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Human Cytomegalovirus Immediate-Early Messenger RNA in Blood of Pregnant Women with Primary Infection and of Congenitally Infected Newborns Maria Grazia Revello,1 Daniele Lilleri,1 Maurizio Zavattoni,1 Mauro Stronati,2,a Lina Bollani,2 Jaap M. Middeldorp,3 and Giuseppe Gerna1

1 Servizio di Virologia and 2Divisione di Patologia Neonatale e Neonatologia, Istituto di Ricovero e Cura a Carattere Scientifico, Policlinico San Matteo, Pavia, Italy; 3Department of Pathology, Free University of Amsterdam, Amsterdam, The Netherlands

Human cytomegalovirus (HCMV) immediate-early messenger RNA (IEmRNA) in sequential blood samples from 32 pregnant women with primary infection and from 14 congenitally infected newborns was qualitatively investigated by nucleic acid sequence–based amplification. IEmRNA was detected in 100%, 75%, 36.3%, 22.2%, and 0% of samples collected 1, 2, 3, 4–6, and 16 months after onset of primary HCMV infection, respectively, showing 83.7% sensitivity and 92.2% specificity, compared with results of quantitative DNAemia (detection of viral DNA in blood). In infected newborns, IEmRNA was positive in 100% of samples collected 1–7 days (median, 1.5 days) and in 46.4% of samples collected 27–260 days (median, 88 days) after birth, showing 75.7% sensitivity and 100% specificity, compared with DNAemia results. IEmRNA was not detected in HCMV-immune individuals with remote or recurrent HCMV infection or in uninfected newborns. IEmRNA determination appears to be a valuable tool for early diagnosis of both primary and congenital HCMV infection.

Diagnosis of primary human cytomegalovirus (HCMV) infection in immunocompetent individuals mostly relies on serologic tests, whereas HCMV recovery from urine is still the reference standard for the diagnosis of congenitally infected newborns [1]. The presence of viral DNA in blood has been shown to be a very sensitive and specific diagnostic marker of primary infection in immunocompetent subjects [2] and of congenital HCMV infection in newborn infants [3]. In addition, reports indicate that the detection of immediate-early (IE) mRNA (IEmRNA) in blood by nucleic acid sequence–based amplification (NASBA) is the most sensitive approach to early detection of HCMV infection in immunocompromised patients, and it reflects active viral infection [4, 5]. The aim of this study was to compare the presence of HCMV IEmRNA in the blood of immunocompetent subjects during primary infection and in

Received 26 March 2001; revised 11 June 2001; electronically published 29 August 2001. Presented in part: 17th annual Clinical Virology Symposium, Clearwater, Florida, 29 April to 2 May 2001 (poster 526). Financial support: Ministero della Sanita`, Ricerca Corrente, Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo, Pavia, Italy (grant 80208). a Present affiliation: Neonatologia e Terapia Intensiva Neonatale, Ospedale Carlo Poma, Mantova, Italy. Reprints or correspondence: Dr. Maria Grazia Revello, Servizio di Virologia, IRCCS Policlinico San Matteo, 27100 Pavia, Italy (mg.revello@ smatteo.pv.it). The Journal of Infectious Diseases 2001; 184:1078–81 䉷 2001 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2001/18408-0019$02.00

neonates with congenital infection with the detection of viral DNA in blood (DNAemia) or virus detection in urine.

Subjects, Materials, and Methods Patients and control subjects. One hundred sequential blood samples were collected from 32 pregnant women (mean age, 30 years; range, 16–37 years) with documented primary HCMV infection. The women were enrolled retrospectively among pregnant women attending the Center for ToRCH (Toxoplasma gondii, rubella virus, cytomegalovirus, and herpes simplex virus) Screening in the Department of Virology (IRCCS Policlinico San Matteo). A diagnosis of primary HCMV infection was made on the basis of IgG seroconversion (n p 14 ), the presence of virus-specific IgM and viral DNA in blood (n p 12), or the presence of declining levels of virus-specific IgM (n p 6 ). In addition, symptoms (fever, asthenia, or malaise) and/or elevated liver enzymes were detected in 19 women at routine examinations performed during pregnancy. Onset of the infection was defined by the appearance of symptoms or by seroconversion. IgG and IgM antibody levels in sequential serum samples allowed for the identification of seroconversion within a period of Ⳳ1 week. In 26 women, primary HCMV infection occurred during pregnancy, whereas, in the remaining 6, a periconceptional infection (Ⳳ2 weeks from presumed conception) was diagnosed. The outcome was as follows: 10 women transmitted the infection and 15 did not, 3 pregnancies were terminated, and, for the remaining 4, the outcome was unknown. In addition, 40 sequential blood samples were obtained from 14 newborns with congenital HCMV infection diagnosed by virus isolation from urine collected within 2 weeks after birth. Three newborns (1 with cho-

