the cell-mediated immune response(CMI) in preventing maternal to fetal transmission of HCMV is questionable. In fact, although CMI is themajor factor responsible for HCMV control, it is unlikelythat CMI plays an important role in determining differ-ences between seropositive and seronegative women as it

has been shown that in seropositive pregnant womenHCMV-induced lymphocyte proliferation is dramaticallydepressed during the second and third trimester of normalpregnancy while levels of antibodies to HCMV remainedunchanged [12, 13].

The role of the humoral immune response is also ques-tionable. Antibodies do not protect from infection sincethe infection generally resolves before antibody levels rise.Yet administration of antibodies protects animals fromdisease. Similarly, newborns that are breast-fed by im-mune mothers may get protected from infection. HCMVcan be transmitted despite the presence of passively

acquired antibody, although HCMV disease may be at-tenuated by antibody administration or by vaccinationwith the Towne vaccine [for review, see 14]. Therefore,the humoral immune response does not protect frominfection but does protect from severe disease. Recently,Fowler and coworkers [3] reported a 1.2 and 12.9% trans-mission in seropositive and seronegative women, respec-tively, indicating that preconceptional maternal immuni-

8/8/2019 000024929


HCMV Diagnosis in Pregnancy Intervirology 1998;41:149–157 151

ty is protective against congenital HCMV infection de-creasing the risk of infection by 90%. Taking into consid-eration the situation described above, the prepregnancyserological status of the mother is extremely important. Inthis respect two possibilities exist: (1) The woman is long-term seropositive (approximately 50–70% of the cases inindustrialized countries). What she could experience dur-

ing pregnancy is a viral reactivation which occurs in thepresence of specific immunity. The risk of transmission isapproximately 1% and even when the virus is transmittedit is linked to a less virulent outcome, as previouslypointed out. Therefore, in this case there is a tacit agree-ment that no laboratory testing for HCMV needs to becarried out. (2) The woman is seronegative for HCMVand is thus susceptible to primary infection. In this case atest for HCMV-specific IgG should be performed at leasttwice (2nd and 4th month of pregnancy). If the seronega-tivity persists, a further follow-up might not be necessary.

On the other hand, if seroconversion is detected in a fol-low-up serum sample and the diagnosis of a primaryHCMV infection is established, prenatal diagnosis shouldbe offered to the mother (see below).

For determining the serological status for HCMV, IgGtesting can be performed using one of the numerous testsavailable on the market today. Sensitivity and specificityas well as the correlation between the results obtainedwith different procedures and the agreement between theresults obtained by the same procedure carried out withkits from different companies are reasonable. The prob-lem remains of the high cross-reactivity with other mem-

bers of the Herpesviridae family [for review, see 15]. Withthe introduction on the market of kits which use well-selected recombinant antigens instead of entire viral par-ticles or infected cell lysates, the problem of cross reactivi-ty should become much less important.

Diagnosis of HCMV Infection in Pregnant Women

with Unknown Prepregnancy Serological Status

Knowledge of the prepregnancy serological status forHCMV is desirable. However, this is still uncommon. In

the case of a pregnant woman whose preserological statusis unknown, the diagnosis of HCMV infection is a com-plex problem and several different questions need to beaddressed.

Diagnosis of an active HCMV infection in pregnantwomen can be obtained searching for the virus or its com-ponents (antigens or nucleic acids) in urine, saliva, cervi-cal swabs and other secretions [16, 17]. Although during

an active HCMV infection the virus can be detected inany secretion (p.e. by ‘shell vial’ assay), the duration andquantity of virus excretion may vary widely, and may beintermittent. Therefore, detecting an infectious virus is anunreliable approach for laboratory diagnosis of acuteHCMV infection particularly during pregnancy [12]. Al-ternatively, HCMV-specific IgM can be determined as a

sensitive and specific indicator of primary HCMV infec-tion in immunocompetent subjects. While low or border-line titers are characteristic for HCMV reactivation inboth immunocompetent and immunocompromised sub-

