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MAJOR ARTICLE Cytomegalovirus (CMV)–Encoded UL144 (Truncated Tumor Necrosis Factor Receptor) and Outcome of Congenital CMV Infection Ravit Arav-Boger,1,2 Catherine A. Battaglia,3 Tiziana Lazzarotto,4 Liliana Gabrielli,4 Jian C. Zong,2 Gary S. Hayward,2 Marie Diener-West,3 and Maria P. Landini4 1 Department of Pediatrics, Division of Infectious Disease, Johns Hopkins Hospital, 2Molecular Virology Laboratories, Sidney-Kimmel Cancer Research Center, Johns Hopkins University School of Medicine, and 3Department of Biostatistics, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland; 4Department of Clinical and Experimental Medicine, Clinical Unit of Microbiology, St. Orsola Malpighi General Hospital, University of Bologna, Bologna, Italy Background. Cytomegalovirus (CMV) is the most common congenital infection in humans. The effect of viral strains on the outcome of congenital CMV is debated. We evaluated whether UL144 polymorphisms in amniotic fluid from CMV-infected Italian women were associated with terminations of pregnancy, subsequent disease in their offspring, or viral load. Methods. The study was nested within a prenatal CMV program. We sequenced the UL144 gene from 66 amniotic-fluid samples, without knowledge of pregnancy outcome. We performed data analyses on 56 samples for which all information was available. Results. Genotype C was associated with termination of pregnancy (P p .03 ). Genotype B was associated with fewer terminations of pregnancy (P p .003 ). A possible association was found between genotype C and symptomatic disease in newborns (odds ratio, 8.81 [95% confidence interval, 0.48–164.02]; P p .05 ). There was no association between specific genotype and the viral load in amniotic fluid. Symptomatic newborns who had the most common UL144 genotype (B) were more likely to have higher viral loads than were asymptomatic infants (P p .003). Conclusions. UL144 polymorphisms may be associated with the outcome of congenital CMV infection. Larger studies should be conducted to confirm this association, before genotype analysis can be used, along with other factors, in considering terminations of pregnancy. Cytomegalovirus (CMV) is the most common congenital infection in humans; it affects ∼1% of newborns in the United States and 0.2%–2.2% of newborns in other countries [1, 2]. A wide spectrum of disease severity has been documented in congenitally infected infants; the majority of them are asymptomatic, but ∼10% have disseminated disease and central nervous system (CNS) Received 28 October 2005; accepted 3 March 2006; electronically published 7 July 2006. Potential conflicts of interest: none reported. Financial support: United Cerebral Palsy Research and Education Foundation; Johns Hopkins Clinician Scientist Award (to R.A.-B.); Ministry of Public Health, Istituto Superiore di Sanità, AIDS Program; Ministry of University, Scientific and Technological Research; St. Orsola Malpighi General Hospital; University of Bologna (grants to T.L., L.G., and M.P.L.). Reprints or correspondence: Dr. Ravit Arav-Boger, Dept. of Pediatrics, Div. of Infectious Diseases, Johns Hopkins Hospital/Park 256, Baltimore, MD 21287 ([email protected]). The Journal of Infectious Diseases 2006; 194:464–73 2006 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2006/19404-0008$15.00 464 • JID 2006:194 (15 August) • Arav-Boger et al. involvement [3–6]. Most of the symptomatic infants who do not die have long-term CNS sequelae, such as mental retardation, seizures, and hearing loss [6, 7]. The estimated annual societal cost of supporting children with congenital CMV sequelae approaches $2 billion (1991 US dollars) [8]. Available evidence suggests that both host and viral factors—including maternal immunity, CMV DNA load in amniotic fluid, and CMV strain— may contribute to the outcome of congenital CMV infection [1–11]. Viral loads have recently been suggested to be good predictive and prognostic markers of disease severity, but it remains unknown which genes influence viral loads [12, 13]. Genetic variability in human CMV isolates has been well established [14–16]. Whether this variability contributes to the outcome of CMV infection in general and to congenital CMV in particular is debated [16]. In the past, we