Update on the prevention, diagnosis and management of cytomegalovirus infection during pregnancy

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1 REVIEW /j x Update on the prevention, diagnosis and management of cytomegalovirus infection during pregnancy T. Lazzarotto 1, B. Guerra 2, L. Gabrielli 1, M. Lanari 3 and M. P. Landini 1 1) Department of Haematology, Oncology and Laboratory Medicine, Operative Unit of Clinical Microbiology, 2) Department of Obstetrics and Gynaecology, St Orsola Malpighi General Hospital, University of Bologna, Bologna and 3) Operative Unit of Paediatrics and Neonatology, La Scaletta Hospital, Imola-Bologna, Italy Abstract Human cytomegalovirus (CMV) is the leading cause of congenital infection, with morbidity and mortality at birth and sequelae. Each year approximately 1 7% (Rev Med Virol 2010; 20: 311) of pregnant women acquire a primary CMV infection. Of these, about 30 40% transmit infection to their fetuses. The risk of serious fetal injury is greatest when maternal infection develops in the first trimester or early in the second trimester. Between 10 and 15% of congenitally infected infants are acutely symptomatic at birth and most of the survivors have serious long-term complications. Until a few years ago, laboratory testing was not possible to precisely define the maternal immune status, the recent development of advanced serological tests (IgG avidity test, IgM immunoblot and neutralizing antibody testing) allow us to identify, among pregnant women with suspected CMV, those with primary infection who are therefore at high risk of transmitting CMV to the fetus. This is done with the use of a screening test. As most maternal infections are asymptomatic, the only way to disclose primary infection is to implement specific serological testing as early in pregnancy as possible (before week of gestation). Given the high risk of mother fetus transmission and fetal damage, prenatal diagnosis is recommended to women with primary CMV infection contracted in the first half of pregnancy and in case of fetal abnormalities suggestive of infection. The correct interpretation of serological and virological tests followed by appropriate counselling by an expert physician is an effective tool to reduce the number of unnecessary pregnancy terminations by over 70% (Am J Obstet Gynecol 2007; 196: 221.e1). Keywords: Amniocentesis, cytomegalovirus, pregnancy, prevention, serological diagnosis Article published online: 25 April 2011 Clin Microbiol Infect 2011; 17: Corresponding author: T. Lazzarotto, Department of Haematology, Oncology and Laboratory Medicine, Operative Unit of Clinical Microbiology, Laboratory of Virology, Policlinico S. Orsola Malpighi, Via Massarenti n. 9, Bologna, Italy tiziana.lazzarotto@aosp.bo.it Introduction Human cytomegalovirus (CMV) is an endemic and ubiquitous beta-herpesvirus that leads to congenital infection in % of all live births [1,2]. The overall birth prevalence of congenital CMV infection was reported to be 0.64% (95% CI ), but varied considerably among different study populations [3]. Congenital CMV infection is the leading non-genetic cause of deafness in children: more than half of babies born with symptomatic infection and 10% of asymptomatic newborns will develop mild to severe neurosensory hearing loss, which is progressive in 50% of cases [4]. Infection with CMV can occur in pregnant women by non-primary infection, namely reactivation of the latent virus or reinfection with a different strain, or by primary infection. Mother child transmission is mainly the result of primary maternal CMV infection, which carries a risk of transmission varying from 14.2% to 52.4% (combined prevalence 32.4%) [3], the transmission rate is lower, 36%, during the first Clinical Microbiology and Infection ª2011 European Society of Clinical Microbiology and Infectious Diseases

2 1286 Clinical Microbiology and Infection, Volume 17 Number 9, September 2011 CMI trimester compared with third-trimester infection, 77.6% [5]. Cases of CMV transmission as the result of non-primary infection have been reported in 1.4% ( %) of cases, i.e. at a much lower rate than those resulting from primary infection [3]. The extent of fetal/newborn injury, namely severe brain damage, is correlated to the gestational epoch in which vertical transmission occurs: the most severe is correlated to primary maternal infection contracted in the first 2 months of pregnancy [6]. Only 10 15% of congenitally infected babies present symptoms of infection at birth and these infants have a perinatal mortality rate of around 10% with 70 80% of surviving babies presenting major neurological sequelae [7]. The prognosis of symptomatic infants is very poor, with the majority suffering from severe mental impairment and/or hearing loss. Despite infection, 85 90% of babies have no symptoms at birth