jects, HCMV IgM titers are usually high during primaryHCMV infection [12, 18, 19]. However, the correlationamong results obtained with different commercial kits ispoor [20–24]. The use of recombinant HCMV proteins orpeptides has proven to be a promising tool in improvingserological diagnosis. The analysis of the humoral im-mune response elicited during natural infection has clear-

ly identified highly immunogenetic proteins [25–30]. Anew generation of serological tests containing a well stud-ied cocktail of genetically defined HCMV antigens will beon the market soon and should make IgM detection morereliable [31, 32]. However, it is to be expected that we willcontinue to deal with a high number of discordant resultsand a golden standard would be desirable. With this inmind, we devised a novel Western blot (new WB) test foranti-HCMV IgM detection which contains viral structuralpolypeptides, significant portions of recombinant p150(ppUL32) and significant portions of the most immuno-genic nonstructural protein p52 (ppUL44) and UL57

[33]. The new WB was evaluated in latently infectedblood donors, pregnant women and transplant recipientswith ongoing HCMV infection. The new WB proved to bemore sensitive and specific than traditional WB or con-ventional enzyme immunoassays and, furthermore, cor-related well with the consensus of different ELISA kits[34]. These results favor the new WB as a reliable refer-ence test in case of discordant or borderline results. A rep-resentative new WB test is shown in figure 1.

As shown in table 3, 17.9% of pregnant women whowere IgM-positive by the new test and IgM-negative bytraditional ELISA had congenitally infected newborns

(unpublished data). When the virus is found in a secretionand/or IgM are found in the serum, the diagnosis shouldtake into consideration the differentiation between pri-mary and recurrent infection.

8/8/2019 000024929


+


Fig. 1. Representative examples of serum reactivity with the newWB. Numbers on the right of the figure represent molecular weight(!1,000) of viral (vp) or recombinant (rp) proteins. CKS is the

Escherichia coli CMP-KDO synthetase which represents the negativecontrol; Ì is the IgM heavy chain and represents the positive control.Numbers on top of the gel correspond to IgM-positive (1–5) and IgM-negative (6, 7) human sera from pregnant women.

Differentiation between Primary and Nonprimary

HCMV Infection

In HCMV-infected pregnant women primary infectioncan be identified by serology demonstrating (1) serocon-version, (2) high titers of HCMV-specific IgM, (3) oligo-clonal humoral immune response and (4) presence of lowavidity antibodies.

Searching for the Oligoclonal Antibody Response by

Immunoblotting

Primary infections are usually characterized by im-mune reactivity to one or very few proteins, while in non-

primary infections the reactivity is much broader. In par-ticular, early during primary HCMV infection antibodiesspecific for ppUL83 (pp65) and/or ppUL44 (p52) aredetectable. Later specificity of antibody response broad-ens and additional proteins are recognized. In contrast,serum samples from patients with an ongoing nonprimaryinfection show a much broader reactivity with at least 4–5proteins being detected [35].

Table 3. IgM serology in the mother and congenital HCMVinfection

IgM serology

ELISA new WB

Pregnantwomen

Infectedfetuses/infants

+ 16 2 (12.5)+ – 1 0– + 28 5 (17.9)– – 32 0

Figures in parentheses represent percentage.

Avidity of CMV-Specific IgG

Another serological procedure that is used for identifi-cation of primary viral infections is the determination of IgG avidity, exploiting the functional affinity of IgG class

antibody. Initially following primary infection, antibodiesare of low avidity, and they progressively maturate ac-quiring higher avidity during the ensuing weeks andmonths (fig. 2). Determination of IgG avidity for distin-guishing primary from nonprimary infections has beenestablished for RNA and DNA viruses such as rubella,varicella zoster, human herpesvirus 6, hepatitits C andmeasles virus and HCMV [35–42]. Our own experiencewith anti-HCMV IgG avidity testing using a commercial-ly available kit shows that reliable distinction betweenprimary and nonprimary HCMV infection in both preg-nant women and solid organ transplant recipients is possi-

ble. In fact 88.6% of primary infections and no nonprima-ry infection revealed low avidity IgG to HCMV. In partic-ular, a low IgG avidity is a marker of primary infection for18–20 weeks after onset of symptoms in immunocompe-tent subjects [43]. Similar results were obtained by Gran-geot-Keros and coworkers [44].

