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

Binda, Sandro; Caroppo, Simona; Didò, P.; Prímache, V.; Veronesi, Licia; Calvario, Agata; Piana, Andrea; Barbi, Maria

doi:10.1016/j.jcv.2003.11.012

Cited by 43 publications

(33 citation statements)

References 6 publications

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“…The threshold values of the CMV DNA-specific PCR in DBS reported here, following any of our two DNA extraction protocols, was in the same range as those (e.g., 2,000 or 4,000 genome copies/ml) reported by two other groups (3,14). These threshold values are much higher than those obtained using fresh whole-blood samples; indeed, the latter values usually range from 50 to 500 genome copies/ml.…”

supporting

confidence: 80%

Evaluation of Cytomegalovirus (CMV) DNA Quantification in Dried Blood Spots: Retrospective Study of CMV Congenital Infection

et al. 2007

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We compared two protocols for extracting DNA from dried blood spots for cytomegalovirus (CMV) DNA detection and quantification by real-time PCR. Both extraction methods were reliable for the retrospective diagnosis of CMV congenital infection. Quantification of CMV DNA was valuable after normalization of viral loads with albumin gene PCR amplification results.Human cytomegalovirus (CMV) is the main organism responsible for congenital infection and permanent deafness in young children in developing countries (13). Policies for screening during pregnancy and at birth have not been implemented in European countries or in the United States, essentially because there is no well-established treatment for pregnant women or for newborns with CMV infection (7). Retrospective diagnosis of congenital infection has been achieved by PCR detection of the CMV DNA in dried blood spots (DBS) stored on perinatal Guthrie cards (2,5,6,8,9,14,16,17). Only one protocol (heat DNA extraction, followed by nested PCR) has been extensively evaluated in a clinical setting, with excellent sensitivity and specificity compared to that of viral isolation in the urine (1, 2). However, lower sensitivities (63 and 71%) were reported using the same protocol (5, 16). Alternative methods based on either phenol-chloroform or silica extraction protocols have been proposed, but their sensitivities (81% and 100%, respectively) were studied only with small numbers of patients (9, 14).In the current study, we compared two DNA extraction protocols (phenol-chloroform versus silica-based technology) followed by quantitative in-house real-time CMV DNA-specific PCR amplification (12). Indeed, knowing the CMV DNA load in DBS could provide unique insights regarding the pathogenesis and outcome of CMV congenital infection, especially the relationship between viral load and the risk of hearing loss (4, 10).DNA extraction using a whole DBS cut into thin strips with single-use scissors was performed using two protocols. In protocol one, the strips were submerged twice in 1.5 ml of washing solution (10 mM NaCl, 10 mM EDTA) for 30 min at room temperature. Then, 150 l of lysis buffer (0.32% NaOH) was added onto the strips, and the lysate was recovered after centrifugation (at 10,000 ϫ g for 2 min) and supplemented with 30 l of neutralization solution (1 M Tris, pH 7.5) before DNA extraction with a QIAamp DNA blood mini-kit (QIAGEN, Courtaboeuf, France). Protocol two was performed as described previously (15). Briefly, the strips were submerged in 400 l of extraction buffer and incubated at 56°C for 1 h. The supernatant was recovered after centrifugation and purified by phenol-chloroform extraction, followed by ethanol precipitation.CMV DNA-specific PCR amplification and human albumin PCR amplification were carried out with in-house real-time PCR assays in duplicate (11,12). The normalized value of the CMV DNA load was expressed as the number of CMV genome copies per 10 5 cells. The 45% and 95% sensitivity values of the assays were calculated with a nonlinear regression...