but 8 15% of them will suffer delayed injury [2,7]. Cytomegalovirus can also be transmitted to the fetus when primary maternal infection occurs before conception. In 2006 Revello et al. [8] reported that preconception primary CMV infection (3 months before the last menstrual period) carries a low risk of intrauterine transmission (8.7%) and one of 12 newborns examined at birth was found to be subclinically infected. In another study, Daiminger et al. [9] reported no cases of intrauterine transmission in three women in whom primary CMV infection occurred 2 8 weeks before their last menstrual period. The risk of intrauterine transmission after preconceptional primary CMV infection appears to be significantly lower than that associated with primary infection during pregnancy. The transmission rate is considerably higher in the case of periconceptional CMV infection. Revello et al. [10] showed that in periconceptional infection (4 weeks after the last menstrual period) the virus was transmitted in 30.8% of cases. Daiminger et al. [9] reported a 45% transmission rate of CMV infection in the periconceptional period (between 1 week before and 5 weeks after last menstrual period) and recently, Hadar et al. [11] found a vertical transmission rate of 25% in primary periconceptional CMV infection (4 weeks before last menstrual period and up to 3 weeks after the expected date of the missed menstrual period). The outcome of periconceptional infection with regard to symptomatic disease in the fetus or newborn was difficult to assess. Prospective studies designed to investigate the transmission rate and outcome in women with preconceptional and periconceptional primary CMV infection are needed. Prevention It is well known that primary infections in the mother have a much greater clinical impact on the fetus than reactivated infections or exogenous reinfections [12]. Prevention is difficult because the virus is ubiquitous and infection is common. Screening No country performs systematic screening for CMV infection among pregnant women as universal screening for CMV primary infection. The time for screening would be at the beginning of pregnancy. Arguments against screening include the fact there is no effective vaccine and no treatment that has been proven effective. Nevertheless, in certain areas, largely uncontrolled serological screening is increasingly performed with the objective of prenatal diagnosis. Hygienic intervention Although controversial, there are some practices that might minimize the risk for congenital infection. The saliva and urine of infected children are significant sources of CMV infection among women who are pregnant. Prevention behaviours (i.e. hand washing whenever there is contact with a child s saliva or urine, not sharing drinking glasses or eating utensils with young children, and not kissing young children on the mouth or cheek) appear to be generally acceptable. Education of pregnant women about the implication of acquiring CMV infection is vital. In a study where seronegative mothers with a child in group day care were instructed on measures to prevent CMV transmission, pregnant mothers had a significantly lower rate of CMV infection when compared with non-pregnant mothers attempting conception [13]. Recently, Vauloup-Fellous et al. [14] reported that hygiene counselling given during pregnancy reduces the CMV seroconversion rate. Hygienic intervention for CMV-seronegative pregnant women in both Europe and the United States is now appropriate. Active vaccine The development of a CMV vaccine is one of the highest health priorities, but availability of a licensed vaccine to prevent CMV infection in seronegative individuals is not imminent. In 2009, Pass et al. published the first results of a phase 2, randomized, double-blind, placebo-controlled clinical trial of vaccine consisting of recombinant CMV envelope glycoprotein B with MF59 adjuvant. The authors conclude that

3 CMI Lazzarotto et al. CMV infection in pregnancy 1287 CMV glycoprotein B vaccine has the potential to decrease incident cases of maternal and congenital CMV infection but future studies, such as a phase 3 clinical trial, are needed to confirm the efficacy of this vaccine [15]. Prenatal treatment Currently, no therapeutic options during pregnancy are available except for clinical trials. Recent data in the literature have focused on the efficacy of preventive administration of CMV immunoglobulins [16] or antiviral drugs (valacyclovir) [17] to pregnant women with primary CMV infection to reduce the rate of vertical transmission and improve neonatal outcome. The first multicentre study (CHIP study: Congenital HCMV Infection Prevention Clinical Trials.gov ID NCT ) was designed as a randomized, double-blind, placebo-controlled, prospective trial (phase 2) for the evaluation of the efficacy of CMV-specific hyperimmune