Virology

Perhaps because isolation of HCMV or detection of antigenemia in blood of normal hosts is rarely successful,the study of maternal viremia or antigenemia received lit-tle attention until very recently [45, 46]. The results indi-

cate that antigenemia, viremia and DNAemia can bedetected during pregnancy, particularly during primaryinfection. Although promising results have been reported,further data are required to understand the pathogenesisof maternal HCMV infection. A proposed algorithm forthe follow-up of pregnant women whose prepregnancystatus for HCMV is unknown is presented in figure 3.

8/8/2019 000024929



Fig. 2. Cograduation graph showing thecorrelation between the avidity index andthe number of weeks after primary HCMVinfection in pregnant women. Correlationanalysis of the joint distribution of the twoseries of values gave a correlation coefficientof 0.8726, indicating a strong associationbetween the two parameters.

Fig. 3. Proposed algorithm for HCMV monitoring of pregnant women with unknown prepregnancy serologicalstatus for HCMV.

8/8/2019 000024929


HCMV-infected mothers


Table 4. Virus isolation and PCR on amniotic fluid fromHCMV-infected pregnant women

Positive results onamniotic fluid by

virus isolation PCR

Transmitters (n = 16) 11 16Nontransmitters (n = 79) 0 16

Test evaluationSensitivity a 70 100Specificity b 100 80Positive predictive value c 100 50Negative predictive value d 94 100

a (Number of true positives/total number of transmitters) !100.b (Number of true negatives/total of nontransmitters)!100.c (Number of true positives/number of true positives + number of false positives)!100.d (Number of true negatives/number of false negatives + number of true negatives)!100.

Prenatal Diagnosis

In cases where primary HCMV infection is diagnosedduring pregnancy, obvious concerns arise over potentialeffects of intrauterine transmission to the developingfetus. Although transmission of HCMV from mother to

fetus may occur any time during pregnancy, severe neuro-logical complications are more likely to arise when theinfection occurs during the first half of gestation [10, 11,47, 48]. If a primary HCMV infection is diagnosed in apregnant woman, should prenatal diagnosis of HCMV beoffered? This is an important issue and several consider-ations should be made. For approximately 70% of womenwith primary infection the results will indicate the ab-sence of fetal infection and this will prevent an unneces-sary abortion of uninfected fetuses and help the women tocontinue their pregnancies with a high level of confidence.In those cases with documented viral transmission some

women will choose termination of pregnancy. Others willopt to continue their pregnancies. A careful follow-up of these women may help to establish parameters that willidentify severely infected fetuses. At present there is notsufficient evidence to predict a fatal outcome. However,abnormalities may be detected with fetal ultrasound scan-ning as part of a routine prenatal care suggesting a possi-ble intrauterine infection. Findings in the fetus that

should alert the clinician to the possibility of intrauterineHCMV infection include the presence of oligo- or polyhy-dramnios, nonimmune hydrops, fetal ascites, intrauterinegrowth retardation, microcephaly, hydrocephalus, intra-cranial calcifications, pleural or pericardial effusion, he-patosplenomegaly, intrahepatic calcifications or pseu-domeconium ileus. Other intrauterine infections (herpes

simplex virus, varicella zoster virus, HIV, rubella, syphilisand toxoplasmosis) can cause similar clinical findings andshould be considered in the differential diagnosis.

