Cytomegalovirus DNA detection in Guthrie cards: a powerful tool for diagnosing congenital infection.

BACKGROUND: A simple and reliable diagnosis of congenital cytomegalovirus infection is necessary both for clinical and epidemiological purposes. This could be accomplished through the demonstration of cytomegalovirus (CMV) DNA in blood spots (DBS) on Guthrie cards. OBJECTIVES: (1) To assess the sensitivity and specificity of the method (DBS test) in diagnosing congenital CMV infection compared with viral isolation and (2) to evaluate the applications of the test to the late diagnosis of congenital CMV. STUDY DESIGN: The method was tested on the cards of (1) 509 babies examined through viral isolation within their third week of life (72 positive cases) and (2) 191 children studied after 3 weeks of life (25 days to 5 years). Blood was eluted from Guthrie cards and heat extracted. The products of a nested polymerase chain reaction (PCR) amplifying one region in the CMV glycoprotein B (gB) gene were detected by agarose gel electrophoresis. RESULTS: DBS test was positive in all 72 congenitally infected babies and in four of the 437 negative at cytomegalovirus isolation (sensitivity 100%, specificity 99%). Infection in 16 of the 92 infants with a late viral isolation was demonstrated to be congenital by the test, which also detected congenital infection in 18 of 83 children in whom viral culture was not performed (13 with and five without symptoms). Fifty-six additional control cases tested negative. CONCLUSIONS: DBS test is a reliable assay for diagnosing congenital cytomegalovirus infection and could be used as an alternative to viral culture. It is able to reveal whether ascertained CMV infection is congenital or postnatal at an age when viral isolation is not able to do so. It can assess the role of risky procedures such as transfusion and it can ascertain the etiology of morbid conditions diagnosed late or of controversial origin.     

Neonatal screening for congenital cytomegalovirus infection and hearing loss.

BACKGROUND: Congenital cytomegalovirus infection causes 20-30% of congenital sensorineural hearing loss (SNHL) cases. Early identification of CMV attributable cases and their successful treatment are often hampered by the late appearance of the damage in a high proportion of children both symptomatic and asymptomatic at birth. OBJECTIVE: To discuss the feasibility of a screening program aimed at finding congenitally infected babies followed by their audiological monitoring. STUDY DESIGN: Opinion-review article. RESULTS AND CONCLUSIONS: Frequency and severity of hearing loss due to congenital CMV suggest it maybe worthwhile setting up neonatal screening campaigns. Structures where SNHL can be diagnosed and treated exist already in many countries as part of universal neonatal audiological screening schemes. A test based on viral DNA extraction from neonatal dried blood spots on Guthrie cards and its amplification by means of a nested PCR (DBS test) seems to offer the best characteristics for use in screening. Clearly it will be necessary to calculate whether the costs of screening, diagnosis and follow-up, and the financial and emotional burden on the families of infected children, are up to the potential gain.     

CMV gB genotypes and outcome of vertical transmission: study on dried blood spots of congenitally infected babies.

BACKGROUND: The role of the virulence of the infecting cytomegalovirus (CMV) strain in the transmission of the virus from mother to fetus and the outcome of the fetal infection has not received much attention yet. Molecular analysis of the gene coding for the surface glycoprotein B (gB) has been used to investigate the relationship between genotype and virulence in groups of immunosuppressed patients. OBJECTIVES: (1) to assess the prevalence of different gB genotypes in babies with congenital CMV infection; (2) to investigate the possible relationship between genotype and severity of congenital CMV disease; (3) to evaluate the possibility of using dried blood on Guthrie cards (DBS) for genotyping. STUDY DESIGN: CMV DNA was extracted from DBS and from urine/saliva samples collected in the first two weeks of life of 98 congenitally infected babies, half of which were symptomatic at birth. Genotyping was performed through RFLP analysis of the region corresponding to the cleavage site of the gB protein. RESULTS: The most prevalent genotype was gB1 (42%) followed by gB3 (26%), gB2 (19%) and gB4 (13%). Rates of disease and CNS damages were higher among children infected by gB1 (35%, 17%) and gB3 (31%, 28%) than in those infected by gB2 and gB4 (20%, 17% and 13%, 15%, respectively). These differences however did not reach the statistical significance. The parallel typing of DBS and urine/saliva strains gave a full concordance of results. CONCLUSIONS: All four major CMV gB genotypes (gB1-4) can cause a congenital infection but none seems to be associated to the development and the severity of disease. The possibility of using the neonatal DBS for genotyping opens a way to the examination of large numbers of cases of congenital CMV infection.     