globulin Cytotect Ò, (Biotest AG, Dreieich, Germany) administration in pregnant women with the acute phase of primary CMV infection for prevention of intrauterine transmission. This experimental study began in February 2010 in Italy, enrolment ended in March 2011 and the study will be completed in December A second multicentre study (Cytotect Ò study) was designed as a phase 3 prospective trial in congenital CMV infection for the evaluation of the efficacy of CMV-specific hyperimmune globulin Cytotect Ò, (Biotest AG) administered in pregnant women who seroconverted in the first trimester to protect against transmission of the virus to the unborn child and thereby to prevent injury to the child. This experimental study began in 2008 in Germany. In January 2011 the interim analysis showed an indication of the efficacy of Cytotect Ò when the treatment group was compared with the control group. The interim analysis was conducted on 7000 of the pregnant women planned to complete the study ( news/newsdetails.cfm?newsid= ). A third multicentre study ( index.php?option=com_content&view=article&id=45&itemid= 57) was designed as a randomized double-blind placebo-controlled prospective trial evaluating the efficacy of valacyclovir (the oral pro-drug of acyclovir), The drug is given orally to pregnant women in cases of confirmed fetal CMV infection (amniotic fluid PCR-positive) and with extracerebral signs visible on ultrasound that can be attributed to the infection to reduce the number of cases with unfavourable outcomes (children symptomatic at birth) and of medically indicated terminations of pregnancy. This experimental study began in September 2009 in France and the study will be completed in June Diagnosis of Maternal Infection Today, laboratory techniques represent a decisive approach for diagnosis of CMV infection in pregnancy. Clinical findings Most CMV infections encountered in pregnant women are asymptomatic even during the acute stage. About 25% of pregnant women with primary infection are reported to be symptomatic. Even in cases with symptoms, the manifestations are non-specific and mild, such as persistent low fever (60.2% of cases), fatigue (48.8%) and headache (26.5%) [18]. Laboratory tests may sometimes disclose atypical lymphocytosis and slightly raised transaminase levels. Clinical diagnosis of CMV infection is unreliable. Serological findings The gold standard of serological diagnosis of primary CMV infection is maternal seroconversion or the presence of serum anti-cmv specific IgM antibodies combined with low avidity anti-cmv IgG antibodies. The diagnosis of primary CMV infection is straightforward if seroconversion to CMV-specific antibodies, i.e. the de novo appearance of virus-specific antibodies in the serum of a pregnant woman who was previously seronegative, is detected. In fact, if consecutive blood samples are available, the presence of anti-cmv IgM and IgG antibodies in a previously IgG-negative individual provides determination of seroconversion and CMV primary infection. Currently, routine antenatal screening for CMV of pregnant women has never been recommended by any public health authority in any country so diagnosis via seroconversion is only occasionally achieved. Testing for anti-cmv IgM antibodies is the most widely used and appropriate procedure for screening pregnant women [19]. Anti-CMV IgM antibodies are a good indicator of acute or recent infection but are not always correlated with primary infection. This is because pregnant women can produce IgM during reactivations or reinfections. In addition, virus-specific IgM may persist for months after natural infection and anti-cmv IgM antibodies have been detected in some pregnant women 6 9 months after the end of the acute phase of primary infection. False-positive results are common and may arise in patients with other viral infections (e.g. parvovirus B19, Epstein Barr virus) or autoimmune diseases or as the result of interference by rheumatoid factor of the IgM class, and finally false-positive results may also be ascribed to the laboratory methods used [19 21]. Hence, the detection of IgM in the serum of pregnant women may

