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Clinical Microbiology Reviews, October 2002, p. 680-715, Vol. 15, No. 4
0893-8512/02/$04.00+0     DOI: 10.1128/CMR.15.4.680-715.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Diagnosis and Management of Human Cytomegalovirus Infection in the Mother, Fetus, and Newborn Infant

Servizio di Virologia, IRCCS Policlinico San Matteo, 27100 Pavia, Italy

SUMMARY

INTRODUCTION

EPIDEMIOLOGY OF VERTICAL HCMV TRANSMISSION

PATHOGENESIS OF CONGENITAL INFECTION

DIAGNOSIS OF PRIMARY INFECTION DURING PREGNANCY

Serology

Seroconversion.

IgM assays.

Recombinant IgM assays.

Interpretation of positive IgM results.

IgG avidity assay.

Neutralizing antibody.

Conclusions.

Detection of Virus and Viral Products in Maternal Blood

Viremia.

Antigenemia.

DNAemia.

RNAemia.

Endotheliemia.

Virus and viral products in blood of immunocompetent persons as an aid for diagnosis of primary infection.

Clinical Signs and Symptoms

Diagnosis of Recurrent Infection in the Mother

Maternal Prognostic Markers of Fetal Infection

COUNSELING

DIAGNOSIS OF CONGENITAL INFECTION IN THE FETUS

Fetal Blood

Amniotic Fluid

Fetal Prognostic Markers of HCMV Disease

DIAGNOSIS OF CONGENITAL INFECTION IN THE NEWBORN

TREATMENT OF CONGENITAL INFECTION

PREVENTION OF CONGENITAL INFECTION

Live Attenuated Vaccines

Recombinant Virus Vaccines

Subunit Vaccines

Peptide Vaccines

DNA Vaccines

PERCEPTION OF THE PROBLEM

UNIVERSAL SEROLOGY SCREENING

CRUCIAL IMPACT OF CORRECT COUNSELING

PRENATAL DIAGNOSIS: IS THERE A ROLE?

PRECONCEPTIONAL AND PERICONCEPTIONAL HCMV INFECTIONS

CONGENITAL INFECTION FROM IMMUNE MOTHERS AND CLINICAL OUTCOME

CONGENITAL INFECTION IN TWINS

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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Human cytomegalovirus (HCMV) is the vernacular name of human herpesvirus 5, a highly host-specific virus of the Herpesviridae family. HCMV is the largest virus in the family and is morphologically indistinguishable from other human herpesviruses. HCMV, like all herpesviruses, undergoes latency and reactivation in the host. Although HCMV has been shown to infect a broad spectrum of cells in vivo (246), the only cells that are fully permissive for HCMV replication in vitro are human fibroblasts. In these cells, virus replication results in the formation of intranuclear and intracytoplasmic inclusion bodies (Fig. 1A), with the former full of nucleocapsids (Fig. 1B) and the latter containing several dense bodies (Fig. 1C). Nucleocapsids acquire the envelope from the nuclear membrane or cytoplasmic vacuoles (Fig. 1D).

View larger version (144K): [in this window] [in a new window]   FIG. 1. HCMV replication in human embryonic lung fibroblast cell cultures. (A) HCMV-infected human fibroblast 120 h postinfection (following immunoperoxidase staining with human antibodies), showing intranuclear (IN) and intracytoplasmic (IC) inclusion bodies. (B to D) Electron microscopy of HCMV-infected human fibroblasts. (B) Horseshoe-shaped intranuclear inclusion (IN). (C) Dense bodies (arrows). (D) Maturing virus particles at the level of the nuclear membrane.

In the following years, HCMV also showed its pathogenic properties in organ transplant recipients, patients with AIDS, and cancer patients, while it gained the leading position among infectious agents responsible for mental retardation, intellectual impairment, and deafness.

Presently, HCMV infection is mostly controlled in immunocompromised patients by available antiviral drugs, yet it continues to maintain its role as the most dangerous infectious agent for the unborn infant. Thus, HCMV infection is still a major health problem, warranting strong preventive measures.

The major scope of this review will be to analyze and update the diagnostic and prognostic implications of primary HCMV infections in pregnancy in the mother, fetus, and newborn. Special emphasis will be given to less-investigated issues, such as detection of virus and viral products in the blood of the mother during primary HCMV infection, the presence of clinical signs and symptoms in the mother, prenatal diagnosis of congenital infection in amniotic fluid and fetal blood, maternal and fetal prognostic markers of HCMV infection and disease, and the impact of counseling. Measures of treatment and prevention of congenital HCMV infection will be mentioned briefly. The last part of the review will deal with the most controversial issues, in particular, how the problem of HCMV infections in pregnancy is perceived by the scientific community and public health authorities. Is preconception serologic screening justified, and should HCMV-seronegative women be prospectively monitored? What are the limits of prenatal diagnosis (false-positive and false-negative results)? What is the role of preconceptional and periconceptional infections in HCMV transmission to the fetus? Are reactivated infections significant in transmission of virus to the fetus?

Finally, one additional goal is to focus the attention of the scientific community on the problem of congenital HCMV infection and to appeal for international collaboration. We truly need to develop and implement consensus strategies for prevention of congenital HCMV infection, ideally through a vaccine.

EPIDEMIOLOGY OF VERTICAL HCMV TRANSMISSION

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The term vertical transmission is used here to indicate HCMV transmission from mother to fetus during pregnancy, thus excluding virus transmission from mother to newborn infant. Due to latency following primary infection and periodic reactivation of HCMV replication causing recurrent infections, in utero transmission of HCMV may follow either primary or recurrent infections (4, 75, 237, 261, 264). It is commonly recognized that primary HCMV infections are transmitted more frequently to the fetus and are more likely to cause fetal damage than recurrent infections (75). In addition, it seems that primary infection occurring at an earlier gestational age is related to a worse outcome (50, 264).