JID 2001;184 (15 October)

HCMV IEmRNA in Primary and Congenital Infections

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Table 1. Comparison of human cytomegalovirus (HCMV) immediate-early mRNA (IEmRNA) and DNA results for sequential blood samples from pregnant women with primary infection and from congenitally infected newborns.

Patient group, DNAemia results Pregnant women (n p 32) Positive Negative Total no. of samples Newborns (n p 14) Positive Negative Total no. of samples

No. of samples with positive IEmRNA results

No. of samples with negative IEmRNA results

41 b 4 45

8 47 55

a

8 7 15

c

25 0 25

Total no. of IEmRNA samples

Sensitivity, %

49 51 100

83.7

33 7 40

75.8

Specificity, %

Concordance, %

92.2 88

100 80

NOTE. DNAemia, detection of HCMV DNA in blood. a Samples were obtained from 8 patients on days 50–227 (median, 73 days) after onset of illness. b Samples were obtained from 4 patients on days 42, 49, 60, and 156 after onset of illness. c Samples were obtained from 7 patients on days 35–185 (median, 87.5 days) after birth.

rioretinitis and bilateral deafness and 2 with hepatosplenomegaly and elevated liver enzymes) were symptomatic at birth. Furthermore, single blood samples were obtained as controls from 95 HCMV-infected, IgG-positive, IgM-negative healthy volunteers; 22 HCMV-positive IgM-negative breast-feeding women excreting HCMV in breast milk; and 14 uninfected newborns of mothers who had primary HCMV infection during pregnancy. Overall, 140 sequential blood samples from 46 subjects (32 mothers and 14 newborns) and 131 blood samples from as many control subjects were examined. Virologic and serologic assays. The presence of HCMV in blood was determined by prospective quantitation of antigenemia and DNAemia and by retrospective detection of IEmRNA in blood samples stored at ⫺80⬚C. Antigenemia was quantitated by counting the number of peripheral blood leukocytes positive for pp65 [6]. Viral DNA was quantitated in 10 mL of whole blood by polymerase chain reaction (PCR) in the presence of 100 copies of an internal control and in parallel with a series of external standards [2]. Single-step PCR consistently amplified samples containing 110 genome equivalents (GEs), whereas samples containing 1–10 GEs were detected by nested PCR and were assigned an arbitrary value of 5 GEs. HCMV IEmRNA was determined by the NASBA technique, following the manufacturer’s (Organon Teknika) instructions. In brief, nucleic acids from 100 mL of whole blood were isolated by the method of Boom et al. [7]. System control (SC) RNA (∼8000 cRNA copies) was added to samples before nucleic acid isolation as a positive control for RNA isolation, amplification, and detection. The SC RNA could be distinguished from wild-type (wt) RNA by insertion of a 134-nt fragment [4, 5]. SC and wt IEmRNAs were amplified with a primer that contained a T7 promoter and a reverse primer. Amplification products were detected by electrochemiluminescence with a common capture probe coupled to magnetic beads and by 2 specific (wt- and SC-specific) ruthenium-labeled oligonucleotide detection probes [4, 5]. IEmRNA NASBA sensitivity was ∼70 copies/10 mL of whole blood. Virus-specific IgG and IgM were determined by ELISAs developed in house [8]. For early HCMV isolation and identification, clinical specimens (urine or human milk) were inoculated onto confluent monolayers of human embryonic lung fibroblasts grown in shell vials. Cultures were fixed 24 h after inoculation and were stained with a monoclonal antibody to HCMV IE protein p72 [9].

Statistical analysis. The duration of DNAemia versus IEmRNAemia in congenitally infected newborns was compared by use of the Mann-Whitney U test.