have studied several genes as potential virulence factors, including UL144, UL146, UL147, US28, and glycoprotein B (gB), and we found that only polymorphisms in UL144, a cytokine receptor gene, were associated with outcome [11]. Our interest in UL144 was piqued because of its consistent presence in all clinical isolates but its frequent deletion, along with up to 18 other open reading frames, in laboratory-adapted strains [17]. Moreover, UL144 is a potential candidate for a pathogenesis marker because it is a truncated tumor necrosis factor (TNF)–like receptor [18]. Interest in this gene locus is reflected in recent reports from other parts of the world, including France, Japan, and China [19–21]. We and others [11, 14, 19, 22] have identified 3 major genotypes in UL144—A, B, and C, of which B is the most common, as well as several relatively rare recombinants of A/B and A/C. Uncommon genotypes of UL144 have been detected more frequently in newborns with severe congenital CMV disease than in asymptomatic newborns [11]. To extend our initial observations to a different geographic area, we evaluated, in a retrospective study, whether polymorphisms of UL144 in amniotic fluid from pregnant Italian women who contracted CMV during pregnancy were associated with termination of pregnancy or with the outcome of infection in their offspring. We also examined the possible relationship between UL144 genotypes and viral loads. PATIENTS AND METHODS The subjects involved in the present study were among those enrolled in a prenatal diagnosis program for CMV in Italy [10]. Samples of amniotic fluid were obtained during 1990–2003 from women who were identified as being at risk of transmitting CMV and who agreed to participate in the program. On the basis of published data, all pregnant women were counseled regarding the risk of vertical transmission, possible fetal and neonatal damage, and the availability of fetal investigations. Sixty-six women agreed to participate in the study and underwent amniocentesis. After written informed consent was obtained, 20 mL of amniotic fluid was collected by transabdominal amniocentesis under continuous ultrasound guidance at 21–23 weeks of gestation [10]. On the basis of clinical and virological results (e.g., viral loads), women decided either to continue their pregnancies to delivery or to terminate their pregnancies. CMV infection in newborns was determined by the isolation of CMV from urine during the first week of life. Infected newborns were classified as having symptomatic disease or asymptomatic infection on the basis of the presence of physical and laboratory findings. One or more of the following findings determined symptomatic disease: retardation of intrauterine growth; hepatosplenomegaly; petechiae/purpura; thrombocytopenia (!100,000 platelets/mm3); jaundice with direct bilirubin levels of 13 mg/dL; alanine aminotransferase levels of 180 U/L; pneumonia; neurologic involvement, including microcephaly, lethargy/hypotonia, poor sucking, seizures, and sensorineural defects (i.e., chorioretinitis and deafness); and abnormal neuroimaging results (periventricular hyperechogenicity, intracranial calcifications, ventriculomegaly, and hyperechogenicity of lenticolostriatal vessels). In aborted fetuses, macroscopic and histologic tissue examination was performed for multiple tissues. The shell-vial procedure [23] was used to isolate CMV from samples of amniotic fluid and urine. Inoculated cells were fixed 24–48 h after inoculation and were stained using an indirect immunofluorescence assay with a monoclonal antibody reacting with the CMV IE1 and EA genes. The methods for DNA extraction and quantitative polymerase chain reaction (PCR) have been described elsewhere [10, 24]. DNA from samples of amniotic fluid was used for the amplification of UL144. The PCR conditions and primers have been published elsewhere [11]. PCR products were gel purified and sequenced using the BigDye Terminator Cycle Sequencing Kit (Perkin-Elmer Applied Biosystems). An ABI 310 automated sequencer (Applied Biosystems) was used to analyze the sequencing products. The sequencing alignment and phylogenetic analyses used the multiple-alignment algorithms in the Megalign program package for proteins (Lasergene version 7; DNAStar). There were no identifiers on any