Initially, the role of recurrent maternal infections in causing congenital infections was supported by three independent reports describing congenital infections in consecutive pregnancies (62, 145, 260). In all three reports, the first newborn was severely affected and the second one was subclinically infected. Molecular epidemiological studies indicated that in each of three pairs of congenitally infected siblings, the viruses were identical to each other when examined by restriction fragment length polymorphism analysis (265). However, the first convincing evidence of the possible transmission of HCMV from immune mothers to the fetus came from a prospective study showing that 10 congenitally infected infants were born to immune mothers within a group of 541 infants of women who were seropositive before pregnancy (261), with a prevalence of 1.9%. Subsequently, similar findings were observed in a geographic area where nearly the entire population was immune to HCMV during childhood, and the prevalence of congenital infection was found to be 1.4% (237).

In 1985, Stagno and Whitley (259) estimated the maternal risk of acquiring either primary or recurrent HCMV infection in pregnancy as well as the risk of intrauterine transmission to the offspring in two groups of women of low or high socioeconomic status. Their estimates showed that the risk of primary maternal infection was about three times higher among the higher-income susceptible women (45%), compared to 15% in the lower-income group (Fig. 2). In both groups, transmission to the fetus occurred in about 40% of cases, with delivery of about 10 to 15% symptomatic and 85 to 90% asymptomatic congenitally infected newborns. Among the asymptomatic newborns, about 10% developed sequelae, while about 90% of infants that were asymptomatic at birth developed normally. On the other hand, the rate of congenital infections from recurrent maternal infection was 0.15% in the higher-income group of pregnant women, who were 55% immune, and 0.5 to 1% in the lower-income group, which was 85% immune, i.e., 3 to 7 times higher. However, the rate of clinically apparent disease was low and similar (0 to 1%) in both groups.

View larger version (48K): [in this window] [in a new window]   FIG. 2. Characteristics of HCMV infection in pregnancy. (From S. Stagno and R. J. Whitley [259], used with permission.)

In conclusion, the true frequency and clinical importance of congenital HCMV infections from recurrent maternal infections remain to be determined in long-term prospective studies. However, primary HCMV infection continues to be the major viral cause of congenital infections, with significant morbidity. Recent findings related to the potential role of recurrent maternal infection in symptomatic congenital infection complicate but will not interfere with efforts aimed at developing a safe and efficacious vaccine.

PATHOGENESIS OF CONGENITAL INFECTION

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In the case of primary maternal infection, the antiviral immune response begins proximate to virus transmission to the fetus, whereas in the case of recurrent infection, virus transmission occurs in the presence of both humoral and cell-mediated immune responses. As a result, viremia occurs as a rule only in primary infections (216), whereas it is either absent or undetectable in recurrent infections of the immunocompetent host (216) and common in recurrent infections of immunocompromised patients (67, 137, 147, 179). Since, following primary HCMV infection, intrauterine transmission occurs in only 30 to 40% of cases, an innate barrier seems to partially prevent vertical transmission (4, 50, 110, 264). In addition, a similar event seems to occur among infected newborns, less than 15% of whom show clinically apparent infection, in the great majority of cases resulting from primary maternal infection (4, 50, 75, 264, 293). Finally, in reactivated maternal infections, the risk of symptomatic congenital infection is even markedly lower (4, 110, 264), as shown by the few symptomatic infants reported in the past to have been born to mothers who were immune before pregnancy. In fact, although existing immunity does not prevent transmission of the virus to the fetus, reactivated infections are less likely to cause damage to the offspring than primary infections (75).

Multiple mechanisms of immune evasion for HCMV could relate to the pathogenic role of the virus. Recently, expression of immune evasion genes US3, US6, and US11 of HCMV in the blood of solid organ transplant recipients has been investigated, showing that, after clinical recovery, transcripts of these genes remain detectable, indicating that persistent low viral activity may have implications for long-term control of HCMV infection (106).

Little is still known about the mechanisms of HCMV transmission to the fetus. It has been reported that about 15% of women undergoing primary infection during the first months of pregnancy abort spontaneously, showing placental but not fetal infection (110, 124). Subsequently in the course of pregnancy, placental infection has been shown to be consistently associated with fetal infection (177).

Understanding the mechanisms of HCMV transmission to the fetus implies elucidation of some major steps in placental development (44, 48). The development of the placenta requires differentiation of specialized epithelial stem cells, referred to as cytotrophoblasts, in both floating villi, where they fuse into multinucleate syncytiotrophoblasts covering the villous surface, and anchoring villi, where they aggregate into columns of single cells invading the endometrium and the first third of the myometrium (interstitial invasion). While the syncytiotrophoblast is in direct contact with maternal blood, mediating transport of multiple substances to and from the fetus, the cytotrophoblast columns also invade maternal arterioles (endovascular invasion) by replacing endothelial and smooth muscle cells and thus generating a hybrid cell population of fetal and maternal cells inside uterine vessels (Fig. 3).

View larger version (52K): [in this window] [in a new window]   FIG. 3. Diagram of a longitudinal section that includes a floating and an anchoring chorionic villus at the fetal-maternal interface near the end of the first trimester of human pregnancy. The anchoring villus (AV) functions as a bridge between the fetal and maternal compartments, whereas the floating villus (FV), containing macrophages (M, Hofbauer cells) and fetal blood vessels, is bathed by maternal blood. Cytotrophoblasts in the anchoring villus (zone I) form cell columns that attach to the uterine wall (zones II and III). Cytotrophoblasts then invade the uterine interstitium (decidua and first third of the myometrium; zone IV) and maternal vasculature (zone V), thereby anchoring the fetus to the mother and accessing the maternal circulation. Zone designations mark areas in which cytotrophoblasts have distinct patterns of stage-specific antigen expression, including integrin and HLA-G. Decidual granular leukocytes (DGLs) and macrophages (M) in maternal blood and fetal capillaries in villous cores are indicated. Areas proposed as sites of natural HCMV transmission to the placenta in utero are numbered 1, 2, and 3. (From Fisher et al. [74], used with permission.)