4 1288 Clinical Microbiology and Infection, Volume 17 Number 9, September 2011 CMI simply be a starting point for further diagnostic investigations [22]. Immunoblotting with purified native viral proteins and purified recombinant proteins (structural and non-structural) has been shown to be an effective method to confirm the presence of CMV IgM antibodies in serum with a high sensitivity (100%) and specificity (98.6%) [23]. In addition, analysis of the virus-specific IgM response to individual structural and non-structural CMV proteins allows the detection of fairly typical reactive profiles to distinguish primary from nonprimary infection. Moreover, serum IgM from women who transmit CMV infection reacts with a higher number of antigen bands than serum IgM from women who do not transmit the infection, probably because transmitting women have a higher viral load. (p <0.0001) [23]. The anti-cmv IgG avidity test is currently the most reliable commercial procedure to identify primary infection in pregnant women [24]. Antibody avidity indicates the strength with which a multivalent antibody binds to a multivalent antigen. During the first weeks following primary infection, IgG antibodies show a low avidity for the antigen, but they progressively and slowly mature, initially acquiring a moderate and then a high avidity. This process reflects the maturation of the immune response and the high avidity antibodies are maintained for many years. For this reason, low avidity CMV-IgG antibodies are found only after primary antigenic stimulation and usually last for approximately weeks after the onset of CMV infection [25,26]. A low avidity index indicates the presence of low-avidity IgG antibodies in serum during an acute or recent primary CMV infection [27,28]. A high avidity index during the first weeks of gestation could be considered a good indicator of past infection [29]. The determination of anti CMV antibody avidity carried out before weeks of gestation is therefore a helpful tool to identify all pregnant women who may give birth to an infected newborn (100% sensitivity). If the IgG avidity index is determined later during pregnancy (after weeks of gestation), the sensitivity is drastically reduced (62.5%) [25]. Interpreting test results is important because serological tests vary from one laboratory to another so the method used and its reference values must be carefully assessed. The value of CMV IgG avidity helps to interpret a positive result for CMV IgM; the limits are that the kinetics of IgG avidity depend on the kit used and with some of these kits, the results may depend on CMV IgG concentration [30,31]. Recently, Revello et al. [30] reported the results of a comparative evaluation of eight Conformité Européenne-marked CMV IgG avidity assays and concluded that commercial kit performances for IgG avidity determination are variable. In this study the time frames chosen for the disaggregation in three groups (<3 months, 3 6 months, >6 months) do not allow a sound estimate of assay accuracy. The conclusions obtained on this panel may then be difficult to translate in practice and to compare with the results of previous studies and with the package inserts of commercial CMV IgG avidity assays. Moreover, dating the infection may be difficult in cases for which the IgG avidity index is reported as moderate/intermediate/grey-zone. Particular problems arise with large ranges of reference values of moderate avidity because the larger the range, the less specific the results are. In our experience using the Radim CMV IgG avidity assay (Cytomegalovirus IgG avidity EIA WELL; RADIM, Rome, Italy), moderate avidity results combined with specific CMV- IgM antibodies constitute a reliable marker of a recent primary infection especially when pregnant women are screened for CMV infection for the first time only after the first trimester and in the absence of previous serological data [22]. We tested 380 sera that were positive for CMV IgG and IgM collected from 380 pregnant women with ascertained primary CMV infections. Pregnant women were randomized into three groups. In the first group the primary CMV infection was diagnosed before 12 weeks of gestation and among the 99 pregnant women of this group, 70 had a low avidity and 29 a moderate avidity for CMV IgG, with 16 of the 70 (23%) and one of the 29 (3.4%) transmitting a congenital CMV infection to the newborn/fetus. In the second group, 240 primary CMV infections were diagnosed between 13 and 24 weeks of gestation: 180 of 240 pregnant women had a low avidity and 60 had a moderate IgG avidity index, with 57 of 180 (31.6%) and 14 of 60 (23.3%) transmitting congenital CMV infection to the newborn/fetus. Similar data were obtained in the third group, in whom the diagnosis of primary CMV infection was established after 24 weeks of gestation (T. Lazzarotto, unpublished data). Today, we believe that moderate CMV IgG avidity must be interpreted with caution and additional studies with other commercial CMV IgG avidity assays are urgently needed to understand the significance of IgG moderate/intermediate/ grey-zone avidity in confirming the diagnosis of CMV primary infection during pregnancy. In 2004 Macé et al. [24] demonstrated that the best algorithm for the diagnosis of primary CMV infection in pregnant women was the determination of the anti-cmv antibody avidity only in serum samples positive for IgM antibodies. This procedure eliminates a number of samples with moderate avidity index and IgM-negative results, which are difficult to manage clinically.