Prenatal diagnosis of fetal infection may lead to treat-ing the mother with ganciclovir, an agent that inhibitsHCMV replication. Ganciclovir is currently administeredto newborn infants congenitally infected with HCMV andencouraging results have been obtained [49]. However,treatment of the affected neonate probably comes too late,as much of the virus-induced damage may already haveoccurred before delivery.

For these reasons we think that prenatal HCMV diag-nosis should be offered to mothers with documented pri-mary HCMV infection. It is thought that HCMV is trans-mitted when infected leukocytes cross the placental bar-rier to reach the fetal circulation via umbilical cord vessels[50, 51]. However, other routes may be equally accessibleto viral transmission. Even if in vitro data do not substan-tiate this hypothesis [52] one possibility is that the virusmay first infect placental tissues and later amniotic cells.Consequently, infected amniotic cells would be ingestedby the fetus, after which the virus could replicate in theoropharynx and invade the fetal circulation to reach tar-

get organs. The tubular epithelium within the kidneyappears to be a major site of viral replication. By eithermechanism of infection, the fetus would excrete HCMVvia urine into the amniotic fluid. The amniotic fluidtherefore seems a logical choice of bodily fluid for the pre-natal diagnosis of HCMV transmission. In amniotic fluidthe virus can be found by culture and/or by PCR [53–57].

Table 4 shows recent results we obtained comparingvirus isolation and PCR for detecting HCMV in theamniotic fluid. Only by the combination of both tech-niques a 100% sensitivity and 100% specificity can be

achieved. However, several variables should be carefullyconsidered that potentially affect the reliability of prena-tal diagnosis of HCMV infection. (1) False-negative re-sults (by culture or PCR) were obtained from amnioticfluid up to 20 weeks of gestation. Because of that theremay be a gestational age threshold for fetal excretion of virus into the amniotic fluid [58–61]. (2) A minimumtime interval between maternal infection and amniotic

8/8/2019 000024929



fluid sampling of 6–9 weeks is necessary for reliable detec-tion of HCMV in the amniotic fluid [59]. (3) More thanone aliquot of amniotic fluid should be tested [60, 62].(4) Fetal blood taken by cordocentesis could represent apathological material of choice, but recent data indicate asensitivity and specificity of both antigenemia and PCRcarried out on this material lower that isolation and PCR

on amniotic fluid [62].

Conclusions

While research continues on the development of anHCMV vaccine and clinical trials are programmed for thebeginning of 1998 with a Toledo/Towne recombinantstrain [63] prevention of congenital HCMV infectionshould include the following. (1) Women of childbearingage at risk should be identified and educated about the

potential effects of HCMV on the unborn child. (2) Preg-nant women who are found to be HCMV-seronegativeshould be counselled about the importance of hygiene,especially if they are routinely exposed to young children.

(3) An IgG test should be performed at the 2nd and 4thmonth of pregnancy. (4) Pregnant women with an un-known serological status for HCMV should be screenedwith a reliable IgM test (confirmed by blot if necessary)followed, in the case of positivity, by an avidity assay. (5)Pregnant women with a primary HCMV infection shouldbe encouraged to obtain a prenatal diagnosis. (6) In

approximately 70% of women the results of the prenataldiagnosis will indicate an absence of fetal infection andwill prevent an unnecessary termination. (7) Very recentresults [Lazzarotto et al., manuscript in preparation] sug-gest that viral load in the amniotic fluid as detected byquantitative PCR can discriminate severely infected fe-tuses.

Acknowledgements

The authors are grateful to Professor P. Dallacasa and Professor

B. Guerra (Bologna, Italy) for having agreed to present unpublisheddata obtained during a collaborative study. This work was partiallysupported by the Italian Ministry of Public Health, the Italian Minis-try of Education and the University of Bologna.

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

References

1 Ivarsson SA, Lernmark B, Svanberg L: Ten-year clinical, developmental, and intellectualfollow-up of children with congenital cytomeg-

alovirus infection without neurologic symp-toms at one year of age. Pediatrics 1997;99:800–803.