That placenta behaves as a reservoir in which HCMV replicates prior to being transmitted to the fetus has been experimentally shown in the guinea pig, which, as in humans, has a hemomonochorial placenta with a single trophoblast layer separating fetal from maternal circulation (162). In experimental infection of the guinea pig with species-specific CMV, the virus disseminates hematogenously to the placenta, from which it is transmitted to the fetus in about 25% of cases. The guinea pig CMV also persists in placental tissues long after virus has been cleared from blood (108). Recently, a greater understanding of the human placenta has been achieved by using two in vitro models for the study of trophoblast populations lying at the maternal-fetal interface, villous explants and isolated cytotrophoblasts (72-74). These data, coupled with immunohistochemical studies of in vivo HCMV-infected placentas (177, 247) and recent findings on HCMV latency (121, 252), have led to new hypotheses for routes of transmission of HCMV to the fetus in primary and reactivated maternal HCMV infection.

During primary infection of the mother, leukocytes carrying infectious virus (79, 81, 211, 216) may transmit HCMV infection to uterine microvascular endothelial cells (E. Maidji, E. Percivalle, G. Gerna, S. Fisher, and L. Pereira, Abstr. 8th International Cytomegalovirus Workshop, abstr. p. 31, 2001). These cells are in direct contact with cytotrophoblasts of anchoring villi invading maternal arterioles and forming hybrids of maternal-fetal cells (Fig. 3). Infected cytotrophoblasts may in turn transmit the infection to underlying tissues of villous cores, including fibroblasts and fetal endothelial cells (247), thus spreading to the fetus. An alternative model of transmission, in the case of primary maternal infection, is spreading of infection to the villous stroma by infected maternal leukocytes through breaches of the syncytiotrophoblast layer (126, 134). A further hypothesis has been raised suggesting possible transportation of the virus as antibody-coated HCMV virions by a process of transcytosis through intact syncytiotrophoblasts similar to that advocated for transport of maternal IgG to the fetus (74). Finally, syncytiotrophoblasts may be directly infected, but the infection proceeds slowly and remains predominantly cell associated (126) until infected cells are eliminated during the physiological turnover (251). This hypothesis therefore excludes transmission through virus replication in syncytiotrophoblasts.

In the case of congenital HCMV infection following recurrent maternal infection, it must be considered that the placenta is a hemiallograft inducing local immunosuppression in the uterus (74, 227). This may cause reactivation of latent virus in macrophages of the uterine wall, with HCMV transmission to the invading cytotrophoblasts. Then, virus could spread in a retrograde manner to anchoring villi and subsequently to the fetus (177). In this regard, HCMV establishes a true latent infection in CD14⁺ monocytes, which can be reactivated upon allogeneic stimulation of monocyte-derived macrophages from healthy blood donors (252). Reactivation of latent HCMV is dependent on the production of gamma interferon in the differentiation process (253). These data await confirmation by other laboratories.

As a consequence of placental infection, HCMV impairs cytotrophoblast differentiation and invasiveness, as shown in vitro (74). This could explain early abortion occurring in women with primary infection. In addition, HCMV infection impairs cytotrophoblast expression of HLA-G, thus activating the maternal immune response against the cytotrophoblast subpopulation expressing this molecule (74).

DIAGNOSIS OF PRIMARY INFECTION DURING PREGNANCY

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By far the major role in transmitting HCMV infection to the fetus is played by primary infections of the mother during pregnancy. In fact, the rate of vertical transmission was found to be 0.2 to 2.2% in previously seropositive mothers undergoing recurrent infection during pregnancy (28, 258) and 20 to 40% in pregnant women with primary infection (258, 259). Thus, the ratio of transmitting to nontransmitting mothers is on the order of 1:100 between those with recurrent and those with primary infection. In this respect, diagnosis of primary infection during pregnancy is a major task of the diagnostic virology laboratory. It may be achieved in the majority of cases through concurrent analysis of the following factors: serum antibodies, virus detection in blood, and clinical signs and symptoms.

IgM assays. Several serologic assays have been used in the past to detect HCMV-specific IgM antibodies both in whole serum and serum fractions obtained by sucrose density gradient centrifugation or column chromatography. These include complement fixation, anticomplement immunofluorescence, indirect hemagglutination, and radioimmunoassay (220). More recently, enzyme-linked immunosorbent assays (ELISAs) have been more widely used in both the indirect ELISA (146, 234) and the capture ELISA format with either labeled antigen or antibody (235, 279). The indirect ELISA shows the following potential sources of error when performed on whole serum: (i) competitive inhibition due to the presence of specific IgG; (ii) interference due to rheumatoid factor of the IgM class (IgM-RF) or to IgM-RF reactive only with autologous complexed IgG; and (iii) interference due to IgM antibody reactive with cellular antigens (38). All these interfering factors could be readily eliminated by mixing serum samples with anti-human gamma chain serum (38).

However, following the development of ELISA technology, most initial IgM indirect ELISAs were replaced by IgM capture assays based on selective binding of IgM antibody to the solid phase. In capture ELISAs, while IgG does not interfere, IgM-RF may cause false results by competing with viral IgM for anti-IgM binding sites on the solid phase, complexing with specific IgG, which in turn binds viral antigens, reacting directly with the labeled viral antibody, and mutual interference with antinuclear antibody. More precisely, in capture ELISAs, the presence of the sole IgM-RF (or IgG-RF) does not cause false-positive results, which have been observed to occur in serum samples containing both IgM-RF and IgG-antinuclear antibody (180). Initially, capture ELISAs with enzyme-labeled antigen appeared to be the most promising assays (235, 279). However, after a few years, it was recommended, for specificity control of test results, that human serum samples be tested in parallel with viral and cell control labeled antigens (185). In addition, false-positive results due to the presence of both RF and antinuclear antibody, as reported above, could be avoided in capture ELISAs employing labeled F(ab')₂ fragments of specific antibody instead of the IgG fraction (180).

In order to avoid false-positive results, we developed a capture ELISA IgM assay (213) with a mixture of viral antigen and mouse monoclonal antibody to the nonstructural HCMV major DNA-binding proteins (pp52 or ppUL44) as a detector system. This phosphoprotein is prominent in HCMV-infected cells (77, 97) and is known to be recognized primarily by human IgM during the convalescent phase of a primary HCMV infection (150). According to this approach, antinuclear antibody of the IgM class bound to the solid phase will not give false reactions because only IgM antibody reactive to pp52 are recognized by the specific monoclonal antibody.

