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

Barbi, Maria; Binda, Sandro; Prímache, V.; Caroppo, Simona; Didò, P.; Guidotti, Paola; Corbetta, Carlo; Melotti, D.

doi:10.1016/s1386-6532(00)00089-5

Cited by 147 publications

(134 citation statements)

References 10 publications

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“…This difference can be explained as follows: (i) only 50 l of blood is extracted from a whole DBS compared to the 200 to 500 l extracted from fresh blood, and (ii) whatever the DNA extraction protocol used, it is less efficient when performed with DBS than with fresh blood. However, even if the in vitro sensitivity of CMV PCR in DBS is relatively low, its clinical sensitivity was 100% in our study and ranges from 80 to 100% in the literature (1,2,9,14). CMV DNA quantification with DBS has not been reported so far.…”

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confidence: 61%

“…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).…”

mentioning

confidence: 99%

“…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).…”

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confidence: 99%

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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: 61%

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confidence: 99%

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confidence: 99%

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

et al. 2007

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“…Retrospective diagnosis of congenital HCMV infection has been achieved by detecting HCMV DNA in dried blood samples applied to perinatal cards (Guthrie cards). Conventional end-point polymerase chain reaction (PCR) has been used to amplify HCMV DNA from perinatal cards of children with sensorineural hearing loss 4,5 and malformations of cortical development. 6,7 Newborn blood is routinely collected and dried on perinatal cards as part of state newborn screening programs.…”

mentioning

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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“…Standardization of PCRs with improved ease of use has resulted from the availability of commercial master mixes that include hot-start Taq polymerase and novel formulations to enhance amplification (20). These properties were utilized in the development of applied mPCRs that are simple to prepare and can be validated and individualized for the clinical situation to maximize efficacy for diagnostic use (12,29,42,43,48).There is wide range of putative agents implicated in congenital infections (4,8,24,25,33,34) and clinical samples such as amniotic fluid and neonatal blood may be limited. The aim of this study was to develop and validate mPCRs that would facilitate this testing.…”

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confidence: 99%

Development of Multiplex PCRs for Detection of Common Viral Pathogens and Agents of Congenital Infections

McIver

Jacques²,

Chow

et al. 2005

J Clin Microbiol

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Potential causes of congenital infection include Toxoplasma gondii and viruses such as cytomegalovirus (CMV), enterovirus, hepatitis C virus, herpes simplex virus types 1 and 2 (HSV-1 and -2), human herpesvirus types 6, 7, and 8, lymphocytic choriomeningitis virus, parvovirus, rubella virus, and varicella-zoster virus. Testing for each of these agents using nucleic acid tests is time consuming and the availability of clinical samples such as amniotic fluid or neonatal blood is often limited. The aim of this study was to develop multiplex PCRs (mPCRs) for detection of DNA and RNA agents in the investigation of congenital infection and an mPCR for the viruses most commonly requested in a diagnostic virology laboratory (CMV, Epstein-Barr virus, enterovirus, HSV-1, HSV-2, and varicella-zoster virus). The assays were assessed using known pathogenpositive tissues (cultures, placentae, plasma, and amniotic fluid) and limits of detection were determined for all the agents studied using serial dilutions of plasmid targets. Nested PCR was performed as the most sensitive assay currently available, and detection of the amplicons using hybridization to labeled probes and enzyme-linked immunosorbent assay detection was incorporated into three of the four assays. This allowed detection of 10 to 10 2 copies of each agent in the samples processed. In several patients, an unexpected infection was diagnosed, including a case of encephalitis where HSV was the initial clinical suspicion but CMV was detected. In the majority of these cases the alternative agent could be confirmed using reference culture, serology, or fluorescence methods and was of relevance to clinical care of the patient. The methods described here provide useful techniques for diagnosing congenital infections and a paradigm for assessment of new multiplex PCRs for use in the diagnostic laboratory.Nucleic acid testing has allowed more sensitive and specific detection of infectious agents and is being increasingly adopted by diagnostic laboratories. The technology is particularly useful in virology as it can replace conventional culture methods that are often expensive and labor intensive, detect fastidious organisms such as hepatitis C virus (HCV), detect low-copynumber agents such as herpes simplex virus (HSV) in cerebrospinal fluid, and improve turn-around times for detection of treatable agents such as herpesviruses (30,42,48,50). In the clinical and diagnostic setting, accurate and rapid diagnosis of the causative agent of disease is paramount. Testing for various agents using multiple primer sets in multiplex PCR (mPCR) reactions is an innovation that offers significant benefits in costs, time and accurate diagnosis (20,35). Furthermore, for any given clinical syndrome there a number of candidate agents that may be implicated, particularly with regard to congenital infection.In the diagnostic setting, standardization of assays, use of quality controlled (usually commercially available) reagents, extensive validation of the assays used, and sensitive detection ...

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

Cited by 147 publications

References 10 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

Development of Multiplex PCRs for Detection of Common Viral Pathogens and Agents of Congenital Infections

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