5 CMI Lazzarotto et al. CMV infection in pregnancy 1289 The CMV microneutralization assay carried out early in pregnancy can identify all pregnant women who will transmit CMV to their offspring [32]. After seroconversion the first neutralizing titres appear after an average of 15 weeks (range weeks), with a progressively increasing titre. The absence of neutralizing antibodies in a serum sample from a pregnant woman containing CMV IgG and IgM may indeed provide additional evidence of recent primary infection [32]. Eggers et al. [32] showed that the combined application of the microneutralization assay and avidity assay identified all primarily infected pregnant women who gave birth to congenitally infected infants. Serological diagnosis of primary CMV infection is then reliable. In pregnant women who are CMV seropositive before pregnancy the serological diagnosis of a non-primary CMV infection may be documented by a significant increase in CMV-specific IgG antibody titre with or without the presence of specific IgM antibodies and high IgG avidity. It is recommended that the consecutive blood samples be processed in the same run to avoid analytical variability. In women whose pre-pregnancy serological status is unknown the presence of high titres of serum-specific IgG and IgM anti-cmv combined with high IgG avidity during the first weeks of gestation may be indicative of a non-primary CMV infection. To date, serological diagnosis of non-primary CMV infection is difficult and often unreliable because no optimal diagnostic methods are available. Virological findings Virological tests play a secondary role in the diagnosis of primary CMV infection in pregnant women and can only support serological diagnosis [22]. Cytomegalovirus may or may not be detected in maternal blood (DNAemia) in pregnant women undergoing primary infection at the time of serological diagnosis. However, positive detection of virus in blood is not associated with a greater risk of infection or fetal/neonatal injury [22]. In most women with non-primary infection, DNAemia is negative. In pregnancy, during both primary and non-primary infection, CMV may be cleared in body fluids. Isolation of virus in urine or cervical secretions is a poor indicator of the risk of intrauterine transmission and the severity of fetal/neonatal damage. Virological diagnosis of CMV infection is often unreliable. Management of CMV Infection During Pregnancy As pregnancy does not usually affect the clinical course of infection, which is usually symptom-free in immmunocompetent subjects, laboratory tests are the best means of establishing diagnosis. It is up to the physician to decide on the basis of maternal status before conception whether to test for CMV in pregnancy and which tests to prescribe and then to interpret the results accurately. Women seropositive for CMV before conception IgG-specific antibodies in serum disclosed at the first test during pregnancy (week 8 10) is indicative of past infection and no further investigation is required. There is tacit agreement among the international community that no laboratory testing for CMV is needed, unless indicated by particular clinical conditions such as abnormal ultrasonographic findings. Even though it does not offer full protection, acquired immunity will defend the mother from primary infection in pregnancy which carries a much greater risk of fetal damage. Any reinfection or reactivation of infection carries the same risk as pregnancy itself. For these reasons, the information on maternal status before conception precludes the need for screening during pregnancy [33]. Women seronegative for CMV before conception Non-immune pregnant women at the early prenatal visit (week 8 10) are at risk of acquiring primary infection. First and foremost they must be informed of hygiene and behaviour measures (avoiding direct contact with organic materials, close contact with pre-school children and frequent thorough hand-washing) to reduce the chance of infection [13]. Women with daily household or occupational contact with children <3 years old are at high-risk of acquiring primary CMV infection during pregnancy. Recommendations to prevent CMV infection during pregnancy have been edited by the European Congenital CMV Initiative (ECCI; and the Centers for Disease Control and Prevention (CDC; cmv/index.html). Seronegative pregnant women must also undergo periodic serological testing. Although there are no universally accepted guidelines, a second testing should be done at weeks of pregnancy and no later than is reasonable to implement fetal investigations in the case of seroconversion. A positive result allows for the optimal timing of prenatal diagnosis (20 21 weeks of gestation) considering the time limit for termination of pregnancy in several countries [34]. If the mother continues to be seronegative, serological follow-up testing can be limited or confined to one more test at weeks to select newborns at risk of congenital infection in the case of late seroconversion.