Different levels of specificity were determined with this assay. General specificity, determined on a series of unselected IgM-negative serum samples from an adult population, was 100%. Stringent specificity, evaluated on a series of potentially interfering serum samples from patients who had Epstein-Barr virus-related infectious mononucleosis, autoimmune diseases, or rheumatoid factor or who had been treated with radioimmunotherapy based on the use of mouse monoclonal antibody, was 96.3%. Finally, clinical specificity, determined on a series of IgM-negative serum samples drawn prior to onset of primary HCMV infection, was 100%. Thus, the overall specificity was 98.9% (363 of 367 IgM-negative serum samples tested). The sensitivity, assayed on 277 IgM-positive serum samples, was 100%. Comparison of the results obtained by this assay with those given by enzyme-labeled antigen showed that the HCMV p52-specific IgM antibody response paralleled that obtained by using enzyme-labeled antigen, thus representing a major component of it, i.e., a major part of the antibody response within the IgM class. In addition, this study showed that, while HCMV-specific IgM drops sharply in titer in normal subjects within 2 to 3 months after onset of infection and is virtually undetectable within 12 months, in immunocompromised patients such a response persists much longer.

Thus, in pregnant women, detection of HCMV IgM antibody may be related to a primary infection occurring during pregnancy when the IgM titer falls sharply in sequential blood samples. The presence of low, slowly decreasing levels of IgM may indicate a primary infection initiated some months earlier and possibly prior to pregnancy. These findings are basically in agreement with previous reports describing a broad HCMV IgM antibody response (111, 188).

An additional risk of HCMV IgM ELISA is a false-positive result due to primary Epstein-Barr virus infection acting as a potent B-cell stimulator and resulting in the production of HCMV IgM antibody in HCMV-immune individuals (53). Dual HCMV and Epstein-Barr virus infection has also been reported (59).

Recombinant IgM assays. Besides the lack of standards for HCMV IgM serology, the high level of discordance among commercial assays for detection of HCMV-specific IgM (156) has been attributed to the lack of standardization of the viral preparations used. More recently, in an attempt to improve the specificity of conventional ELISAs and to overcome the discordant results given by commercial kits based on use of crude viral preparations, HCMV IgM immunoassays have been developed based on recombinant HCMV proteins or peptides. The HCMV-coded proteins reactive with IgM antibody are both structural and nonstructural (39, 144, 151, 213, 223, 224, 280). Major structural proteins include pp150 (UL32), pp65 (UL83), and pp38 (UL80a), while nonstructural proteins include pp52 (UL44) and p130 (UL57). Vornhagen et al. (281) developed a recombinant HCMV IgM ELISA for Biotest (Biotest AG, Dreieich, Germany) with only peptides derived from nonstructural proteins pp52 (amino acids 297 to 433) and p130 (amino acids 545 to 601). In particular, it was found that the indicated portion of the UL57 gene product is a dominant IgM antigen which may be superior in both sensitivity and specificity to fragments from other HCMV proteins for detection of IgM antibodies during primary HCMV infection.

Recombinant proteins and their fragments have been studied in a Western blot or immunoblot assay for their reactivity to IgM-positive serum samples prior to being included in an ELISA. The group of M. P. Landini, in close association with Abbott Laboratories (Abbott Park, Ill.), developed two versions of the HCMV IgM immunoblot assay with both recombinant proteins or peptides and viral proteins from purified virus preparations (152, 155, 158). In the new version of the assay (152), the viral section of a slot blot contains the entire viral proteins pp150 (UL32), pp82, pp65 (UL83), and pp28 (UL99) purified by gel electrophoresis, while the recombinant section contains only portions of pp150 (amino acids 595 to 614 and 1006 to 1048), p130 (amino acids 545 to 601 and 1144 to 1233), pp52 (amino acids 202 to 434), and pp38 (amino acids 117 to 383).

A preliminary evaluation of the new immunoblot assay indicated that 13 of 80 (16%) IgG- and IgM-negative serum samples and as many as 38 of 200 (19%) IgG-positive, IgM-negative serum samples did react with one or more of the viral or recombinant proteins, while 126 of 126 (100%) IgM-positive serum samples reacted variably. In order to render these highly nonspecific results interpretable, an algorithm for reading of test results had to be introduced. Thus, only serum samples reactive with at least one viral and one recombinant protein or serum samples reactive with at least three recombinant protein bands were considered positive for IgM. By using this approach, a sensitivity of 100% and specificity of 98.6% were reached with respect to the consensus of two of the most used commercial ELISAs (Behring AG, Marburg, Germany, and DiaSorin, Saluggia, Italy).

This assay was used as a reference test for development of the Abbott AxSYM CMV IgM microparticle enzyme immunoassay, with microparticles coated with the indicated portions of three structural (pp150, amino acids 595 to 614 and 1006 to 1048; pp65, amino acids 297 to 510; and pp38, amino acids 117 to 373) and one nonstructural (p52, amino acids 202 to 434) protein. This assay, when compared to a consensus given by three commercial HCMV IgM immunoassays (discordant results were resolved by immunoblot), showed a relative sensitivity, specificity, and agreement of greater than 95%. In addition, the assay was able to detect seroconversion very early and displayed a higher positive reactivity rate than the commercial assays tested on pregnant women (168). The level of cross-reactivity was 3.3%. The diagnostic utility of the AxSYM IgM assay in detecting low levels of IgM antibody (not detected by other commercial assays) in some serum samples is stressed by the finding that some of these serum samples contain low-avidity IgG (154), a marker of primary HCMV infection (see IgG Avidity Assay).

At least one additional approach has been reported, with a combination of two HCMV peptides derived from pp150 (UL32, amino acids 1011 to 1048) and pp52 (UL44, amino acids 266 to 293) for IgM detection and a combination of peptides from pp150 (amino acids 1011 to 1048), pp28 (amino acids 130 to 160), and gB (amino acids 60 to 81) for optimal IgG detection (107). Sensitivity was 96.4% for the IgM assay with respect to a viral lysate-based ELISA.