Evaluation of DNA extraction methods for dried blood spots in the diagnosis of congenital cytomegalovirus infection

BackgroundDried blood spots (DBS) may be valuable in the diagnosis of congenital cytomegalovirus (CMV) infection. However, the 2007 European Quality Control for Molecular Diagnostics (QCMD) proficiency testing programme showed that CMV DNA detection in DBS was lacking sensitivity in a considerable number of participating laboratories.ObjectiveTo compare DNA extraction methods for DBS for detecting CMV. Sensitivity and applicability of the methods for high-throughput usage were assessed.Study designGuthrie cards were spotted with CMV DNA-positive whole blood (n = 15). DNA was extracted from the DBS using different extraction methods, followed by CMV amplification by means of real-time PCR.ResultsSignificant differences between the extraction methods with respect to the sensitivity were found. Optimal sensitivity was achieved when samples were tested in triplicate, demonstrating that the methods in general operated around their detection limits. Triplicate testing using the protocol by Barbi et al. [Barbi M, et al. Cytomegalovirus DNA detection in Guthrie cards: a powerful tool for diagnosing congenital infection. J Clin Virol 2000;17:159–65], representing the most sensitive methods, resulted in sensitivities of 100%, 86%, and 50% for DBS with CMV DNA loads of 5–4, 4–3, and 3–2 log₁₀ copies/ml, respectively. This indicates that sensitivity limitations apply in the clinically relevant concentration range. Few methods appeared suitable for 96-well format high-throughput testing.DiscussionWhen considering universal neonatal screening for congenital CMV infection, an assay which is both sensitive and applicable for high-throughput testing is required. The protocol by Barbi et al. and the BioRobot Universal System appear appropriate candidates currently available for 96-well format application in neonatal screening using DBS.     

Evaluation of different cytomegalovirus (CMV) DNA PCR protocols for analysis of dried blood spots from consecutive cases of neonates with congenital CMV infections

Two protocols for the extraction of cytomegalovirus (CMV) DNA and two methods for the amplification of CMV DNA in dried blood spots were evaluated for the retrospective diagnosis of congenital CMV infection. During the period from 1996 to 2006, a urine screening program detected 76 congenitally infected neonates. Stored Guthrie cards with blood from 55 cases and 12 controls were tested. Two spots of dried blood were cut from each card and evaluated in two centers. CMV DNA was extracted from a whole single spot. Center 1 used phenol-chloroform extraction and ethanol precipitation followed by a conventional PCR. Center 2 used the NucliSens easyMAG automated DNA/RNA extraction platform (bioMérieux) followed by a real-time PCR. For evaluation of the extraction method, DNA extracted from each blood spot was evaluated by the amplification method used by the collaborating center. The sensitivities were 66% for center 1 and 73% for center 2. None of the controls were positive. A sensitivity as high as 82% could be obtained by combining the most sensitive extraction method (the phenol-chloroform procedure) with the most sensitive PCR method (real-time PCR). The detection rate was not influenced by the duration of storage of the spots. The sensitivity was higher with blood from congenitally infected cases due to a primary maternal CMV infection, regardless of the protocol used. However, the difference reached significance only for the least-sensitive protocol (P = 0.036).     

Congenital cytomegalovirus infection in the Netherlands: birth prevalence and risk factors