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confidence: 80%

Evaluation of Cytomegalovirus (CMV) DNA Quantification in Dried Blood Spots: Retrospective Study of CMV Congenital Infection

et al. 2007

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“…It should be noted that the sensitivity in our assay (1600 copies/ml) is not as low as that described in the nested PCR method (400 copies/ ml). 15 Nevertheless, a real-time PCR method has analytical and clinical benefits, and it may be applicable to other laboratory assays targeting low-level DNA in blood card samples.…”

Section: Discussionmentioning

confidence: 99%

“…Finally, retrospective studies will provide valuable epidemiological information to further define the spectrum of neurological deficits attributable to HCMV infection and the percentage attributable to HCMV infection. Although perinatal card testing is considered a retrospective diagnostic test, Binda and colleagues 15 have recently proposed dried blood spot testing for congenital HCMV infection for the purpose of newborn screening. This application may allow timely diagnosis and treatment, even in the absence of concurrent signs or symptoms, and may improve long-term outcome.…”

Section: Discussionmentioning

confidence: 99%

Diagnosis of Human Congenital Cytomegalovirus Infection by Amplification of Viral DNA from Dried Blood Spots on Perinatal Cards

Scanga

Chaing

Powell

et al. 2006

The Journal of Molecular Diagnostics

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Congenital human cytomegalovirus (HCMV) infection affects 1% of children and is the most common infectious cause of sensorineural hearing loss. Due to the difficulty of diagnosing deafness and other neurological disorders in infants , affected individuals may not be recognized until much later when active infection has resolved and culture is no longer informative. To overcome this problem , congenital HCMV infection was diagnosed retrospectively by testing residual blood samples collected from newborns and dried on perinatal cards as part of the North Carolina Newborn Screening Program. We modified the Qiagen method for purifying DNA from dried blood spots to increase the sample size and recovery of the lysate. A multiplex , real-time TaqMan polymerase chain reaction assay on an ABI 7900 instrument measured a highly conserved segment of the HCMV polymerase gene and the APOB human control gene. HCMV DNA was detected in blood dried on perinatal cards from all seven infants with culture-proven congenital infection , and all 24 negative control cases lacked detectable HCMV DNA. Our findings suggest that it is possible to diagnose congenital HCMV infection using dried blood collected up to 20 months earlier.

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“…Virological studies were not conducted during the neonatal period, so the time or route of CMV infection cannot be specified. Regrettably, examination of dried umbilical cords or Guthrie cards were not done (2,15). However, it is possible that these three infants had congenital infections, as hepatitis and hepatomegaly are among the symptoms of congenital CMV infection.…”

Section: Discussionmentioning

confidence: 99%

Association of Cytomegalovirus with Infantile Hepatitis

Shibata

Kitajima

Kawada

et al. 2005

Microbiology and Immunology

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Infantile hepatitis is occasionally seen in apparently healthy children. In most cases, the etiology of the infection is uncertain. However, cytomegalovirus (CMV), Epstein‐Barr virus (EBV), human herpesvirus‐6 (HHV‐6), human herpesvirus‐7 (HHV‐7), human parvovirus B19, and TT virus (TTV) are considered to be associated with hepatitis in children. The objective of this study was to investigate the correlations between these viruses and infantile hepatitis. Twenty‐six children from 1 to 24 months old (median age, 7 months) who had liver dysfunction of unknown etiology were enrolled in this study. Plasma samples were examined by a real‐time PCR assay for CMV, EBV, HHV‐6, HHV‐7, parvovirus B19, and TTV DNA. The DNA of CMV was detected in the plasma of four patients (15.4%) and was detected significantly more often in the patient group than in the control group. The CMV‐infected patients were 1 to 3 months old, which was significantly younger than the remaining patients. The serological findings did not always correlate with the results of the real‐time PCR assay. The DNA of TTV was detected in four patients (15.4%), while human parvovirus B19 DNA was detected in three (11.5%). However, the detection frequencies of these viral DNAs were not significantly different from those in the control groups, and some of these patients had co‐infections. These results indicate that CMV might be one of the major pathogens responsible for infantile hepatitis; however, serological tests have limited utility for the diagnosis of CMV infection in young children.

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

Cited by 43 publications

References 6 publications

Evaluation of Cytomegalovirus (CMV) DNA Quantification in Dried Blood Spots: Retrospective Study of CMV Congenital Infection

Evaluation of Cytomegalovirus (CMV) DNA Quantification in Dried Blood Spots: Retrospective Study of CMV Congenital Infection

Diagnosis of Human Congenital Cytomegalovirus Infection by Amplification of Viral DNA from Dried Blood Spots on Perinatal Cards

Association of Cytomegalovirus with Infantile Hepatitis

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