Although the development of immunoassays based on use of recombinant viral proteins or peptide epitopes represents major progress towards standardization of serological assays, these assays do not appear to be reliable from the diagnostic standpoint due to exceedingly high sensitivity and somewhat low specificity. In a recent study, 10 of 42 (23.8%) potentially cross-reactive or interfering serum samples were scored IgM-positive with a commercial ELISA based on use of recombinant HCMV antigens, whereas two commercial ELISAs based on use of viral lysates detected zero and one positive sample, respectively, in the same panel of problematic serum samples (46). Indeed, false-positive results still represent the major pitfall of HCMV IgM serology. In this respect, in a recent retrospective review of 325 consecutive pregnant women referred to our laboratory over a 2-year period because of a positive IgM result and a suspicion of primary HCMV infection, as many as 188 (57.8%) were found to be IgM negative by two different in-house-developed capture ELISAs in the absence of primary infection (207).

Interpretation of positive IgM results. Once the specificity of a positive IgM result has been verified, the interpretation of the clinical significance of IgM antibody present in the serum of a pregnant woman begins. We must recall that the IgM antibody response, which is currently detected in primary HCMV infections of both immunocompetent and immunocompromised patients, may also be detected during recurrent infections of the immunocompromised person, but generally not in the immunocompetent host. Thus, IgM detection in the serum of a pregnant woman is likely to be a reliable marker of a primary HCMV infection. However, IgM can reveal different clinical situations which can be related to the acute phase of a primary HCMV infection, the convalescent phase of a primary HCMV infection, or the persistence of IgM antibody.

The kinetics of the HCMV-specific IgM antibody response during primary infection may vary greatly among individuals and depends substantially on the test or commercial kit used for testing. However, in general, high to medium levels of IgM antibody (peak titers) can be detected during the first 1 to 3 months after the onset of infection (acute or recent phase), after which the titer starts declining (convalescent or late phase) (Fig. 4; M. G. Revello and G. Gerna, unpublished data). By using two capture ELISAs, it was shown that of nine immunocompetent individuals, four became negative for IgM within 6 months, three within 12 months, while two remained IgM positive for more than a year after the onset of primary infection (213). A recent study compared the sensitivities of the same two in-house-developed IgM capture assays based on use of viral lysates (213) and a commercially available recombinant IgM assay (168). The kinetics of the IgM antibody response as determined on 213 sequential serum samples from 76 pregnant women with primary HCMV infection was grossly overlapping (Fig. 5), showing a low-level IgM antibody response persisting for several months (M. G. Revello, G. Gorini, M. Parea, and G. Gerna, unpublished data).

View larger version (24K): [in this window] [in a new window]   FIG. 4. (A) Kinetics of IgG, IgM, and neutralizing (Nt) antibody (Ab) response as well as IgG avidity index (AI) in a pregnant woman with primary HCMV infection. (B) Kinetics of infectious virus and different virus products in the blood of the same pregnant woman as in A during the convalescent phase of a primary HCMV infection. Ag, antigenemia; Vir, viremia; DNA, DNAemia; IE mRNA, immediate-early mRNA; +, positive; -, negative; GE, genome equivalents; PBL, peripheral blood leukocytes. (M. G. Revello and G. Gerna, unpublished data.)

View larger version (12K): [in this window] [in a new window]   FIG. 5. Kinetics of IgM antibody response in 76 pregnant women with primnary HCMV infection as determined in 213 sequential serum samples by using two in-house-developed capture assays in parallel. IgM assays were based on the use of (thin line) virus lysate (213) and (thick line) a commercial recombinant IgM assay (168). (M. G. Revello, G. Gorini, M. Parea, and G. Gerna, unpublished data.)

IgG avidity assay. When the presence of HCMV-specific IgM antibody in the serum of a pregnant woman cannot be directly related to a primary infection during pregnancy, an IgG avidity assay can help distinguish primary from nonprimary HCMV infection. This assay is based on the observation that virus-specific IgG of low avidity is produced during the first months after onset of infection, whereas subsequently a maturation process occurs by which IgG antibody of increasingly higher avidity is generated. Only IgG antibody of high avidity is detected in subjects with remote or recurrent HCMV infection. Avidity levels are reported as the avidity index, expressing the percentage of IgG bound to the antigen following treatment with denaturing agents, such as 6 M urea. The utility of the assay in diagnosing a primary infection has been reported for a variety of viruses (7, 17, 102, 123, 125, 142, 283). Measurement of IgG avidity is also of value in determining the duration of primary HCMV infection (20, 23, 101, 156, 207).

We have shown that mean avidity index values relevant to serum samples collected less than 3 months after onset of primary infection were 21% ± 13%, whereas mean avidity index values for serum samples from subjects with remote HCMV infection were 78% ± 10% (207). Thus, the presence of high IgM levels and a low avidity index are highly suggestive of a recent (less than 3 months) primary HCMV infection. In a recent study, an avidity index above 65% during the first trimester of pregnancy could reasonably be considered a good indicator of past HCMV infection, whereas in all women with a low avidity index (50%), there was a risk of congenital HCMV infection. The risk increased with the gestational age at the time of testing (20). That is, only 2 of 12 (16.7%) women with a low avidity index during the first trimester of pregnancy transmitted the infection to the fetus, whereas in utero infection of the fetus was found in 6 of 15 (40.0%) women with a low avidity index detected during the second or third trimester of pregnancy (20), approaching the transmission rate reported by several groups (110, 131, 264, 293). A negative predictive value of 100% was found when the avidity index was determined to be high or moderate before 18 weeks of gestation (157, 169). On the other hand, when the avidity index was calculated at 21 to 23 weeks of gestation, it failed to identify some women who transmitted the virus, with a negative predictive value of 90.9% (169).

Figure 6 shows the maturation of HCMV-specific IgG avidity in 560 sequential serum samples from 176 immunocompetent individuals with primary HCMV infection (M. G. Revello and G. Gerna, unpublished data). It can be observed that in the interval between 4 and 6 months after the onset of infection, while most avidity index values are intermediate, a minor portion are either low (<30%) or high (>50%). This implies that in some pregnant women examined during the first trimester of pregnancy, a low avidity index may be related to a primary infection acquired prior to conception (false-positive result with respect to primary infection during pregnancy), while a high avidity index observed in the second trimester of pregnancy does not necessarily exclude a primary infection acquired during pregnancy. Recently, the ability of three IgG avidity assays to detect a primary HCMV infection was found to approximate 100%, whereas the ability to exclude a recent infection was shown to range from 96% to 32%. These data indicate that standardization of the assay is urgently needed (22).