6 1290 Clinical Microbiology and Infection, Volume 17 Number 9, September 2011 CMI Negative IgG IgM Serological screening Positive 6 12 weeks of gestation Hygiene and behaviour measures Periodic serological testing IgG+ IgM+ or IgG IgM+ IgG+ IgM Past infection NO No seroconversion Seroconversion Advancedd serological testing No further investigation BBefore f 16 weeks k of gestation Serological screening at weeks of gestation* Recommendations for future pregnancy Primary infection Non primary infection Past infection No seroconversion* Prenatal diagnosis Ultrasonographic evaluation No further investigation weeks of gestation Positive Negativee Fetal infection PPV: 100% No fetal infection NPV: ~95% FIG. 1. Recommendations for management of cytomegalovirus (CMV) infection during pregnancy. *Represents, the serological screening at weeks of gestation. s Represents, the recommendations for future pregnancy. NPV, negative predictive value; PPV, positive predictive value. Women whose pre-pregnancy serological status is unknown Diagnosis of CMV infection is more complex in women who are unaware of their serological status before pregnancy. As most infections are asymptomatic, the only way to disclose primary infection is to implement specific serological testing as early in pregnancy as possible (before week of gestation). Recommendations for management of CMV infection during pregnancy are shown in Fig. 1. Diagnosis of Fetal Infection Prenatal diagnosis The fetal compartment can be studied by invasive and noninvasive prenatal diagnostic investigation. Ultrasound has the advantage of not being invasive and will disclose any structural or growth abnormalities caused by CMV infection, but its sensitivity is poor and it correctly identifies no more than 20% of infected babies, even in a selected population [35,36]. In addition, a structural abnormality may be disclosed a long time after the initial negative tests and borderline structural changes detected early in pregnancy could be temporary [35,36]. Moreover, when fetal infection status is unknown, ultrasound abnormalities predict symptomatic congenital infection in only a third of cases [35]. The diagnostic protocol, both invasive (amniocentesis or cordocentesis) and non-invasive (ultrasonography) prenatal diagnostic tests, has to be offered to women at high risk of transmitting CMV to the fetus, in particular to pregnant women who develop a primary infection or an undetermined type of infection during the first half of gestation and in case of abnormal findings suggestive of congenital infection (such as intrauterine growth retardation, hydrops or ascites, hyperechogenic bowel, pleural or pericardial effusion, hepatosplenomegaly, intrahepatic calcifications, pseudomeconium ileus, and central nervous system abnormalities) [26,37]. Prenatal diagnosis provides the best means for diagnosing fetal infection. The specificity is usually very good (100%) and the sensitivity depends on: the kind of samples used (amniotic fluid > fetal blood); the technique used (real-time PCR > viral culture); the number of techniques used; the timing of the procedure with respect to (i) the onset of maternal infection, there is a 6 8-week window between maternal infection and antenatal procedure [26,37], and (ii) the gestational age, false-negative results are common when amniocentesis is carried out before week 20 of gestation [38]. Little virus is shed by the fetal kidney because of limited diuresis early on. Amniocentesis The amniotic fluid (AF) is the most appropriate material for the diagnosis of fetal CMV infection. The risk of CMV transmission during antenatal diagnostic procedures performed in

7 CMI Lazzarotto et al. CMV infection in pregnancy 1291 the presence of maternal DNAemia does not seem to be major as maternal CMV DNAemia is not a significant risk factor for iatrogenic CMV transmission to the fetus during amniocentesis [39]. The AF is subjected to a direct search for CMV in culture and for the viral genome by real-time PCR. If both techniques are negative then fetal infection can be ruled out with a high degree of certainty (specificity = 100% and negative predictive value = 94.2%). Positive results in AF identify CMV-infected fetuses (positive predictive value = 100%) but do not discriminate those infants who will have symptoms at birth [40]. If results are