Congenital cytomegalovirus (CMV) infection is the most common congenital viral infection worldwide. The sequela encountered most frequently is hearing impairment, affecting approximately one out of five infants congenitally infected. Data on the birth prevalence and risk factors of congenital CMV infection in the Netherlands are scarce. The aim of this study was to determine the birth prevalence of congenital CMV in the Netherlands. A sample of 6,500 dried blood spots (DBS) from infants born in the Netherlands was tested anonymously for CMV DNA. The sample was stratified by the number of live births in different regions of the Netherlands of the year 2007. Additionally, on a regional level, risk factors for congenital CMV were analyzed. The birth prevalence of congenital CMV in the Netherlands was 0.54% (35/6,433, 95%CI 0.36-0.72). Congenital CMV infection was significantly higher in regions with more than 15% young children (0-5 years) compared with regions with a lower proportion of young children (OR 5.9, 95%CI 1.4-25.2). Congenital CMV infection was significantly higher in regions with more than 30% immigrants compared with regions with a lower proportion of immigrants (OR 2.2, 95%CI 1.1-4.6). This association was strongest for regions with more than 30% non-Western immigrants (OR 3.3, 95%CI 1.5-7.5). Based on the knowledge of the natural history of congenital CMV infection, approximately 1,000 children are born with congenital CMV infection in the Netherlands annually, of whom eventually approximately 180 children (0.1% of all newborns) will be affected by long term sequelae, with hearing loss being the symptom encountered most frequently.     

Modification of CMV DNA detection from dried blood spots for diagnosing congenital CMV infection.

BACKGROUND: Detection of viral DNA in dried blood spots using the Guthrie card (DBS test) is a reliable and practical method of diagnosing congenital cytomegalovirus (CMV) infection. The test lends itself to epidemiological studies to establish the prevalence of the infection, but also to neonatal screening for secondary prevention of sequelae. These applications would be facilitated if it were possible to use smaller samples and do the test on pools of individual cases. OBJECTIVE: To ascertain whether doing the test on smaller, pooled samples still accurately identifies neonates with congenital CMV infection. STUDY DESIGN: We tested DBS from: (A) 39 laboratory reference cases; (B) 156 neonates suspected of having congenital CMV infection; (C) 119 children examined for the retrospective diagnosis of congenital CMV; (D) mock specimens prepared with known amounts of viral DNA. RESULTS: The test using only one third of the usual amount of dried blood was 100% sensitive and specific compared to the standard DBS test (A) and to viral isolation (A and B). Pools of three single cases gave the same results as viral isolation (B) and the small-sample test (B and C). All the versions of the test gave a detection limit of 400 copies/ml. CONCLUSIONS: The modified procedure can accurately diagnose congenital CMV infection. It achieves savings in both the patient material and the costs of testing.     

Prevalence of congenital cytomegalovirus infection in Slovenia: a study on 2,841 newborns

Human cytomegalovirus (CMV) is the most frequent cause of congenital infection in humans. In the first prevalence study of congenital CMV infection in Eastern and Central Europe, all neonates born in a 22‐month period in two Slovenian maternity units (total of 2,841 newborns) were screened prospectively for congenital CMV infection by a real‐time polymerase chain reaction (PCR) in urine. In all newborns with positive screening results, plasma and dried blood spots (DBS) collected at birth were tested additionally for CMV DNA. Congenital CMV infection was confirmed by virus isolation from a urine sample collected within the first 2 weeks of life. Congenital CMV infection was identified in four out of 2,841 newborns tested (incidence 0.14%; 95% CI, 0.05–0.39%). In four newborns with confirmed congenital infection, the concentration of CMV DNA in urine ranged from 4.68 to 8.18 log₁₀ copies/ml, all four newborns had detectable CMV DNA in plasma taken at birth (1.26–3.34 log₁₀ copies/ml) and two out of four had detectable CMV DNA in DBS collected during newborn metabolic screening. None of the four newborns with confirmed congenital CMV infection was symptomatic. The study showed that the prevalence of congenital CMV infection at birth in Slovenia is among the lowest in the world and that CMV DNA PCR testing of urine is a suitable and affordable real‐time screening strategy for congenital CMV infection. If it is performed in 24 mini‐pools, the cost of screening is 1.4 €/newborn and the cost of detecting a single newborn with congenital CMV infection 1,000 €. J. Med. Virol. 84:109–115, 2011. © 2011 Wiley Periodicals, Inc.     