View larger version (20K): [in this window] [in a new window]   FIG. 6. Kinetics of IgG avidity index (maturation of HCMV-specific IgG) in 560 serum samples from 176 pregnant women with primary HCMV infection. (M. G. Revello, and G. Gerna, unpublished data.)

When we tested 89 serum samples from 22 pregnant women with primary HCMV infection with the same neutralizing assay, we found neutralizing antibodies in 9 of 20 (45%) serum samples collected within 30 days, 20 of 23 (87%) serum samples collected within 30 to 60 days, and in all 46 (100%) serum samples collected >60 days after onset (Fig. 4) (M. G. Revello and G. Gerna, unpublished data). Thus, the absence of neutralizing antibody in a serum sample from a pregnant woman containing HCMV IgG and IgM may indeed provide additional evidence of recent primary infection. In contrast, the presence of neutralizing antibody is of no help in interpreting a positive IgM result.

Conclusions. The most definitive diagnosis of primary HCMV infection in a pregnant woman is by detection of seroconversion, i.e., the appearance of HCMV-specific IgG antibody during pregnancy in a previously seronegative woman (Fig. 7). When this result cannot be achieved, detection of IgM antibody during pregnancy as well as during follow-up (whenever possible) can be used to determine clinically significant primary HCMV infection. Further testing by the IgG avidity test may be of great help in both confirming and clarifying the clinical significance of IgM antibody. When, at the end of the diagnostic algorithm, a primary HCMV infection is either diagnosed or suspected, prenatal diagnosis should be offered to a pregnant woman to verify whether the infection has been transmitted to the fetus. However, prior to performance of prenatal diagnostic procedures, the diagnosis of primary infection may be further confirmed or substantially supported by performing assays for detection of virus or virus products in the blood of the mother (Fig. 7).

View larger version (28K): [in this window] [in a new window]   FIG. 7. Schematic of diagnosis of primary HCMV infection in pregnancy, including both serologic and virologic approaches. AI, avidity index; Ag, antigenemia; Vir, viremia; DNA, DNAemia; IE mRNA, immediate-early mRNA; NT, neutralization test; Ab, antibody; pos, positive.

During the last decade, several methods have been developed to detect and quantify HCMV in blood. The most widely used assays include determination of viremia, i.e., infectious HCMV in blood; determination of antigenemia, i.e., number of pp65-positive peripheral blood leukocytes; quantification of HCMV DNA in whole blood (DNAemia), leukocytes (leuko-DNAemia), or plasma; determination of immediate-early and late mRNA (RNAemia); and search for the presence of circulating cytomegalic endothelial cells (CEC) in blood. An extended review of the methodological aspects and clinical applications of different assays for quantitation of HCMV has been published recently (24).

Viremia. Conventional methods for determination and quantitation of viremia are time-consuming because they are based on the appearance of cytopathic effect and include determination of 50% tissue culture infectious doses and plaque assays. These methods have been replaced by the "shell vial" assay, which provides results within 24 h. Following its introduction in the early 1980s (98), the assay was later rendered quantitative based on the assumption that each p72-positive fibroblast in a human fibroblast monolayer is infected by a single leukocyte carrying infectious virus (93). The shell vial monolayer is stained with either the immunofluorescence or the immunoperoxidase technique and a monoclonal antibody reactive with the HCMV major immediate-early protein (93). Then, the number of positive nuclei is counted (Fig. 8A). Since it was shown that a single monoclonal antibody may not identify virus strains with mutations in the relevant epitope of the major immediate-early protein, virus identification in our laboratory is performed with a pool of monoclonal antibodies reactive to different epitopes of p72 (G. Gerna, E. Percivalle, and M. G. Revello, unpublished data).

View larger version (80K): [in this window] [in a new window]   FIG. 8. (A) Viremia, indicating the presence in a shell vial monolayer of HCMV p72-positive fibroblast nuclei following cocultivation with peripheral blood leukocytes carrying infectious virus and immunostaining by fluorescein-conjugated p72-specific monoclonal antibody. (B) Antigenemia ex vivo, showing immunofluorescent staining with a pool of monoclonal antibodies of pp65-positive peripheral blood polymorphonuclear leukocytes from a patient with AIDS and disseminated HCMV infection. (C) Antigenemia in vitro, showing pp65-positive polymorphonuclear leukocytes from a healthy blood donor following cocultivation with HCMV-infected human umbilical vein endothelial cells and immunofluorescent staining with the same pool of pp65-specific monoclonal antibodies used in B. (D) Circulating cytomegalic endothelial cell with a pp65-positive leukocyte (arrow). Immunofluorescent staining was done with a pool of pp65-specific monoclonal antibodies.

Antigenemia. The antigenemia assay (Fig. 8B) detects and quantifies peripheral blood leukocytes, mostly polymorphonuclear leukocytes and, to a much lesser extent, monocytes/macrophages, which are positive for the HCMV lower matrix phosphoprotein pp65 (105, 209). This HCMV protein, which was initially believed to be the major immediate-early protein p72 (214, 278), is transferred to polymorphonuclear leukocytes from infected permissive cells via transitory microfusion events between two adhering cells (81, 211). The antigenemia assay has been optimized (92) and standardized (83) by using in vitro-infected leukocytes (Fig. 8C). The methodological aspects of this assay have been reviewed recently (24).

Experience obtained with transplant recipients has shown that antigenemia becomes positive earlier than viremia but later than DNAemia at the onset of infection, and it becomes negative later than viremia but earlier than DNAemia in the advanced stage of a systemic infection (89); high antigenemia levels are often associated with HCMV disease; the assay is widely used for monitoring of HCMV infections and antiviral treatment (25, 78, 116, 166, 172, 277); and during ganciclovir treatment of primary HCMV infections, antigenemia levels may increase for up to 2 to 3 weeks despite the efficacy of treatment as shown by the disappearance of viremia, prompting clinicians to erroneously change antiviral drugs (26, 94, 117, 183). A major advantage of the antigenemia assay is rapidity in providing results in a few hours, while major disadvantages are the limited number of samples processed per test run and the subjective component in slide reading (24).