Results HCMV IEmRNA determination in control groups. To assess the general specificity of the HCMV IEmRNA assay, 131 blood samples obtained from as many healthy subjects were tested for both IEmRNA and viral DNA and were found to be negative by both assays. Thus, overall specificity of HCMV IEmRNA assay was 100%. HCMV IEmRNA in primary infections. In total, 45 and 49 of 100 blood samples were positive for IEmRNA and DNA, respectively (table 1). Twelve samples gave discrepant results (i.e., 8 were DNA-positive and IEmRNA-negative, and 4 were DNA-negative and IEmRNA-positive). Of the 8 DNA-positive IEmRNA-negative samples, 6 contained 5 GEs, and 2 contained 10 GEs. Thus, overall concordance was 88% (88/100 samples). Table 2 shows the kinetics of IEmRNA, viral DNA, and pp65 antigenemia in sequential blood samples grouped according to the time interval after onset of primary infection. HCMV IEmRNA and DNA were detected in comparable proportions of blood samples examined throughout follow-up. In particular, both assays exhibited the highest sensitivities in samples obtained during the first 2 months after the onset of primary infection, whereas the detection rate decreased comparably at later times, although viral DNA apparently persisted slightly longer than IEmRNA. Antigenemia was positive in 80% and 38.1% of samples examined during the first and second months after the onset, respectively, and in none of the samples examined afterwards. Viral DNA was detected in ⭓1 blood sample from 26 (81.2%) of 32 pregnant women examined during follow-up. On the other hand, IEmRNA was detected in 21 of 26 DNA-positive and in 2 of 6 DNA-negative subjects. Thus, altogether, 23 (71.9%) of 32 women were positive for IEmRNA. The two assays in

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Table 2. Human cytomegalovirus (HCMV) detection in 100 sequential blood samples from 32 pregnant women with primary HCMV infection and in 40 sequential samples from 14 congenitally infected newborns. Subject group, days after onset or after birth (median) Pregnant women 4–30 (21) 31–60 (51) 61–90 (80) 91–180 (130) 1181 (210) Newborns 1–7 (1.5) 8–90 (45) 91–180 (107) 1181 (210)

No. of samples positive/no. tested (%) pp65 Antigenemia

DNAemia

8/10 (80) 8/21 (38.1) 0/5 (0) ND ND

10/10 20/28 10/22 8/27 1/13

(100) (71.4) (45.5) (29.6) (7.7)

4/8 (50) 2/5 (40) ND ND

12/12 (100) 14/15 (93.3) 5/8 (62.5) 2/5 (40)

a

IEmRNAemia 10/10 21/28 8/22 6/27 0/13

(100) (75) (36.4) (22.2) (0)

12/12 (100) 10/15 (66.7) 3/8 (37.5) 0/5 (0)

NOTE. DNAemia, detection of HCMV DNA in blood; IEmRNAemia, detection of HCMV immediate-early RNA in blood; ND, not done. a Two additional newborns found to be DNA positive at birth were not tested for IEmRNA because of insufficient specimens.

combination detected HCMV nucleic acids in 28 (87.5%) of 32 individuals after primary HCMV infection. HCMV IEmRNA in congenital HCMV infections. As shown in table 1, IEmRNA was detected in 25 of 33 DNApositive blood samples (75.7% sensitivity) and in 0 of 7 DNAnegative blood samples (100% specificity). Overall concordance was 80% (32/40). Viral DNA was detected in all of the 14 blood samples collected from the 14 HCMV-infected neonates during the first week of life (median, 1.5 days; range, 1–7 days). Similarly, IEmRNA was detected in all of 12 blood samples from as many newborns still available for testing (table 2). However, when sequential blood samples were considered, the NASBA assay showed a slightly lower sensitivity, compared with PCR. In particular, of the 28 blood samples obtained 8 to 1181 days (median, 83 days) after birth, only 13 (61.9%) of 21 DNApositive samples were also positive for IEmRNA (table 2). The 13 positive and 7 negative concordant samples were collected 27–170 days (median, 37 days) and 31–260 days (median, 158 days) after birth, respectively, whereas the 8 discordant PCRpositive, NASBA-negative samples were obtained from 7 patients 35–185 days (median, 87.5 days) after birth (i.e., at an intermediate time between concordant positive and negative samples). Mean DNA levels were 211 GEs (range, 5–2000 GEs) and 9.8 GEs (range, 5–10 GEs) in the 2 groups of concordant and discordant results, respectively.