of the samples for which PCR and sequence data were available. To assess bivariate relationships among genotype, viral load, and either symptomatic disease or termination of pregnancy, analyses were performed using x2 and Fisher’s exact tests. A test-based 95% confidence interval (CI) was constructed for the unadjusted odds ratio (OR) when cell counts were small in the cross-tabulation table. A correction of 0.5 was added to each cell count when a zero cell was present. Unadjusted and adjusted ORs for exposure and associated 95% CIs were obtained using simple and multiple logistic regressions, respectively. All exposure covariates that were significantly associated with outcome in the bivariate analyses (P ! .05 ) were included in the multivariate analysis. Analyses to assess differences in the relative frequency of genotypes over time were obtained using the x2 goodness-of-fit test. All statistical analyses were performed using Stata software (version 8.0; StataCorp). RESULTS Sixty-six samples of amniotic fluid were obtained from 66 pregnant women during 1990–2003 and were available for genotype analysis. We were able to amplify the UL144 genotype in 56 of 66 samples (GenBank accession numbers DQ275474–DQ275528). All 10 samples from which UL144 was not amplified were obtained from mothers of asymptomatic newborns. Data analysis was therefore performed on the 56 samples typed. Of the enrolled women, 25 terminated their pregnancies, 1 had an intrauterine death, and 30 had an uneventful delivery. Of the 25 women who decided to terminate CMV-Encoded UL144 in Congenital Infection • JID 2006:194 (15 August) • 465 Table 1. Detailed results of the study population. Amniotic fluid Patient UL144 genotype Symptoms, autopsy findings Late sequelae Virus isolation qPCR result, copies/mL Isolation of CMV in urine during the first week of life 11 12 A1 B Asymptomatic Asymptomatic … Bilateral hearing loss … Positive !9.4 ⫻ 102 Positive 1.0 ⫻ 103 14 15 16 17 A1 A1 A1 A2 Asymptomatic Asymptomatic Asymptomatic Petechiae, thrombocytopenia … … … Monolateral hearing loss Positive … … Positive 1.0 ⫻ 103 1.2 ⫻ 104 !9.4 ⫻ 102 1.9 ⫻ 105 Positive Positive Positive 18 19 B C/B Hepatitis ToP, disseminated infection, cerebral ventriculomegaly, hyperechogenic bowel … … Positive Positive 1.8 ⫻ 106 1.4 ⫻ 106 Positive 20 B Cerebral ventriculomegaly, diffused microcalcified cerebral areas Bilateral hearing loss, psychomotor retardation Positive 1.8 ⫻ 106 Positive 21 C ToP, disseminated infection, hyperechogenic cerebral areas … Positive 6.5 ⫻ 105 NA 22 B Diffused microcalcified cerebral areas Positive 9.0 ⫻ 105 Positive 23 B ToP, disseminated infection, DandyWalker syndrome Monolateral hearing loss, psychomotor retardation … Positive 5.5 ⫻ 105 NA 24 25 B A/C Hepatitis, high C-reactive protein Thrombocytopenia, diffused microcalcified cerebral areas … Strabismus Positive Positive 1.8 ⫻ 106 1.7 ⫻ 106 Positive 26 A/C ToP, disseminated infection, cerebral ventriculomegaly, hyperechogenic bowel, pneumonitis, hepatitis ToP, disseminated infection … Positive 4.0 ⫻ 104 NA NA NA Positive Positive NA Positive 28 C … Positive 3.1 ⫻ 105 29 30 C B ToP, disseminated infection ToP, disseminated infection, cerebral ventriculomegaly, pneumonitis … … Positive Positive 1.7 ⫻ 105 1.0 ⫻ 106 31 B … Positive 3.0 ⫻ 106 NA 32 C ToP, disseminated infection, cerebral ventriculomegaly, hepatosplenomegaly, pneumonia Diffused microcalcified cerebral areas, cerebral ventriculomegaly, hepatosplenomegaly Bilateral hearing loss, psychomotor retardation Positive 2.9 ⫻ 105 Positive 33 34 B A1 Intrauterine growth restriction ToP, disseminated infection, pneumonitis, agenesis of corpus callosum, hepatomegaly IQ !70 … Positive Positive 4.0 ⫻ 105 1.1 ⫻ 106 Positive NA 35 36 B A1 Hepatitis ToP, disseminated infection, hepatosplenomegaly, pneumonia … … Positive Positive 3.7 ⫻ 105 9.1 ⫻ 105 Positive 37 38 39 40 B C B A1 Hyperechogenic bowel Intrauterine deatha ToP, disseminated infection ToP, disseminated infection … … 3.7 ⫻ 103 Positive … … … … Positive Positive !9.4 ⫻ 102 NA NA 11.0 ⫻ 106 NA 41 42 A2 A1 Diffused microcalcified cerebral areas ToP, disseminated infection, pneumonia … … Positive Positive 1.0 ⫻ 105 1.1 ⫻ 105 Positive 43 44 A2 A1 ToP, disseminated infection, spleen, lung ToP, disseminated infection, pneumonia, diffused