positive, investigation of AF is completed by CMV-DNA load evaluation. Low viral load in AF is consistently found to be associated with asymptomatic congenital infection. The presence of viral loads <10 3 copies/ml in the AF, sampled at the appropriate times (at weeks of gestation and when the time interval between onset of maternal infection is 6 8 weeks) are a good indicator that rules out fetal damage at birth and subsequent exacerbation of infection with the onset of sequelae like hearing loss and delayed psychomotor development [40]. Although the highest median values of CMV-DNA in AF tend to indicate an increased risk of severe infection, high viral loads may be associated with symptomatic or asymptomatic congenital infections indeed, a correlation between the high CMV load in AF and fetal/neonatal outcome has not been demonstrated [40]. Cordocentesis A PCR on fetal blood taken at weeks gestation has a sensitivity for congenital infection lower than that obtained with AF (sensitivity range, % and specificity about 100%) [18,26]. Studies of prenatal diagnosis of fetal CMV infection found that no assay for detection of virus or virus components in fetal blood was sensitive enough to significantly improve prenatal diagnosis of intrauterine transmission of the virus. The sensitivity of PCR on fetal blood reaches 100% when the fetal blood is taken after 30 weeks of gestation [36]. The sensitivity of specific IgM detection in fetal blood obtained after 20 weeks of gestation ranged from 55.5% to 80%. Moreover, past studies demonstrated that the presence of CMV-specific IgM at significant levels represented a prognostic marker of congenital CMV disease [18,26]. More recently, other studies have evoked the prognostic value of fetal viraemia/viral load and level of specific IgM but this remains controversial. It has been proposed that platelet count gives a better indication [36,41]. Virus isolation from urine and/or saliva in the first 2 weeks of life Negative result Uninfected newborn No further testing is required New data demonstrated that the determination of multiple markers (haematological, biochemical and virological markers) in fetal blood following virus detection in AF, is predictive of perinatal outcome in fetuses with CMV infection [42]. Further studies in a larger number of symptomatic cases should be performed to verify the prognostic efficacy of determination of multiple parameters in fetal blood. Diagnosis of Infection in Newborns At birth, it is essential to use appropriate tests for the diagnosis of CMV congenital infection. The standard for the diagnosis of congenital CMV infection in newborns remains viral isolation in the urine or saliva within the first 2 weeks of life (Fig. 2). But even urinary real-time PCR is a reliable, rapid and convenient method to diagnose congenital CMV infection. Detection of specific IgM in neonatal serum also discloses congenital infection, but IgM antibodies are only present in between 20 and 70% of infected babies [18]. All infected babies undergo follow-up monitoring at 1, 3, 6 and 12 months of life and annually thereafter until school age (Fig. 3). Conclusion Positive result Newborn with CMV congenital infection Asymptomatic Symptomatic Long-term follow-up (neurodevelopmental, ophthalmologic, and audiologic evaluation) Therapy IF neurologic involvement and/or abnormal sensorineural findings FIG. 2. Diagnosis of cytomegalovirus (CMV) infection in newborn. Cytomegalovirus remains the main infectious cause of congenital abnormalities in the central nervous system caused by intrauterine infection in humans, with an average incidence of approximately 1% of live births. Although the diagnosis of congenital CMV infection is still complex, major goals have been achieved in recent years including: more reliable IgM tests for screening pregnant women whose pre-pregnancy serological status for CMV is