2 Natali A, Valcavi P, Medici MC, Dieci E, Mon-tali S, Chezzi C: Cytomegalovirus infection inan Italian population: Antibody prevalence, vi-rus excretion and maternal transmission. NewMicrobiol 1997;20:123–133.

3 Fowler KB, Stagno S, Pass RF: Congenitalcytomegalovirus (CMV) infection risk in futurepregnancies and maternal CMV immunity. 6thInternational Cytomegalovirus Workshop, Per-dido Beach Resort, 1997.

4 Conboy T, Pass RF, Stagno S, Alford CA,Myers GJ, Britt WJ, McCollister FP, SummersMN, McFarland CE, Boll TJ: Early clinical

manifestations and intellectual outcome inchildren with symptomatic congenital cyto-megalovirus infection. J Pediatr 1987;111:343–348.

5 Williamson WD, Desmond MN, La Fevers N,Taber LH, Catlin FI, Weaver TG: Symptomat-ic congenital cytomegalovirus. Disorders of language, learning, and hearing. Am J DisChild 1982;136:902–905.

6 Williamson WD, Percy AK, Yow MD, GersonP, Catlin FI, Koppelman ML, Thurber S:Asymptomatic congenital cytomegalovirus in-

fection. Audiologic, neuroradiologic, and neu-rodevelopmental abnormalities during the firstyear. Am J Dis Child 1990;144:1365–1368.

7 Williamson WD, Demmler GJ, Percy AK, Cat-lin FI: Progressive hearing loss in infants withasymptomatic congenital cytomegalovirus in-fection. Pediatrics 1992;90:862–866.

8 Williamson WD: The longitudinal assessmentof congenitally infected infants. Semin PediatrNeurol 1994;1:58–62.

9 Grose C, Weiner CP: Prenatal diagnosis of con-genital cytomegalovirus infection: Two de-cades later. Am J Obstet Gynecol 1990;163:447–450.

10 Yow MD, Williamson DW, Leeds LJ, Thomp-son P, Woodward RM, Walmus BF, Lester JW,

Six HR, Griffiths PD: Epidemiologic charac-teristics of cytomegalovirus infection in moth-ers and their infants. Am J Obstet Gynecol1988;158:1189–1195.

11 Stagno S, Pass RF, Cloud G, Britt WJ, Hender-son RE, Walton PD, Veren DA, Page F, AlfordCA: Primary cytomegalovirus infection in

pregnancy. Incidence, transmission to fetus,and clinical outcome. JAMA 1986;256:1904–1908.

12 Britt WJ, Alford CA: Cytomegalovirus; inFields BN, Knipe DM, Howley PM (eds):Fields Virology. Philadelphia, Lippincott-Raven, 1996, vol 2.

13 Gehrz RC, Christianson WR, Linner KM,Conroy MM, McCue SA, Balfour HH Jr: Cyto-megalovirus-specific humoral and cellular re-sponses in human pregnancy. J Infect Dis1981;143:391–395.

14 Plotkin SA, Starr SE, Friedman HM, GonczolE, Brayman K: Vaccines for the prevention of human cytomegalovirus infection. Rev InfectDis 1990;12:S827–S838.

15 Landini MP, Mach M: Searching for antibodiesspecific for human cytomegalovirus: Is it diag-nostically useful? Scand J Infect Dis Suppl1995;99:18–23.

16 Gleaves CA, Smith TF, Shuster EA, PearsonGR: Rapid detection of cytomegalovirus inMRC5 cells inoculated with urine specimensby use of low speed centrifugation and mono-clonal antibody to an early antigen. J ClinMicrobiol 1984;19:917–919.

8/8/2019 000024929



17 Prösch S, Kimel V, Dawydowa I, Krüger D:Monitoring of patients for cytomegalovirus af-ter organ transplantation by centrifugation cul-ture and PCR. J Med Virol 1992;38:246- 251.