microcalcified cerebral areas, kidney, pancreas … … Positive Positive 11.0 ⫻ 106 NA 1.7 ⫻ 105 NA 5.2 ⫻ 105 NA NA NA (continued) Table 1. (Continued.) Amniotic fluid Patient UL144 genotype Late sequelae Virus isolation qPCR result, copies/mL Isolation of CMV in urine during the first week of life Bilateral hearing loss Positive 11.0 ⫻ 106 Positive Symptoms, autopsy findings 45 B Diffused microcalcified cerebral areas, chorioretinitis 46 B Asymptomatic … … 3.7 ⫻ 103 Positive 47 48 B B Asymptomatic Asymptomatic … … … … 5.20 ⫻ 102 !9.4 ⫻ 102 Positive Positive 49 50 B B Asymptomatic Asymptomatic … Bilateral hearing loss Positive Positive 1.8 ⫻ 106 4.9 ⫻ 104 Positive 51 52 A1 B Asymptomatic Asymptomatic … … … Positive !9.4 ⫻ 102 Positive 1.3 ⫻ 104 53 C ToP, disseminated infection, pneumonia, kidney, pancreas, liver … Positive 3.0 ⫻ 105 Positive NA 54 B ToP, disseminated infection, pneumonia, kidney … Positive 2.9 ⫻ 106 NA 55 A/B … Positive 1.2 ⫻ 106 NA 56 A1 ToP, disseminated infection, pneumonia, kidney, liver, heart, thymus ToP, disseminated infection, pneumonia, kidney … Positive 2.7 ⫻ 105 NA 57 C … Positive 1.4 ⫻ 105 NA 58 A1 ToP, disseminated infection, pneumonia, cerebral ventriculomegaly, liver, pancreas, kidney Asymptomatic … … !9.4 ⫻ 102 Positive 59 60 A1 B ToP, disseminated infection, kidney ToP, disseminated infection, hepatosplenomegaly, lung, pancreas … … Positive Positive 1.6 ⫻ 105 1.7 ⫻ 105 NA 61 62 B A2 Asymptomatic ToP, lung, liver, kidney … … Positive Positive 2.4 ⫻ 105 4.2 ⫻ 105 Positive 63 68 B A/C Asymptomatic ToP, disseminated infection, lung, pancreas, kidney … … Positive Positive 3.5 ⫻ 105 1.9 ⫻ 105 Positive 69 70 72 73 A1 A2 B B ToP, disseminated infection Asymptomatic Asymptomatic Asymptomatic … … … … Positive … Positive Positive 1.5 6.5 2.5 5.0 ⫻ ⫻ ⫻ ⫻ NA Positive 105 102 105 104 Positive NA NA NA Positive Positive NOTE. CMV, cytomegalovirus; NA, not available; qPCR, quantitative polymerase chain reaction; ToP, termination of pregnancy. a Results at autopsy were fetal hydrops, edema, and hepatosplenomegaly. their pregnancies, 10 did so on the basis of both pathologic ultrasound findings and high viral loads in amniotic fluid, 4 on the basis of both positive CMV results in fetal blood and high viral loads in amniotic fluid, 3 on the basis of borderline ultrasound findings and high viral loads in amniotic fluid, and 8 on the basis of high viral loads in amniotic fluid. Viral and host characteristics of pregnant women who agreed to participate in the study are detailed below; expanded characteristics of both pregnant women and their offspring are described in table 1. All women were white, and the age at the time of amniocentesis was recorded for 30 women with term deliveries and ranged from 20 to 41 years. Of the 56 women who underwent amniocentesis and for whom we had both viral load and genotype data, CMV was present in 45 samples of amniotic fluid (80.4%). We used the viral-load threshold cutoff in amniotic fluid of 1 ⫻ 10 5 copies/mL determined by Lazzarotto et al. [10, 24], and we found that a viral load in amniotic fluid of ⭓1 ⫻ 10 5 copies/mL was present in 39 (69.6%) of the samples. Of the samples of amniotic fluid evaluated, the following distribution of UL144 genotypes was determined: B, 25 (44.6%); A1, 14 (25.0%); C, 7 (12.5%); A2, 5 (8.9%); and A/C recombinant, 3 (5.4%), with 1 (1.8%) each of recombinants A/B and C/B. Each genotype was analyzed in comparison with all other CMV-Encoded UL144 in Congenital Infection • JID 2006:194 (15 August) • 467 Table 2. Association of UL144 genotype and viral load in amniotic fluid with outcome of cytomegalovirus (CMV) infection. Isolation of CMV Amniotic fluid qPCR in amniotic fluid result, copies/mL UL144 recombinant UL144 genotype B UL144 genotype C UL144 genotype A1 genotypes UL144 genotype A2 Association result Non–A/B, !1 ⫻ 105 ⭓1 ⫻ 105 B Non-B C Non-C A1 Non-A1 A2 Non-A2 A/B, A/C, B/C A/C, B/C 2 (94.7) 3 (7.9) 35 (92.1) 14 (36.8) 24 (63.2) 7 (18.4) 31 (81.6) 8 (21.1) 30 (79.9) 4 (10.5) 34 (89.5) 5 (13.2) 33 (86.8) 9 (50.0) 14 (77.8) 4 (22.2) 11 (61.1) 7 (38.9) 0 (0) 18 (100) 6 (33.3) 12 (66.7) 1 (5.6) 17 (94.4) 0 (0) 18 (100) Positive Negative 36 (5.3) Infection, no. (%) Symptomatic Asymptomatic Pearson’s x2 P OR (95% CI) 9 (50.0) 15.49 28.2 2.91 3.79 0.98 0.37 0.11 !.001 !.001 .09 .05 .32 .54 .11 18.00 (3.30–98.27) 40.83 (8.08–206.40) 0.371 (0.12–1.18) 8.81 (0.48–164.02) 0.533 (0.15–1.87) 2.00 (0.21–19.31) 6.07 (0.32–115.58) a a NOTE. CI, confidence interval; OR, odds ratio; qPCR, quantitative polymerase chain reaction. a Before the calculation of OR and CI, 0.5 was added to each cell count. The 2-sided Fisher’s exact test results for genotype C and for recombinant genotypes were 0.08 and 0.13, respectively. Table 3. Association of UL144 genotypes with isolation of cytomegalovirus (CMV) and quantitative polymerase chain reaction (qPCR). Result of amniotic fluid testing Isolation of CMV Positive Negative Pearson’s x2 P qPCR !1 ⫻ 105 copies/mL ⭓1 ⫻ 105 copies/mL Pearson’s x2 P C Non-C B Non-B 6 (85.7) 1 (14.3) 39 (79.6) 10 (20.4) 21 (84.0) 4 (16.0) 24 (77.4) 7 (22.6) 0.15 .70 0.15 .70 0.38 .54 0.38 .54 1 (14.3) 16 (32.6) 8 (32.0) 9 (29.0) 6 (85.7) 33 (67.4) 17 (68.0) 22 (71.0) 0.98 .32 0.98 .32 0.06 .81 0.06 .81 NOTE. Data are no. (%), unless otherwise indicated. genotypes. We considered genotype recombinants A/B, A/C, and C/B to be 1 group in comparison with the separate genotypes A1, A2, B, and C. All 30 live newborns had a positive urine culture; 12 (40%) were symptomatic, and 18 (60%) were asymptomatic. All terminations of pregnancy and the single intrauterine death were defined as symptomatic on the basis of viral loads, ultrasound findings, and macroscopic and histological evidence of multiple organ involvement. We also performed subset analysis to compare symptomatic versus asymptomatic newborns and terminations of pregnancy versus asymptomatic newborns. CMVassociated late sequelae were observed in 9 (30%) of the newborns and included monolateral and bilateral hearing loss, psychomotor retardation, strabismus, and an IQ of !70. We investigated the associations among UL144 genotypes, viral loads, and symptomatic disease (table 2). Culture positivity of and high viral load (⭓1 ⫻ 10 5 copies/mL of virus) in amniotic fluid were significantly associated with symptomatic disease (OR, 18.00 [95% CI, 3.30–98.27]; P ! .001; and OR, 40.83 [95% CI, 8.08–206.40]; P ! .001, respectively). Although genotypes A1, A2, and B occurred in both symptomatic and asymptomatic newborns, genotype C and all recombinants were found exclusively in symptomatic newborns and terminations of pregnancy. A possible association was noted for genotype C and symptomatic disease (OR, 8.81 [95% CI, 0.48–164.02]; P p .05, Pearson’s x2; P p .08, 2-sided Fisher’s exact test). When we grouped recombinant genotypes containing C with genotype C, we found a statistically significant association between these genotypes and outcome (OR, 15.47 [95% CI, 0.86–279.01]; P p .01, 2-sided Fisher’s exact test). The probability of having genotype B was 61.1% in asymptomatic newborns, compared with 36.8% in symptomatic newborns (P p .09). A statistically significant association was determined between the presence of genotype C CMV and termination of pregnancy (P p .03 ). There was increased odds of terminating pregnancy when genotype C CMV was detected in amniotic fluid (OR, 8.7 [95% CI, 1.3–59.4]) (table 3). The OR for genotype C, after adjustment for culture positivity (P p .70) and for viral loads of ⭓1 ⫻ 10 5 copies/mL in amniotic fluid (P p .32), was 12.87 (95% CI, 0.92–178.40) and 8.95 (95% CI, 0.76–105.20), respectively. We also found that the percentage of women terminating their pregnancies in the presence of non-B UL144 genotypes was 64.5%, compared with 24.0% for genotype B (P p .003) (table 4). The odds of a woman terminating her pregnancy were reduced by 83% (OR, 0.17 [95% CI, 0.05–0.56]) when genotype B CMV was detected in amniotic fluid. After adjustment for both culture positivity and ⭓1 ⫻ 10 5 copies/mL of DNA amniotic fluid, the adjusted odds that a woman infected with genotype B CMV would terminate her pregnancy, compared with women infected with non–genotype B CMV, were reduced by 90% (OR, 0.10 [95% CI, 0.02–0.42]). In subset analysis of symptomatic versus asymptomatic newborns and terminations of pregnancy versus asymptomatic newborns (table 5), findings were consistent with those of the original analysis, in which symptomatic newborns and terminations of pregnancy were grouped together under the category of “symptomatic” (table 2). Viral