Usefulness of blood and urine samples collected on filter paper in detecting cytomegalovirus by the polymerase chain reaction technique

Yamamoto, Aparecida Yulie; Mussi-Pinhata, Marisa Márcia; Pinto, Patrícia Cristina Gomes; Figueiredo, Luíz Tadeu Moraes; Jorge, Salim Moysés

doi:10.1016/s0166-0934(01)00347-0

Cited by 76 publications

(66 citation statements)

References 15 publications

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“…Johansson et al (9) and Yamamoto et al (16) had detection rates of 81 and 71%, respectively. The highest detection rate was found by Barbi et al (1), whose protocol had a 100% sensitivity and a 99% specificity.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Different Cytomegalovirus (CMV) DNA PCR Protocols for Analysis of Dried Blood Spots from Consecutive Cases of Neonates with Congenital CMV Infections

et al. 2008

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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).Cytomegalovirus (CMV) infection during pregnancy can be transmitted to the fetus, resulting in a congenital infection. Congenital CMV infection (cCMV) occurs in 0.2 to 2.2% of live-born infants. An incidence of 0.62% is found in the Brussels, Belgium, region (11).The standard diagnosis of cCMV relies on the isolation of the virus from urine or saliva collected in the first 2 weeks of life. The diagnosis of cCMV infection in children older than 2 weeks cannot be made or excluded on the basis of viral isolation. Blood is routinely collected from neonates in the first 2 weeks of life and is stored as dried blood spots (DBSs) on Guthrie cards. The detection of CMV DNA on these stored DBSs could be an opportunity to diagnose cCMV during later life, when symptoms suggestive of cCMV develop (3).The aim of this study was to test the clinical sensitivity of CMV DNA PCR with DBSs from consecutive cases of neonates with cCMV infections. We evaluated two methods for the extraction of CMV DNA and two methods for the amplification of CMV DNA from DBSs on Guthrie cards for the retrospective diagnosis of cCMV. MATERIALS AND METHODSThe study protocol was approved by the Committee of Medical Ethics of the UZ Brussel. cCMV infections. From 1996 to 2006 a neonatal screening program (11) detected cCMV in 76 newborns at the Universitair Ziekenhuis Brussels. The diagnosis of cCMV infection was made by isolation of the virus from urine within 7 days of birth. Blood from the first consecutive 57 cases and from the last 4 cases was retrieved and placed on Guthrie cards (no. 903 ...

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Section: Discussionmentioning

confidence: 99%

Evaluation of Different Cytomegalovirus (CMV) DNA PCR Protocols for Analysis of Dried Blood Spots from Consecutive Cases of Neonates with Congenital CMV Infections

et al. 2008

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“…Detection of CMV DNA in DBS has been shown to be a challenge (3,5,6,9,10,35,36,39) due to the small amount of dried blood (50 l per spot) available. Optimizing the DNA extraction step from the DBS has been shown to result in significantly increased sensitivity of CMV DNA detection in DBS.…”

Section: Discussionmentioning

confidence: 99%

Rapid Genotyping of Cytomegalovirus in Dried Blood Spots by Multiplex Real-Time PCR Assays Targeting the Envelope Glycoprotein gB and gH Genes

et al. 2012

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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 l 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. C ytomegalovirus (CMV) is the most common cause of congenital infection worldwide and an important viral pathogen affecting immunocompromised patients (13, 21). Both in congenitally infected newborns and in immunocompromised patients, genotyping of CMV has been used to study potential differences in pathogenicity of specific strains. However, few authors describe a correlation between specific CMV genotypes and severity of disease (29,32,34,40). More important, genotyping of CMV has enabled the discrimination of reactivation of latent virus from reinfection with new CMV strains in transplant patients, allowing a better definition of donor-to-recipient transmission patterns (24). Congenital CMV infections mainly result from recurrent infections among pregnant women (37), comprising both reactivation and reinfection. The discrimination of reactivation from reinfection may give insight into the mother-to-fetus transmission pattern and the possible associations with the outcome of congenital CMV infections.Genotyping of CMV has mainly focused on envelope glycoproteins gB (UL55) and gH (UL75), which play a role in virus entry and are major targets for neutralizing antibody response. The most frequently used methods for genotyping of CMV are nucleotide sequence analysis (27) and restriction fragment length polymorphism of PCR products (25, 28). Recently, real-time PCR-based assays have been used for rapid detection and quantification of CMV gB and gH genotypes (12,15,24,26). However, they have mainly been applied to plasma or other high-volume samples. Also, deep-sequencing-based methods, sensitive in the de...

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“…1 Intrauterine infection occurs in up to 50% of pregnancies following primary maternal infection, and 10% of the infected infants are symptomatic at birth. 2,3 However nearly 10%-15% of the asymptomatic newborns develop late sequelae, particularly sensorineural hearing loss and neurodevelopmental disorders. 4 Genital herpes continues to be a public health problem in both developed and developing countries.…”

Section: Introductionmentioning

confidence: 99%

Molecular detection of cytomegalovirus, herpes simplex virus 2, human papillomavirus 16-18 in Turkish pregnants

Dinç¹,

Bozdayı²,

Bırı³

et al. 2010

Braz J Infect Dis

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Objective: Human cytomegalovirus (CMV) is the most common cause of viral intrauterine infections in the world. Herpes simplex virus type 2 (HSV-2) and human papillomavirus (HPV) are the main agents of viral sexually transmitted diseases, which cause genital ulcers and genital warts, respectively. HPV infection has been linked to the majority of the anogenital malignancies. The aim of this study was to detect the existence of CMV, HSV-2 and HPV type 16-18 in Turkish pregnants by using sensitive molecular assays. Methods: One hundred thirty-four women (18-41 years old; mean age ± SD: 27 ± 8) applied to outpatient clinic of Obstetrics and Gynecology, in between 18th -22nd weeks of their pregnancy and a control group of 99 healthy women (15-39 years old; mean age ± SD: 24 ± 8) were included in the study. Cervical smear samples were used for DNA extraction. CMV, HSV-2 and HPV 16-18 detections were carried out by real time PCR and in house PCR method, respectively. Results: Three patients (3/134; 2.2%) were found to be positive for each HPV and HSV-2. Dual infection with HPV and HSV was found in just one patient. HPV 18 was detected in all positive samples. CMV was found to be positive in two patients (2/134; 1.4 %). Conclusion: HPV, HSV and CMV must be screened due to high prevalence of these viruses in pregnants by using sensitive molecular methods.

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Usefulness of blood and urine samples collected on filter paper in detecting cytomegalovirus by the polymerase chain reaction technique

Cited by 76 publications

References 15 publications

Evaluation of Different Cytomegalovirus (CMV) DNA PCR Protocols for Analysis of Dried Blood Spots from Consecutive Cases of Neonates with Congenital CMV Infections

Evaluation of Different Cytomegalovirus (CMV) DNA PCR Protocols for Analysis of Dried Blood Spots from Consecutive Cases of Neonates with Congenital CMV Infections

Rapid Genotyping of Cytomegalovirus in Dried Blood Spots by Multiplex Real-Time PCR Assays Targeting the Envelope Glycoprotein gB and gH Genes

Molecular detection of cytomegalovirus, herpes simplex virus 2, human papillomavirus 16-18 in Turkish pregnants

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