18 Britt WJ: Infections associated with humancytomegalovirus; in Glaser R, Jones JF (eds):Herpesvirus Infections. New York, Dekker,1994.

19 Landini MP: New approaches and perspectivesin cytomegalovirus diagnosis. Prog Med Virol1993;40:157–177.

20 Gleaves CA, Wendt FF, Dobbs DR, MeyersJD: Evaluation of the CMV-CUBE assay fordetection of cytomegalovirus serological statusin marrow transplant patients and marrow do-nors. J Clin Microbiol 1990;28:841–842.

21 Koerner K, Kilian D, Zimmerman B, NebelSchickel H, Horn J: Comparison of four differ-ent ELISAs and direct immunofluorescence forscreening of blood donors for antibodies tocytomegalovirus. Biotest Bull 1990;4:119–123.

22 Gutierrez J, del Carmen Maroto C, Piedrola G:Evaluation of a new reagent for anti-cytomega-lovirus and anti-Epstein-Barr virus immuno-globulin G. J Clin Microbiol 1994; 32:2603–2605.

23 Lazzarotto T, Dalla Casa B, Campisi B, Lan-dini MP: Enzyme-linked immunosorbent assayfor the detection of cytomegalovirus IgM:Comparison between eight commercial kits,immunofluorescence, and immunoblotting. JClin Lab Anal 1992;6:216–218.

24 Nielsen SL, Sfrensen I, Andersen HK: Kineticsof specific immunoglobulins M, E, A, and G incongenital, primary, secondary cytomegalovi-rus infection studied by antibody-capture en-zyme-linked immunosorbent assay. J Clin Mi-crobiol 1988;26:654–661.

25 Landini MP, Baldassari B, Mirolo G, Ripalti A,La Placa M: Reactivity of cytomegalovirusstructural polypeptides with different sub-classes of IgG present in human serum. J Infect1988;16:163–167.

26 Landini MP: Antibody responses to humancytomegalovirus proteins. Rev Med Virol1992;2:63–72.

27 Landini MP, Guan MX, Jahn G, LindenmeierW, Mach M, Ripalti A, Necker A, LazzarottoT, Plachter B: Large scale screening of humansera with cytomegalovirus recombinant anti-gens. J Clin Microbiol 1990;28:1375–1379.

28 Novak J, Sova P, Krchnak V, Hamsikova E,Zavadova H, Roubal J: Mapping of serological-ly relevant regions of human cytomegalovirusphosphoprotein pp150 using synthetic pep-tides. J Gen Virol 1991;72:1409–1413.

29 Landini MP, Ripalti A, Sra K, Pouletty P:Human cytomegalovirus structural proteins:Immune reaction against pp150 synthetic pep-tides. J Clin Microbiol 1991;29:1868–1872.

30 Kraat YJ, Stals FS, Christiaans MH, Lazzarot-to T, Landini MP, Bruggeman CA: IgM anti-body detection of ppUL80a and ppUL32 byimmunoblotting: An early parameter for recur-rent cytomegalovirus infection in renal trans-plant recipients. J Med Virol 1996;48:289–294.

31 Vornhagen R, Plachter B, Hinderer W, TheTH, Van Zanten J, Matter L, Schmidt CA, Son-neborn HH, Jahn G: Early serodiagnosis of acute human cytomegalovirus infection by en-zyme-linked immunosorbent assay using re-combinant antigens. J Clin Microbiol 1994;32:981–986.

32 Landini MP, Lazzarotto T, Maine GT, RipaltiA, Flanders R: Recombinant mono- and po-lyantigens to detect cytomegalovirus-specificimmunoglobulins M in human sera by enzymeimmunoassay. J Clin Microbiol 1995;33:2532–2542.

33 Lazzarotto T, Maine GT, Dal Monte P, RipaltiA, Landini MP: A novel Western blot test con-taining both viral and recombinant proteins foranticytomegalovirus immunoglobulin M detec-tion. J Clin Microbiol 1997;35:393–397.