DNAemia. Detection and quantification of HCMV DNA in blood has become a major diagnostic tool for transplant recipients. To this purpose, two major approaches have been used, PCR and hybridization techniques. For PCR, two main types of competitors have been used in the quantitative-competitive PCR: homologous competitors containing small deletions or insertions with respect to the target sequence (27, 76), and heterologous competitors having the target sequence for primers as the target nucleic acid but differing in the intervening sequence (84, 90, 95). In addition to in-house-developed methods, a commercially available method has been developed by Roche (Cobas Amplicor CMV monitor test; Roche Molecular Systems, Branchburg, N.J.) for both detection and quantification of HCMV DNA (55, 128). Finally, a new and interesting approach to the quantification of viral DNA is the detection and measurement of PCR products as they accumulate, thus overcoming the limited linear dynamic range of the traditional quantitative PCR. This technique, referred to as real-time PCR, is now being tested (Perkin Elmer, Applied Biosystems, Foster City, Calif.) and is based on the release of fluorescent dye molecules at each PCR cycle, the intensity of which is proportional to the amount of DNA in the sample (127).

Among the hybridization techniques amplifying the signal generated rather than the viral DNA itself, two have become commercially available for quantification of HCMV DNA: the Digene hybrid capture system CMV DNA assay (version 2.0; Abbott Laboratories, Abbott Park, Ill.) and the branched DNA assay (Bayer, Chiron Corporation, Emeryville, Calif.). The hybrid capture system is based on the formation of a DNA-RNA hybrid which is captured by a monoclonal antibody specific for the hybrid and is then reacted with the same monoclonal antibody labeled with alkaline phosphatase. The hybrid is finally detected with a chemiluminescent substrate, whose emission is proportional to the amount of target DNA present in the sample (173). The second-generation hybrid capture system assay has been reported to have increased sensitivity (24) and, thus could be considered for detection of viral DNA in the blood of immunocompetent hosts (see below). The branched DNA assay is based on the use of branched DNA amplifiers (branched probes) containing multiple binding sites for an enzyme-labeled probe. The target DNA sequence binds to the branched DNA molecule, and the complex is revealed by a chemiluminescent substrate whose light emission is directly proportional to the target DNA present in the sample (40, 141).

In immunocompromised patients, HCMV DNA quantification has been shown to be useful for follow-up of disseminated infections and evaluation of the efficacy of antiviral treatment. In addition, it is useful for the diagnosis and local evaluation of the effect of antiviral treatment at special body sites, such as the eye and nervous system (84, 210). Finally a special application concerns its use for prenatal diagnosis of HCMV infection and for quantification of viral DNA in amniotic fluid samples (see below).

RNAemia. Detection of HCMV transcripts in blood is currently considered a marker of HCMV replication in vivo and late viral transcripts in particular are considered to better reflect active HCMV replication and dissemination (100, 148). With reverse transcription-PCR, false-positive results may result from the difficulty in differentiating between RNA- and DNA-derived PCR products in the case of unspliced transcripts (85). Unlike reverse transcription-PCR, detection of mRNAs by the recently introduced nucleic acid sequence-based amplification (NASBA) method, which allows specific amplification of unspliced RNA in a DNA background (42), appears very useful for different populations of transplant recipients (8, 19).

Recently, two retrospective studies, in which preemptive therapy of both solid organ and hematopoietic stem cell transplant recipients was antigenemia guided, monitoring of HCMV pp67 mRNA (a late viral transcript) by NASBA appeared to be a promising tool for initiation and termination of preemptive therapy for solid organ transplant recipients with reactivated HCMV infection (87), whereas monitoring of immediate-early mRNA expression appeared to be a useful parameter for initiation of preemptive therapy in hematopoietic stem cell transplant recipients (86). At this time, prospective studies with NASBA assays are ongoing in transplant recipients, whereas preliminary data on the kinetics of immediate-early mRNA in immunocompetent individuals with primary HCMV infection are already available (208).

Endotheliemia. The term endotheliemia was introduced to indicate HCMV-infected CEC in the peripheral blood of immunocompromised patients. CEC were first described in 1993 by two independent groups (104, 196) and were shown to be endothelial in origin and fully permissive for HCMV replication. CEC are derived from infected endothelial cells of small blood vessels, which progressively enlarge until they detach from the vessel wall and enter the bloodstream. More recently, CEC have been studied in hematopoietic stem cell transplant recipients (232) and in AIDS patients with disseminated HCMV infection (96). In recent years, the introduction of highly active antiretroviral therapy for AIDS patients and the adoption of prophylactic and preemptive therapy approaches for transplant recipients have nearly eliminated CEC from blood of these patient groups. However, CEC may still be found in the blood of fetuses (Fig. 8D) and newborns with symptomatic congenital HCMV infection (M. G. Revello, E. Percivalle, and G. Gerna, unpublished data).

Virus and viral products in blood of immunocompetent persons as an aid for diagnosis of primary infection. Although there is an extensive amount of data obtained from studies with immunocompromised patients (78, 166, 274), very few data are available on the presence of HCMV in the blood of immunocompetent individuals with primary infection (33, 138, 140, 221, 222). In particular, little has been done to assess the diagnostic value of virus detection in the blood of nonimmunocompromised patients. Recently, an investigation was conducted on the peripheral blood leukocytes of 52 immunocompetent individuals (40 pregnant women) with primary HCMV infection by quantitation of pp65 antigenemia, viremia, and leukoDNAemia (216). pp65 antigenemia was detected in 12 of 21 (57.1%), 4 of 16 (25%), and 0 of 10 patients examined 1, 2, and 3 months after onset, respectively. Viremia was detected in 5 of 19 (26.3%) patients during the first month only. Finally, leukoDNAemia was detected in 20 of 20, 17 of 19 (89.5%), and 9 of 19 (47.3%) patients tested 1, 2, and 3 months after onset, respectively. Four (26.6%) of 15 patients were still DNAemia positive at 4 to 6 months, whereas none were positive at >6 months. No assay was positive in any of 20 subjects with remote infection or of 9 subjects with recurrent infection. In addition, virus levels were low by all assays. The conclusion of the study was that primary HCMV infection can be rapidly and specifically diagnosed whenever any of the studied virologic markers is detected in blood. On this basis, dating of the onset of infection can also be attempted (216).