Rapid genotyping of cytomegalovirus in dried blood spots by multiplex real-time PCR assays targeting the envelope glycoprotein gB and gH genes

Genotyping of cytomegalovirus (CMV) is useful to examine potential differences in the pathogenicity of strains and to demonstrate coinfection with multiple strains involved in CMV disease in adults and congenitally infected newborns. Studies on genotyping of CMV in dried blood spots (DBS) are rare and have been hampered by the small amount of dried blood available. In this study, two multiplex real-time PCR assays for rapid gB and gH genotyping of CMV in DBS were developed. Validation of the assays with 39 CMV-positive plasma samples of transplant recipients and 21 urine specimens of congenitally infected newborns was successful in genotyping 100% of the samples, with gB1 and gB3 being the most prevalent genotypes. Multiple gB and gH genotypes were detected in 36% and 33% of the plasma samples, respectively. One urine sample from a newborn with symptomatic congenital CMV was positive for gB1 and gB2. DBS of congenitally infected newborns (n = 41) were tested using 9 μl of dried blood, and genotypes were detected in 81% (gB) and 73% (gH) of the samples, with gB3 being the most prevalent genotype. No clear association of specific genotypes with clinical outcome was observed. In conclusion, the CMV gB and gH PCR assays were found to be rapid, sensitive for detecting mixed infections, and suitable for direct usage on DBS. These assays are efficient tools for genotyping of CMV in DBS of congenitally infected newborns.     

Influence of different extraction methods and PCR techniques on the sensitivity of HCMV-DNA detection in dried blood spot (DBS) filter cards

BackgroundInfection with human cytomegalovirus (HCMV) is the most common congenital virus infection, affecting about 0.5–2% of newborns. Using DBS on Guthrie cards, it is possible to discriminate congenital from postnatal HCMV-infection. However, a recent European trial revealed serious problems in detection of low HCMV-DNA levels from DBS-filter-cards (Barbi et al., 2008).⁷ObjectivesEvaluation of the most sensitive combination of sample size, DNA extraction method and PCR system for the detection of low copy numbers of HCMV-DNA from DBS-filter-cards.Study designWe compared three different manual extraction methods for the detection of HCMV-DNA out of DBS: the QIAmp-blood-Mini-Kit, a heat-extraction-method and traditional phenol–chloroform extraction. Additionally, we tested an automated nucleic acid extraction system (NucliSense EasyMag/Biomerieux). Different punch-sizes of DBS spiked with defined HCMV AD169-DNA copy numbers were analyzed. For detection, we used a quantitative in-house-LightCycler-PCR targeting the gB-region using the hybridisation-probe-format. We compared the sensitivity of the real-time-PCR with IE1Ex4-targeted nested-PCR.ResultsThe highest sensitivity with 200 copies HCMV DNA/ml was achieved using the phenol–chloroform method in combination with the nested-PCR and 6 mm, 3 × 3 mm punches or the whole DBS. The QIAmp-blood-Mini-Kit also showed a very high sensitivity by using the whole DBS and the nested-PCR.ConclusionThese results may have strong implications for retrospective diagnosis of congenital HCMV (cHCMV) infection, since a defined combination of the area of punch, the extraction method, and PCR method determine the probability of detection of viral DNA from DBS according to a logistic model.     

Use of stored dried blood spots for retrospective diagnosis of congenital CMV

The diagnosis of congenital cytomegalovirus infection cannot be made with certainty in children presenting after the perinatal period, unless stored early samples are available for diagnostic testing. This has led to uncertainty in confirming the overall contribution of CMV to hearing loss and neurodevelopmental impairment. The use of dried blood spots (DBSs) to retrospectively diagnose infection in children with compatible symptoms may be helpful diagnostically although there are ongoing uncertainties regarding the stability of viral DNA in cards, the risk of contamination between cards, and sensitivity and specificity in a clinical setting. This report aims to address these areas and evaluate the use of DBS testing in our hands in the United Kingdom to date. Results from testing artificially prepared cards and cards from three populations of children suggest a high specificity for congenital CMV infection and a good sensitivity for cases where sensorineural hearing loss is caused by congenital CMV. J. Med. Virol. 81:1394–1398, 2009. © 2009 Wiley‐Liss, Inc.     