34 Lazzarotto T, Brojanac S, Maine GT, LandiniMP: Search for cytomegalovirus-specific im-munoglobulin M: Comparison between a newWestern blot, conventional Western blot, andnine commercially available assays. Clin DiagnLab Immunol 1997;4:483–486.

35 Landini MP, Mirolo G, Coppolecchia P, ReMC, La Placa M: Serum antibodies to individ-ual cytomegalovirus structural polypeptides inrenal transplant recipients during viral infec-tion. Microbiol Immunol 1986;30:683–695.

36 Hedman K, Rousseau SA: Measurement of av-idity of specific IgG for verication of recent pri-mary rubella. J Med Virol 1989;27:288–292.

37 Kangro HO, Manzoor S, Harper DR: Antibodyavidity following varicella-zoster virus infec-tions. J Med Virol 1991;33:100–105.

38 Ward KN, Gray JJ, Joslin ME, Sheldon MJ:Avidity of IgG antibodies to human herpesvi-rus-6 distinguishes primary from recurrent in-fection in organ transplant recipients and ex-cludes cross-reactivity with other hepesviruses.J Med Virol 1993;39:44–49.

39 Ward KN, Dhaliwal W, Ashworth KL, Clutter-buck EJ, Teo CG: Measurement of antibodyavidity for hepatitis C virus distinguishes pri-mary antibody responses from passively ac-quired antibody. J Med Virol 1994;43:367–372.

40 Tuokko H: Detection of acute measles infec-tions by indirect and Ì-capture enzyme immu-noassays for immunoglobulin M antibodiesand measles immunoglobulin G antibody avid-ity enzyme immunoassay. J Med Virol 1995;45:306–311.

41 Lutz E, Ward KN, Gray JJ: Maturation of anti-body avidity after primary human cytomegalo-virus infection is delayed in immunosup-pressed solid organ transplant patients. J MedVirol 1994;44:317–322.

42 Lutz E, Ward KN, Szydlo R, Goldman JM:Cytomegalovirus antibody avidity in allogenicbone marrow recipients: Evidence for primaryor secondary humoral responses depending ondonor immune status. J Med Virol 1996;49:61–65.

43 Lazzarotto T, Spezzacatena P, Pradelli P,Abate DA, Varani S, Landini MP: Avidity of immunologulin G directed against human cy-tomegalovirus during primary and secondaryinfections in immunocompetent and immuno-compromised subjects. Clin Diagn Lab Immu-nol 1997;4:469–473.

44 Grangeot-Keros L, Mayaux MJ, Lebon P, Frey-muth F, Eugene G, Stricker R, Dussaix E: Val-ue of cytomegalovirus (CMV) IgG avidity in-dex for the diagnosis of primary CMV infectionin pregnant women. J Infect Dis 1977;175:944–946.

45 Balcarek KB, Oh MK, Pass RF: Maternal vire-mia and congenital CMV infection; in Michel-son S, Plotkin SA (eds): Multidisciplinary Ap-proach to Understanding CytomegalovirusDisease. Amsterdam, Elsevier Science Publish-ers, 1993, pp 169–173.

46 Revello MG, Zavattoni M, Sarasini A, Perci-valle E, Simoncini L, Gerna G: Human cyto-megalovirus in blood of immunocompetentpersons during primary infection: prognosticimplications for pregnancy. J Infect Dis 1998;77:1170–1175.

47 Griffiths PD, Baboonian D: A prospectivestudy of primary cytomegalovirus infectionduring pregnancy: Final report. Br J ObstetGynaecol 1984;92:307–315.

48 Nelson CT, Demmler GJ: Cytomegalovirus in-fection in the pregnant mother, fetus, and new-born infant. Clin Perinatol 1997;24:151–160.