8 1292 Clinical Microbiology and Infection, Volume 17 Number 9, September 2011 CMI At the time of diagnosis Laboratory investigations: complete blood count, platelet count, transaminase levels, bilirubin levels (direct and indirect); CMV Real Time PCR in blood and urine; Clinical and neurological examination, height, weight and head circumference; Fundus oculi; Cranial ultrasound and/or computed tomography scan of the brain and/or brain magnetic resonance imaging; Auditory brainstem responses. IF Neurologic symptoms and/or abnormal auditory and/or visual function and/or abnormal cranial ultrasound and/or abnormal computed tomography scan of the brain; and/or brain magnetic resonance imaging. Therapy Pathological signs CMV symptomatic infection Long-term follow-up No pathological signs CMV asymptomatic infection Follow-up at 3 months of life Laboratory investigations; Physical, neurological and anthropometric evaluation; Neurodevelopmental evaluation; Auditory brainstem responses; Ophthalmologic investigation. Follow-up at 6 months of life CMV Real Time PCR in blood and urine; Laboratory investigations; Physical, neurological and anthropometric evaluation; Neurodevelopmental evaluation; Auditory brainstem responses. Follow-up at 12, 24 months of life and yearly untilsixthyear CMV Real Time PCR in blood and urine (at 12 months); Physical, neurological and anthropometric evaluation; Neurodevelopmental evaluation; Ophthalmologic investigation (at 12 months); Hearing assessment. FIG. 3. Long-term follow-up in newborn with congenital cytomegalovirus (CMV) infection. unknown; tests to determine the avidity index of anti-cmv IgG, allowing the diagnosis of a primary CMV infection; and innovative virological tests to detect the virus in amniotic fluid. A fully standardized diagnostic algorithm should lessen the anxiety felt by pregnant women largely as the result of concerns about mis-management. It was recently demonstrated that a correct interpretation of serological and virological tests followed by appropriate counselling by an expert physician is an effective tool in reducing the number of unnecessary pregnancy terminations by over 70% [18]. Despite ongoing research, no vaccine is currently available for use before conception to prevent congenital CMV infection. Prevention must therefore be based on correct hygiene and behaviour. Preventive administration of immunoglobulins or antiviral drugs to pregnant women with primary CMV infection may reduce the rate of vertical transmission and improve neonatal outcome, but these findings await confirmation in randomized studies on large patient cohorts. Acknowledgements Anne Collins edited the English text. This work was supported by grants from St. Orsola-Malpighi General Hospital of Bologna (SA43GABR). Transparency Declaration The authors declare no potential conflicts of interest. References 1. Hyde TB, Schmid DS, Cannon MJ. Cytomegalovirus seroconversion rates and risk factors: implications for congenital CMV. Rev Med Virol 2010; 20: Peckham CS. Cytomegalovirus infection: congenital and neonatal disease. Scand J Infect Dis Suppl 1991; 80: Kenneson A, Cannon MJ. Review and meta-analysis of the epidemiology of congenital cytomegalovirus (CMV) infection. Rev Med Virol 2007; 17: Dahle AJ, Fowler KB, Wright JD, Boppana SB, Britt WJ, Pass RF. Longitudinal investigation of hearing disorders in children with congenital cytomegalovirus. J Am Acad Audiol 2000; 11: Bodéus M, Hubinont C, Goubau P. Increased risk of cytomegalovirus transmission in utero during late gestation. Obstet Gynecol 1999; 93: Pass RF, Fowler KB, Boppana SB, Britt WJ, Stagno S. Cytomegalovirus infection following first trimester maternal infection: symptoms at birth and outcome. J Clin Virol 2006; 35: Boppana SB, Pass RF, Britt WJ, Stagno S, Alford CA. Symptomatic congenital cytomegalovirus infection: neonatal and mortality. Pediatr Infect Dis J 1992; 11: Revello MG, Zavattoni M, Furione M, Fabbri E, Gerna G. Preconceptional primary human cytomegalovirus infection and risk of congenital infection. J Infect Dis 2006; 193: Daiminger A, Bäder U, Enders G. Pre- and periconceptional primary cytomegalovirus infection: risk of vertical transmission and congenital disease. BJOG 2005; 112: Revello MG, Zavattoni M, Furione M, Lilleri D, Gorini G, Gerna G. Diagnosis and outcome of preconceptional and periconceptional primary human cytomegalovirus infections. J Infect Dis 2002; 186: Hadar E, Yogev Y, Melamed N, Chen R, Amir J, Pardo J. Periconceptional cytomegalovirus infection: pregnancy outcome and rate of vertical transmission. Prenat Diagn 2010; 30: Guerra B, Simonazzi G, Banfi A et al. Impact of diagnostic and confirmatory tests and prenatal counseling on the rate of pregnancy termination among women with positive cytomegalovirus

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