Diagnosis of congenital CMV infection via dried blood spots

Cytomegalovirus (CMV) infection is the most frequent congenital infection in humans and can cause permanent damage--particularly neurological--in about 20% of those infected, with or without symptoms at birth. Laboratory diagnosis is essential on account of the relatively non-specific clinical manifestations in symptomatic newborns but also because of the high frequency of asymptomatic cases that are nevertheless at risk of lesions later in life. However, these tests need samples taken within 3 weeks of birth to distinguish congenital infection from the more common, but clinically benign, perinatal infection.Tests for viral DNA have proved a valid means of diagnosing congenital CMV infection in neonatal blood dried on paper (DBS) widely used in screening for metabolic and genetic diseases, as an alternative to the conventional urine culture method. The DBS test is simpler, faster and less costly than viral isolation; in addition the samples can be safely stored for long periods, so diagnosis can be made even after several years. The sensitivity and specificity of the DBS test, compared to the reference method, have been reported to range between 71 and 100% and 99 and 100%, respectively, depending on the different studies and diagnostic criteria applied.The most interesting applications reported so far involve retrospective determination of the impact of congenital CMV in sensorineural deafness, abnormalities of cortical development, neonatal cholestasis and surveys of the prevalence of this infection in various populations. The test might be useful in the future for neonatal screening with a view to treating neonates and so avoiding the damage this disease can cause.     

Congenital cytomegalovirus infection

Cytomegalovirus (CMV) is the most common congenital infection in humans. Congenital CMV infection can follow either a primary or recurrent maternal infection, but the likelihood of fetal infection and the risk of associated damage is higher after a primary infection. Approximately 90% of congenitally infected infants are asymptomatic at birth. Jaundice, petechiae, and hepatosplenomegaly are the most frequently noted clinical triad in symptomatic infants. More frequent and more severe sequelae occur in symptomatic infants, notably psychomotor hearing loss and retardation. Congenital CMV infection can be diagnosed by isolation of the virus from the urine or saliva within the first three weeks of life. Rapid diagnosis can be accomplished by detection of CMV DNA by DNA amplification or hybridization techniques.     

Implementing neonatal screening for congenital cytomegalovirus: addressing the deafness of policy makers

Congenital cytomegalovirus (CMV) infection is an important public health problem with approximately 7 in 1,000 newborns infected and consequently at risk for hearing impairment. Newborn hearing screening will fail to detect this hearing impairment in approximately half of the cases because late onset hearing loss is frequent. Hearing impairment has profound impact on cognitive and social development of children and their families, determining most of the disease burden of congenital CMV infection. The potential value of newborn screening for congenital CMV is increasingly discussed. To date, many experts acknowledge the benefit of antiviral treatment in the prevention of hearing deterioration in newborns with neurological symptoms, and the benefit of early identification of late-onset hearing impairment by means of extensive audiological follow up of infected infants. These opinions imply that the potential of newborn screening for CMV would lie in the identification of the large proportion of asymptomatic congenitally infected newborns at risk for developing late-onset hearing loss. Experience with postnatal antiviral treatment of symptomatic newborns is encouraging, but has not been studied in asymptomatic congenitally infected newborns. A large-scale study on the safety and effectiveness of combined screening and antiviral therapy for congenital CMV infection is the necessary next step to take and should not be delayed.     

Detection of herpes simplex virus DNA in dried blood spots making a retrospective diagnosis possible.

BACKGROUND: Herpes simplex virus (HSV) infections in neonates are associated with life-threatening disease. Early diagnosis and treatment with antiviral therapy has decreased the morbidity, mortality and long-term sequelae in surviving children. The aim of the study was to investigate if herpes simplex virus DNA detection in dried blood spots on filter papers (Guthrie cards) sampled for screening of metabolic diseases may contribute to early diagnosis of neonatal HSV infection and enable pre-emptive therapy. METHODS: For detection of HSV-1 and -2 DNA, two different DNA extraction methods were evaluated. A minimal essential medium (MEM) extraction method was found superior and was used in combination with detection of HSV-1 and -2 DNA by PCR in dried blood spots from children with verified neonatal HSV infection. Cards from 28 children were included. The onset of illness varied from day 0 to 42 days and was the result of different types of maternal infection (27 cases) and an external source (one case). RESULTS: HSV DNA was detected in seven of the 28 Guthrie cards, two were HSV-1 and five were HSV-2 DNA positive. Positive dried blood spot cards were sampled within the interval 5 days before, to 6 days after onset of neonatal herpes. In cases of late onset CNS disease, viremia, was not demonstrable at the age of 3-5 days, the time period when the blood spot cards are normally sampled. CONCLUSION: Viremia, the prerequisite for demonstrating HSV DNA in dried blood spot cards preceded the onset of illness by up to 5 days and lasted at least up to 6 days thereafter. Analysis of HSV DNA in dried blood spot cards may be of value in the diagnostic arsenal for early onset of neonatal herpes and also have a role in the follow up of a child exposed at delivery. As the majority of the later onset neonatal herpes encephalitis cases are missed, a large-scale neonatal screening does not seem appropriate.     