Viremia, i.e., virus recovery from blood, allows diagnosis of primary infection in about 25% of cases during the first month after onset. In fact, HCMV could not be recovered from the blood of 86 blood donors (249) and in only one study was HCMV isolation from the blood of 2 of 35 normal donors reported (56). However, in this case, the interpretation of positive HCMV recovery from the blood of healthy people may hypothetically be referred to the convalescent phase of an unknown asymptomatic primary infection. Antigenemia may allow diagnosis of primary HCMV infection in 50% of patients in the first month and in 25% of patients in the second month after onset of infection. Again, positive antigenemia was never reported in immune healthy subjects or in patients prior to transplantation. Finally, DNAemia and, in particular, leukoDNAemia allow diagnosis of primary HCMV infection in 100% of subjects examined within 1 month after onset of infection and in 98% of those tested within 2 months.

The presence of viral DNA in the leukocytes of healthy people is a more controversial issue. While in three reports viral DNA was found in virtually all seropositive healthy adult volunteers and in most seronegative persons when monocytes (268, 273) or peripheral blood leukocytes (14) were examined, other investigators failed to detect viral DNA by PCR in monocytes (16) or peripheral blood leukocytes (78, 135, 229, 240) or reported low (4 to 6%) positivity rates (34, 249). Recent studies support the concept that viral DNA is not detected in the peripheral blood leukocytes of HCMV-seropositive immunocompetent individuals (216, 294), emphasizing the utility of viral DNA detection in blood as a parameter for diagnosing primary HCMV infection.

More recently, a new virologic parameter has been found to be useful for diagnosis of primary HCMV infection, detection of immediate-early mRNA by the NASBA technology. The clinical specificity of this parameter was first assessed in healthy individuals with remote or recurrent HCMV infection, showing its consistent negativity. Then, immediate-early mRNA was detected in the blood of all subjects with primary HCMV infection in the first month after onset of infection, whereas the proportion of positive subjects declined over time and became negative 6 months after onset of infection (208). Thus, immediate-early mRNA kinetics appears to be comparable to that already reported for viral DNA, which suggests that detection of immediate-early mRNA in the blood of immunocompetent individuals can be considered an additional marker of recent primary HCMV infection. However, if immediate-early mRNA detection appears to be slightly more sensitive than DNA detection in diagnosing the early phase of primary HCMV infection (86), it appears to be slightly less sensitive in detecting the late phase of primary HCMV infection (208).

In a survey conducted on 60 pregnant women with primary HCMV infection, mild clinical symptoms and/or liver function abnormalities were detected in as many as 38 (60%) (207). In a more recent survey (M. G. Revello, and G. Gerna, unpublished data) conducted on 244 pregnant women with primary HCMV infection, clinical symptoms were present in 166 (68.1%), fever (60.2%), fatigue (48.8%), and headache (26.5%) being the most frequent symptoms (Table 1). In addition, 70 (42.1%) women reported three or more symptoms. The high rate of symptomatic primary HCMV infections in pregnancy may be explained by the careful medical interview. Whether the pregnancy-associated immunosuppression might play a critical role remains to be determined.

Diagnosis of recurrent infection can be accomplished by virus isolation or viral antigen or viral DNA detection in clinical samples, such as samples from the genital tract or cervix or urine, other than blood in the absence of concomitant serologic and virologic markers of primary HCMV infection, i.e., HCMV-specific IgM, low-avidity IgG, absence of neutralizing antibodies, and absence of virus and viral markers in blood. In this respect, it is very important to emphasize that, in order to ascertain the diagnosis of a congenital HCMV infection following a recurrent infection of the mother, at least the following requirements must be satisfied: the mother must be defined as immune to HCMV at least 1 year prior to pregnancy; a prepericonceptional HCMV infection (see below) must be excluded; and HCMV should be recovered from the genital tract. Thus, only prospective well-designed and extended epidemiological studies will be able to define the true impact of recurrent maternal infections (either reactivations or reinfections) in determining congenital HCMV infections in the future.

In addition, virus recovery during pregnancy from the cervical tract or urine in both primary and recurrent infections is a poor indicator of risk of intrauterine transmission (258). The neutralizing antibody response has also been investigated as a potential prognostic marker of intrauterine transmission. Lower neutralizing antibody titers were detected in transmitting mothers with primary HCMV infection compared to nontransmitters, suggesting an association of neutralizing activity and intrauterine transmission (29). In the same study, a significant correlation was also observed between neutralizing activity and antibody avidity, thus suggesting that a maturation of antibody avidity is necessary for production of high levels of neutralizing antibodies, while a defect or delay in avidity maturation may play a role in intrauterine HCMV transmission (29).

Symptomatic congenital HCMV infections have been noted in infants born to mothers with prepregnancy anti-HCMV immunity (28). Moreover, intrauterine transmission of HCMV from immune mothers to their infants has been related to reinfection with a different virus strain capable of causing symptomatic infections, as measured by the acquisition of new antibody specificities against epitopes of the glycoprotein H of the reinfecting HCMV strain (30). However, only prospective studies will be able to define the frequency of reinfection in immune pregnant women and its clinical impact on congenital infections.

Finally, a lymphoproliferation assay against HCMV has been reported to provide an early marker of fetal infection after primary HCMV infection in pregnancy (269). In that study, all eight women with positive lymphoproliferative response gave birth to uninfected babies, whereas four of six women with negative responses delivered congenitally infected babies. Those findings suggested that depression of cell-mediated immunity in pregnant women after primary infection may represent a marker of fetal infection.

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Once a diagnosis of primary HCMV infection has been achieved, the woman should receive sufficient information to make informed choices about further testing and options. This step is generally indicated with the term counseling. The term