isolation and loads in amniotic fluid were significantly associated with outcome. There were higher odds of infection with C genotype or C and recombinant genotypes containing C in symptomatic than in asymptomatic newborns (OR, 4.83 and 8.81, respectively) and in terminations of pregnancy than in asymptomatic Table 4. Unadjusted and adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for termination of pregnancy, by genotype. Termination, no. (%) OR (95% CI) Yes No P Unadjusted Adjusteda B Non-B C 6 (24) 20 (64.5) 6 (85) 19 (76) 1 (35.5) 1 (14.3) .003 0.17 (0.05–0.56) 0.10 (0.02–0.42) .03 8.70 (0.97–77.20) 10.9 (0.74–160.40) Non-C 20 (40.8) 29 (59.2) Genotype a Adjusted for isolation of cytomegalovirus and quantitative polymerase chain reaction. CMV-Encoded UL144 in Congenital Infection • JID 2006:194 (15 August) • 469 Table 5. Subset analysis of symptomatic vs. asymptomatic liveborn infants and terminations of pregnancy vs. asymptomatic liveborn infants. Terminations vs. asymptomatic Symptomatic vs. asymptomatic Characteristic OR (95% CI) P OR (95% CI) P Viral isolation qPCR 11.00 (1.16–103.94) 38.50 (3.75–395.40) .036 .002 25.00 (2.76–226.03) 42.00 (6.80–259.44) .004 .000 Genotype B Genotype C 1.27 (0.28–5.87) 4.83 (0.18–128.80) .757 .213 0.19 (0.05–0.71) 11.73 (0.06–222.88) .014 .103 Genotype C and recombinant C genotypes 8.81 (0.38–201.40) .15 20.08 (1.08–371.67) .005 Genotype A1 0.080 (0.004–1.520) .06 0.89 (0.25–3.22) .858 Genotype A2 Recombinant genotypes 3.40 (0.27–42.44) 4.83 (0.18–128.80) .342 .40 1.42 (0.12–16.91) 7.40 (0.37–146.54) .783 .133 NOTE. For the calculation of odds ratios (ORs) and 95% confidence intervals (CIs), 0.5 was added to each cell of the 2 ⫻ 2 table. qPCR, quantitative polymerase chain reaction. newborns (OR, 11.73 and 20.08, respectively). Genotype B was associated with fewer terminations of pregnancy. A graphic display of the association between UL144 genotypes, given symptomatic and asymptomatic disease versus log viral load in amniotic fluid, is shown in figure 1. For all UL144 genotypes, symptomatic disease was associated with higher viral loads in amniotic fluid. We performed a subgroup analysis of the 25 mothers enrolled who had UL144 genotype B, to determine the relationship between symptomatic disease in newborns and the viral load in amniotic fluid. We determined that the percentage of symptomatic newborns with viral loads in amniotic fluid of ⭓1 ⫻ 10 5 copies/mL was 92.9%, compared with 36.4% of those with asymptomatic infection (P p .003). The OR of a symptomatic newborn having ⭓1 ⫻ 10 5 copies/ mL of virus in amniotic fluid was 22.8 (95% CI, 2.1–244.9) times higher than that of an asymptomatic newborn. A similar subgroup analysis could not be performed for genotype C, because there were no asymptomatic newborns with this genotype. No statistically significant associations were found between UL144 genotypes A1, A2, or recombinants A/B, A/C, and C/B and outcomes of pregnancy or virologic parameters (data not shown). To determine whether the relative frequency of genotypes changed over time, the date of amniocentesis was determined. Test dates preceding January 1998 were excluded from this analysis (n p 4). The remaining 52 test dates were then divided into 6-month intervals, beginning with 1 January 1998. Using a x2 goodness-of-fit test, we determined that the distribution of genotypes did not significantly vary over time, thereby providing no evidence that the appearance of uncommon genotypes are responsible for outbreaks of severe disease (P p .759; data not shown). 470 • JID 2006:194 (15 August) • Arav-Boger et al. DISCUSSION Our genotype analysis of UL144 in amniotic fluid from Italian women who contracted CMV during pregnancy suggests that polymorphisms, specifically genotypes B and C, may be associated with the outcome of congenital CMV disease. For the common genotype B, viral load predicted outcome, but infection with one of the uncommon UL144 genotypes was associated with severe disease, irrespective of viral load. In a recent US cohort of newborns with congenital CMV infection, we found and reported that UL144 polymorphism was associated with outcome. Among 23 newborns, 13 of whom were symptomatic and 10 of whom were asymptomatic, genotype B was found in both groups, whereas genotypes C, A1, and