Diagnostic and prognostic value of human cytomegalovirus load and IgM antibody in blood of congenitally infected newborns.

BACKGROUND: Diagnosis of congenital human cytomegalovirus (HCMV) infection relies on virus isolation from urine collected in the first 3 weeks of life. However, very little is known about the presence, levels and duration of HCMV pp65 antigenemia, viremia and DNAemia in congenitally infected newborns. OBJECTIVES: To investigate the diagnostic and prognostic value of HCMV load determination in blood of newborns/infants with congenital HCMV infection. STUDY DESIGN: HCMV pp65 antigenemia, viremia and DNAemia were investigated in 116 sequential peripheral blood leukocytes (PBL) samples from 41 newborns/infants with congenital HCMV infection and in 34 PBL samples from 34 uninfected newborn. Virus-specific IgM were determined in parallel on 145 sequential serum samples. RESULTS: Compared to virus isolation from urine, sensitivities of DNAemia, antigenemia, viremia, and IgM determination were 100, 42.5, 28.2, and 70.7%, respectively. Specificity was 100% for all assays. Antigenemia, viremia and DNAemia levels were significantly higher and persisted longer in newborns with symptomatic infection compared to subclinically infected babies, whereas no difference was observed for virus-specific IgM antibody between the two groups. CONCLUSIONS: (i) determination of viral DNA in blood at birth appears to be a sensitive and specific marker for diagnosis of congenital HCMV infection; (ii) significantly higher levels of HCMV load were detected in infants with symptomatic HCMV infection; and (iii) virus clearance from blood occurs spontaneously both in symptomatic and subclinically infected infants. However, the process takes longer in infants presenting with symptoms at birth.     

Cytomegalovirus DNA detection in dried blood spots and perilymphatic fluids from pediatric and adult cochlear implant recipients with prelingual deafness

BackgroundCongenital cytomegalovirus (CMV) infection is the leading cause of non-genetic congenital hearing loss. The contribution of congenital CMV to prelingual deafness and the pathophysiology is largely unknown.Objective(1) To analyze the prevalence of congenital CMV among cochlear implant (CI) recipients with prelingual deafness. (2) To genotype CMV present in dried blood spots (DBS) and in the inner ear years after birth.Study designChildren and adults with prelingual deafness who received a CI in 2010–2011 were included prospectively. Perilymphatic fluids were collected during CI surgery and, in the pediatric cases, DBS were retrieved for CMV DNA detection. Furthermore, a cohort of children with prelingual deafness who received a CI between 2003 and 2008 were included retrospectively. CMV detection in DBS and perilymph was followed by gB and gH genotyping.ResultsSeventysix pediatric CI recipients were included. Seventy DBS were tested for CMV DNA, resulting in a prevalence of congenital CMV of 14% (10/70). Perilymphatic fluid was available from 29 pediatric CI recipients. One perilymph fluid, of a 21-month old girl with congenital CMV, asymptomatic at birth, was CMV DNA positive. The CMV strain in the perilymph was genotypically identical to the strain present in her DBS (gB1/gH2). Perilymph samples from 21 adult CI recipients were CMV DNA negative.ConclusionsOur study stresses the important contribution of congenital CMV among pediatric CI recipients. Furthermore, our genotyping data support the hypothesis that CMV-related hearing loss is associated with ongoing viral replication in the inner ear up to years after birth.     

