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

BACKGROUND: Congenital 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 DESIGN: Children 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. RESULTS: Seventysix 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. CONCLUSIONS: Our 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.

Rapid susceptibility testing for herpes simplex virus type 1 using real-time PCR.

BACKGROUND: Susceptibility testing of herpes simplex virus type 1 (HSV-1) is traditionally performed by a plaque reduction assay (PRA), but this is labor intensive, time consuming and has a manual read out. OBJECTIVES: The goal of this study was to develop an internally controlled real time PCR-based phenotypical susceptibility test for HSV-1 that is suitable for use in a clinical diagnostic setting. STUDY DESIGN: A DNA reduction assay (DRA) was developed and validated on a test panel of 26 well-characterized isolates of varying susceptibility to aciclovir or foscarnet, including low-level resistant isolates. The DRA consisted of pre-culture of a clinical sample for 48 h and subsequent culture in the presence of antivirals for 24 h. Viral DNA concentration in the culture lysates was measured by an internally controlled quantitative real-time HSV-1 PCR and corrected for cell count and lysis by beta-globin PCR. DRA results were compared to results from PRA and sequence analysis. RESULTS: DRA results were in accordance with PRA results for both aciclovir and foscarnet susceptibility and appeared to have good discriminative value for low-level resistance due to UL30 gene mutations. Although the direct application of DRA in clinical samples appeared not possible, short pre-culture of 48 h was sufficient and ensured results within a clinically relevant time frame of 5 days. CONCLUSIONS: DRA is an accurate, rapid and easy to perform phenotypical susceptibility test for HSV-1.

Real-time PCR versus viral culture on urine as a gold standard in the diagnosis of congenital cytomegalovirus infection.

BACKGROUND: Cytomegalovirus (CMV) infection is the most common cause of congenital infection. Whereas CMV PCR has replaced viral culture and antigen detection in immunocompromised patients because of higher sensitivity, viral culture of neonatal urine is still referred to as the gold standard in the diagnosis of congenital CMV infection. OBJECTIVE: To compare real-time CMV PCR with shell vial culture on urine in the diagnosis of congenital CMV, in a multicenter design. STUDY DESIGN: A series of neonatal urines (n=340), received for congenital CMV diagnostics and routinely assessed with shell vial CMV culture, was retrospectively tested by real-time CMV PCR. RESULTS: The proportion of newborns found to be congenitally infected by real-time CMV PCR was 8.2% (28/340, 95%CI 5.6-11.8%), and 7.4% (25/340, 95%CI 4.9-10.8%) by rapid culture. When considering rapid culture as reference, real-time PCR was highly sensitive (100%), whereas sensitivity of rapid culture was 89.3% when considering real-time PCR as reference. CONCLUSIONS: Our results, supported by analytical and clinical data on CMV DNA detection in neonatal urine, suggest enhanced sensitivity of recent PCR techniques when compared to viral culture. There is considerable rationale to favor real-time CMV PCR as a gold standard in the diagnosis of congenital CMV infection. A large-scale study combining both laboratory and clinical data is required to determine the exact time frame for sampling of neonatal urine when using real-time PCR.

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.

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.