A/C were found exclusively in symptomatic newborns [11]. Of the 5 genes that were sequenced (UL144, UL146, UL147, US28, and gB), only polymorphisms in the UL144 gene appeared to be associated with the outcome of congenital CMV infection during the immediate neonatal period. We hypothesized that human CMV genotypes that have not recently coevolved with the host population are less well tolerated and are therefore associated with a worse outcome. We were, therefore, interested in studying UL144 polymorphisms during pregnancy in a different geographic region, to determine whether they could predict the outcome of congenital CMV infection in newborns. The distribution of genotypes in the Italian population that we report shows, again, that genotype B is the most common genotype in the population (73.9% in the US cohort and 44.6% in the Italian cohort). The distribution of the uncommon genotypes shows some differences (table 6). Among 56 samples in the Italian cohort, 38 were from symptomatic newborns (25 terminations, 1 intrauterine death, and 12 newborns), and 18 Figure 1. Box plots comparing log10 cytomegalovirus load in amniotic fluid (in copies per milliliter) for symptomatic and asymptomatic newborns, by UL144 genotype. For each genotype, symptomatic newborns are shown as black bars on the left, and asymptomatic newborns (when present) are shown as bars shaded on the right. Genotypes for symptomatic newborns were genotype B (n p 14 ), genotype C (n p 7), genotype A1 (n p 8), genotype A2 (n p 4), and recombinant genotypes (n p 5 ). Genotypes for asymptomatic newborns were genotype B (n p 11), genotype C (n p 0), genotype A1 (n p 6), genotype A2 (n p 1 ), and recombinant genotypes (n p 0). were from asymptomatic newborns. Genotypes B, A1, and A2 were found in both symptomatic and asymptomatic newborns, whereas genotypes C and the recombinants A/B, A/C, and C/ B were found exclusively in symptomatic newborns. We note that the newborns in our US study cohort were African American; therefore, the distribution of polymorphisms may have been related to genetic susceptibility between the 2 different populations. Our results show that the odds of having genotype B CMV in the amniotic fluid of asymptomatic newborns was ∼20% higher than that of symptomatic newborns (P p .09 ). For genotype B CMV, a severe outcome was associated with higher viral loads in amniotic fluid, specifically those ⭓1 ⫻ 10 5 copies/ mL (P p .003). We note that there was no association between genotype B and symptomatic disease; therefore, the high viral load could be secondary to the presence of symptomatic disease and may not be related to UL144 genotype. For genotypes other than B, viral loads in amniotic fluid were not predictive of outcome, although our analyses were limited by the small sample sizes of these groups Similar to genotype B, genotypes A1 and A2 appeared in both symptomatic and asymptomatic newborns, but no significant associations were found between outcome and virologic parameters. Genotypes C and all recombinants (A/B, A/C, and C/B) were exclusively found to be associated with severe disease, in either live newborns or terminations of pregnancy. Although the recombinant genotypes were not statistically associated with symptomatic severe disease, a possible association was found for genotype C and symptomatic CMV disease (OR, 8.81 [95% CI, 0.48–164.02]; P p .05, Pearson’s x2; P p .08, 2-sided Fisher’s exact test). We found that genotypes B and C had opposite effects on termination of pregnancy, both of which were statistically significant. Although genotype C was significantly associated with termination of pregnancy (P p .03), fewer terminations were noted for genotype B (P p .003). The odds of termination of pregnancy increased in the presence of non-B and non-C genotypes (OR, 5.76 [95% CI, 1.78–18.67] and OR, 8.7 [95% CI, 0.97–77.92], respectively). When we analyzed the role of possible confounding due to virologic parameters, these associations remained statistically significant. After adjustments for viral load, both ORs moved farther away from the null value, which suggests that the relationship between genotype and viral load may be underestimated without adjustment. There was no association between a specific genotype and the ability to culture CMV from either