Discussion Our previous studies showed that, in immunocompetent individuals and newborns, the sole qualitative detection of HCMV DNA in blood appears to be diagnostic of primary or congenital HCMV infection, respectively [2, 3]. However, viral DNA levels were very low in both groups of patients (with the exclusion of


newborns with symptomatic HCMV infection). Thus, recent reports of IEmRNA detection by NASBA as the most sensitive assay for the diagnosis of HCMV infection in immunocompromised individuals [4, 5] prompted us to assess the diagnostic value of IEmRNA, compared with DNAemia detection. The NASBA format used in this study is not commercially available; however, a comparable level of performance can be obtained by using commercially available reagents (Nuclisens Basic Kit; Organon Teknika) (authors’ unpublished data). Specificity of the NASBA IEmRNA assay initially was assessed in the 3 groups of control subjects tested (healthy individuals with remote or recurrent HCMV infection and uninfected newborns). As for sensitivity, IEmRNA could be detected for a limited period of time in the blood of healthy subjects undergoing primary HCMV infection. In particular, all subjects tested positive for IEmRNA in the first month after onset of infection, whereas the proportion of positive subjects declined over time until becoming negative ⭓6 months after the onset of infection. Thus, IEmRNA kinetics appears to be comparable to that already reported for viral DNA [2], which suggests that qualitative detection of IEmRNA in blood of immunocompetent individuals can be considered to be an additional marker of acute or recent primary HCMV infection. However, compared with DNAemia, IEmRNA detection appears to be slightly less sensitive when late-phase (⭓3 months after onset) blood samples are considered. A similar trend was observed during follow-up of infants congenitally infected with HCMV (see below). In addition, the IEmRNA NASBA assay showed 100% sensitivity in identifying HCMV-infected newborns examined during the first week of life. Similarly, as reported elsewhere [3, 10] and confirmed in the present study, HCMV DNAemia was detected in all newborns with positive virus isolation from urine. However, viral IEmRNA could be detected for a shorter period of time (median, 37 days; range, 27–170 days), compared with DNAemia (median, 87.5 days; range, 37–185 days; P p .04). Thus, in both groups of patients examined, NASBA appeared to be slightly but consistently less sensitive than PCR in latephase blood samples, which was due to the more confined association of IEmRNA with the early stages of HCMV infection in vivo. Finally, it appears to be important to stress that the clinical specificity of both assays was 100%, since neither viral DNA nor IEmRNA was ever found in healthy newborns. These findings are in contrast with those reporting an HCMV DNA positivity rate of 25.1% in healthy newborns [11] and a positive predictive value of PCR in fetal blood (33%) and amniotic fluid (48%) [12]. The latter findings were not confirmed in a recent extended report [13]. In previous studies, IEmRNA detection by NASBA was reported to be superior to DNA detection by PCR in the diagnosis of HCMV infection in immunocompromised patients [5]. However, the greater sensitivity of IEmRNA detection was observed in the early phase of HCMV infection, whereas no data


HCMV IEmRNA in Primary and Congenital Infections

were reported concerning later stages of natural infection. Indeed, after treatment, IEmRNA was reported to precede DNA disappearance in some patients [5]. It seems reasonable to reconcile previous and present apparently contrasting results by comparing early versus late phases of HCMV infection. Indeed, by using an in vitro model developed in our laboratory [14], we have shown that viral DNA detected in polymorphonuclear leukocytes is transferred from HCMV-infected cells, whereas an aliquot of IEmRNA is synthesized in these cells, which indicates an active, albeit abortive, replication of HCMV [15]. Thus, it is possible to speculate that IEmRNA might be a more reliable indicator of active HCMV infection. In conclusion, to our knowledge, this study shows for the first time that IEmRNA detection has the same diagnostic value as DNA determination in primary and congenital HCMV infection. In addition, it provides evidence that IEmRNA and DNA assays in combination may improve both diagnosis and dating of primary HCMV infection in pregnant women, and it confirms and extends previous results concerning the diagnostic value of DNA detection in blood of immunocompetent individuals with primary infection and of congenitally infected newborns. Acknowledgments We thank Lucia Chezzi, Cinzia Zanello, and Luca Dossena, for skillful technical help; Linda D’Arrigo, for revision of the English; and Organon Teknika, for providing nucleic acid sequence–based amplification reagents.

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