DECIBEL study: Congenital cytomegalovirus infection in young children with permanent bilateral hearing impairment in the Netherlands

BackgroundA significant number of asymptomatic newborns infected with congenital cytomegalovirus (CMV) will present with permanent childhood hearing impairment (PCHI) during early childhood.ObjectivesTo investigate the role of congenital CMV infection in causing PCHI in the Netherlands, and assess the efficacy of two different hearing screening strategies and the developmental outcome following each strategy.Study designWe included 192 children with PCHI at the age of 3–5 years, who were offered hearing screening in their first year of life. Dried blood spots from 171 children were available for CMV detection using real-time PCR. The results of eight previously tested samples were also available. Clinical baseline characteristics were collected from medical records and the Child Development Inventory was used to investigate the developmental outcome.ResultsThe rate of congenital CMV among the 179 children was 8% (14/179) and 23% (9/39) among children with profound PCHI. Two of eight CMV-positive children with PCHI at the age of 3–5 years had passed the newborn hearing screening (NHS) test. Developmental outcome measures showed a significantly greater delay in language comprehension in children with both PCHI and congenital CMV infection (the largest in symptomatic children) than in the children with PCHI without congenital CMV infection.ConclusionsCongenital CMV infection is important in the etiology of PCHI. Universal NHS is not a guarantee of normal hearing and development in childhood for children with congenital CMV infection. This is a problem which might be solved by universal congenital CMV screening.     

Evaluation of a New Protocol for Retrospective Diagnosis of Congenital Toxoplasmosis by Use of Guthrie Cards

The aim of this study was to assess the diagnostic value of IgM Western blotting (WB), IgA enzyme immunoassay (EIA), and DNA amplification by real-time PCR on Guthrie cards to retrospectively establish the diagnosis of congenital toxoplasmosis (CT). To this purpose, Guthrie cards were collected from 18 infants born to mothers with primary Toxoplasma gondii infection during pregnancy. Moreover, the analytical sensitivity of T. gondii PCR was assessed by testing mock dried blood specimens set up with several known DNA dilutions. IgM WB was demonstrated to be the most sensitive method. When the results of T. gondii DNA detection and specific IgM recovery were combined, retrospective CT diagnosis by using Guthrie cards was established in 3 out of 6 infected infants (sensitivity, 50%; 95% confidence interval, 26.8% to 73.2%). No positive PCR or serologic results were found in the group of 12 uninfected infants, demonstrating the excellent specificity of the three methods (95% confidence interval, 78.1% to 99.5%). The findings of the present study suggest that, in cases of missed diagnosis of CT at birth, analysis of Guthrie cards for children with compatible clinical findings after the perinatal period, in particular the combination of recovery of specific IgM antibodies and T. gondii DNA amplification, could be helpful. Nevertheless, since suboptimal conditions of storage of dried blood specimens can seriously affect sensitivity, negative results cannot rule out CT diagnosis. In contrast, because of the excellent specificity shown by IgM serologic testing and T. gondii DNA amplification on Guthrie cards, positive results obtained by either of the two methods should be considered diagnostic.     

Is the mixture of human cytomegalovirus genotypes frequent in infants with congenital infection at birth in a high seroprevalence population?

It is still not well known, in a population with high human cytomegalovirus (HCMV) seroprevalence, whether a child with congenital infection harbors multiple viral strains at birth, and whether the prolonged viral excretion in these children is secondary to the persistence of the same viral strain. To verify the genomic diversity of HCMV detected in congenitally infected children, the nucleotide viral sequences from urine and/or saliva obtained at birth from 14 newborns with congenital infection and breast milk obtained from mothers of 5 of these children were analyzed. Among the 14 children, 10 had sequential samples until the median age of 10 months. The viral nucleotide sequences in the breast milk were compared with those identified in the respective children at birth. The differentiation of viral strains was based on the variability of 3 regions of viral genes (UL55/gB, UL144, and UL73/gN). In 13/14 children (92.8%), a single genotype was observed at birth. Different viral genotypes were found in 1 child (7.2%). Among the sequential samples from 10 children, the same genotype obtained at birth was detected in 9/10 (90%), and in 1 of them (10%), a genotype change in the urine was found. More than 1 HCMV strain in milk was observed in 2 mothers (2/5, 40%). In a population with high seroprevalence, a single genotype was found in the majority of infected children. Reinfection did not frequently occur in the first months of life. Maternal reinfection does not seem to be a rare event in transmitter mothers.