49 Whitley RJ, Cloud G, Gruber W, Storch GA,Demmler GJ, Jacobs RF, Dankner W, SpectorSA, Starr S, Pass RF, Stagno S, Britt WJ, AlfordC Jr, Soong S, Zhou XJ, Sherrill L, FitzGeraldJM, Sommadossi JP: Ganciclovir treatment of symptomatic congenital cytomegalovirus in-fection: Results of a phase II study. NationalInstitute of Allergy and Infectious DiseasesCollaborative Antiviral Study Group. J InfectDis 1997;175:1080–1086.

50 Sinzger C, Müntenfering H, Löning T, Stöss H,Plachter B, Jahn G: Cell types infected inhuman cytomegalovirus placentitis identifiedby immunohistochemical double staining. Vir-chows Arch A Pathol Anat Histopathol 1993;423:249–256.

51 Hemmings DG, Kilani R, Nykiforuk C, Preik-saitis J, Guilibert LJ: Permissive cytomegalovi-rus infection of primary villous term and firsttrimester trophoblasts. J Virol 1998;72:4970–4979.

52 Muhlemann K, Menegus MA, Miller RK: Cy-tomegalovirus in the perfused human term pla-centa in vitro. Placenta 1995;16:367–373.

53 Grose C, Weiner CP: Prenatal diagnosis of con-genital cytomegalovirus infection by virus iso-lation from amniotic fluid. Am J Obstet Gyne-col 1990;163:1253–1255.

54 Lynch L, Daffos F, Emanuel D, GiovangrandiY, Meisel R, Forestier F, Cathomas G, Berko-witz RL: Prenatal diagnosis of fetal cytomega-lovirus infection. Am J Obstet Gynecol 1991;165:714–718.

8/8/2019 000024929



55 Lamy ME, Mulongo KN, Gadisseux JF, LyonG, Gaudy V, Van Lierde M: Prenatal diagnosisof fetal cytomegalovirus infection. Am J ObstetGynecol 1992;166:91–94.

56 Weber B, Opp M, Born HJ, Langenbeck U,Doerr HW: Laboratory diagnosis of congenitalhuman cytomegalovirus infection using poly-merase chain reaction and shell vial culture.Infection 1992;20:155–157.

57 Revello MG, Baldanti F, Furione M, SarasiniA, Percivalle E, Zavattoni M, Gerna G: Poly-merase chain reaction for prenatal diagnosis of congenital human cytomegalovirus infection. JMed Virol 1995;47:462–466.

58 Catanzarite V, Dankner WM: Prenatal diagno-sis of congenital cytomegalovirus infection:False-negative amniocentesis at 20 weeks’ ges-tation. Prenat Diagn 1993;13:1021–1025.

59 Donner C, Liesnard C, Brancart F, Rodesch F:Accuracy of amniotic fluid testing before 21weeks’ gestation in prenatal diagnosis of con-genital cytomegalovirus infection. PrenatDiagn 1994;14:1055–1059.

60 Revello MG, Sarasini A, Zavattoni M, Baldan-ti F, Gerna G: Improved prenatal diagnosis of congenital human cytomegalovirus infectionby a modified nested polymerase chain reac-tion. J Med Virol 1998;56:99–103.

61 Nicolini U, Kustermann A, Tassis B, FoglianiR, Galimberti A, Percivalle E, Revello MG,Gerna G: Prenatal diagnosis of congenital hu-man cytomegalovirus infection. Prenat Diagn1994;14:903–906.

62 Lazzarotto T, Guerra B, Spezzacatena P, Va-rani S, Gabrielli L, Pradelli P, Rumpianesi F,Banzi C, Bovicelli L, Landini MP: Prenataldiagnosis of congenital cytomegalovirus infec-tion. J Clin Microbiol 1998;36:3540–3544.

63 Kemble G, Duke G, Winter R, Evans P, SpaeteR: Defined large-scale alterations of the humancytomegalovirus genome constructed by co-transfection of overlapping cosmids. J Virol1996;70:2044–2048.

Documents

000024929