amniotic fluid or urine, which suggests that they all were able to grow well in cell culture. Therefore, we agree with other researchers who reported that all genotypes can be transmitted from mothers to newborns [19]. The results of our study differ, with regard to UL144 genotypes and their association with symptomatic disease, from those of a report by Picone et al. [19], in which a distribution of UL144 genotypes in a cohort of CMV-infected newborns Table 6. Distribution of UL144 genotypes in the US and Italian cohorts. Cohort, UL144 genotype (%) Symptomatic newborns Asymptomatic newborns US (n p 23) B (73.9) A1 (4.3) 7 1 10 0 A2 (0.0) C (17.4) 0 4 0 0 A/C (4.3) 1 0 A/B (0.0) B/C (0.0) Total (%) 0 0 13 (56.5) 0 0 10 (43.5) B (44.6) 14 11 A1 (25.0) A2 (8.9) C (12.5) 8 4 7 6 1 0 A/C (5.4) A/B (1.8) 3 1 0 0 Italian (n p 56) B/C (1.8) Total (%) 1 38 (67.9) 0 18 (32.1) CMV-Encoded UL144 in Congenital Infection • JID 2006:194 (15 August) • 471 and immunocompromised adults (organ or bone-marrow transplant recipients) is described. Although we agree that all UL144 genotypes are transmitted to the fetus, Picone et al. defined 2 groups in their study—severely and nonseverely symptomatic fetuses (defined by the presence of, at most, only 1 extracerebral ultrasound feature)—on the basis of ultrasound findings determined during second- and third-trimester evaluations. There were 23 terminations of pregnancy in that study, all of which were based on abnormal ultrasound results, but histopathologic data were not presented, and viral loads were not determined. Of 11 newborns delivered at term, 9 were asymptomatic and 2 were symptomatic, yet all were grouped as severely symptomatic. Picone et al. [19] concluded that all UL144 genotypes are found in both symptomatic and asymptomatic infections. However, a better classification of outcome would help in the analysis of the results of that study. In the present article, we report a possible association between genotype C and symptomatic disease in newborns (P p .05 , Pearson’s x2) and a significant association between genotype C and termination of pregnancy (P p .03). Additionally, genotype B was associated with fewer terminations of pregnancy (P p .003 ). The limitations of the present study are the small sample size of the nested cohort, which led to wide CIs (i.e., imprecise estimates of ORs). In addition, we acknowledge that, because of the uncommon presence of genotype C and recombinants in the amniotic fluid, we were limited in our interpretations of the association between these genotypes and symptomatic CMV disease. We also recognize potential enrollment bias. Of the pregnant women approached for enrollment into the study, ∼50% refused amniocentesis. It is unclear whether these refusals were based on severity of disease, socioeconomic status, and/or religious considerations. Given the documented risks to the fetus of amniocentesis, is it possible that evidence of more severe disease leads to a pregnant woman’s decision to consent to amniocentesis. The numbers of enrolled women in our Italian cohort support this theory: 38 (67.9%) of the neonates were classified as being symptomatic, compared with only 18 (32.1%) who were classified as being asymptomatic. In addition, we were unable to amplify the specific UL144 genotype in 10 samples of amniotic fluid. The outcome of these 10 pregnancies was asymptomatic infection. It is likely that we were unable to amplify UL144 from these samples because of the low number of DNA copies detected and the use of a less-sensitive PCR sequencing technique. In conclusion, the present results indicate that UL144 genotypes C and B seem to have opposite effects on the outcome of congenital CMV disease. We believe that these findings need to be replicated and confirmed before they can begin to affect clinical practice. Although there may be difficulties in obtaining 472 • JID 2006:194 (15 August) • Arav-Boger et al. a larger cohort of samples, future studies should be conducted to investigate further this controversial relationship between UL144 genotypes and congenital, symptomatic CMV disease. A recent elegant study showed that UL144 binds to the B and T lymphocyte attenuator (BTLA) and may inhibit T cell proliferation [25]. 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