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Dried blood spot real-time polymerase chain reaction assays to screen newborns for congenital cytomegalovirus infection.

ABSTRACT: CONTEXT:Reliable methods to screen newborns for congenital cytomegalovirus (CMV) infection are needed for identification of infants at increased risk of hearing loss. Since dried blood spots (DBS) are routinely collected for metabolic screening from all newborns in the United States, there has been interest in using DBS polymerase chain reaction (PCR)-based methods for newborn CMV screening. OBJECTIVE:To determine the diagnostic accuracy of DBS real-time PCR assays for newborn CMV screening. DESIGN, SETTING, AND PARTICIPANTS:Between March 2007 and May 2008, infants born at 7 US medical centers had saliva specimens tested by rapid culture for early antigen fluorescent foci. Results of saliva rapid culture were compared with a single-primer (March 2007-December 2007) and a 2-primer DBS real-time PCR (January 2008-May 2008). Infants whose specimens screened positive on rapid culture or PCR had congenital infection confirmed by the reference standard method with rapid culture testing on saliva or urine. MAIN OUTCOME MEASURES:Sensitivity, specificity, and positive and negative likelihood ratios (LRs) of single-primer and 2-primer DBS real-time PCR assays for identifying infants with confirmed congenital CMV infection. RESULTS:Congenital CMV infection was confirmed in 92 of 20,448 (0.45%; 95% confidence interval [CI], 0.36%-0.55%) infants. Ninety-one of 92 infants had positive results on saliva rapid culture. Of the 11,422 infants screened using the single-primer DBS PCR, 17 of 60 (28%) infants had positive results with this assay, whereas, among the 9026 infants screened using the 2-primer DBS PCR, 11 of 32 (34%) screened positive. The single-primer DBS PCR identified congenital CMV infection with a sensitivity of 28.3% (95% CI, 17.4%-41.4%), specificity of 99.9% (95% CI, 99.9%-100%), positive LR of 803.7 (95% CI, 278.7-2317.9), and negative LR of 0.7 (95% CI, 0.6-0.8). The positive and negative predictive values of the single-primer DBS PCR were 80.9% (95% CI, 58.1%-94.5%) and 99.6% (95% CI, 99.5%-99.7%), respectively. The 2-primer DBS PCR assay identified infants with congenital CMV infection with a sensitivity of 34.4% (95% CI, 18.6%-53.2%), specificity of 99.9% (95% CI, 99.9%-100.0%), positive LR of 3088.9 (95% CI, 410.8-23 226.7), and negative LR of 0.7 (95% CI, 0.5-0.8). The positive and negative predictive values of the 2-primer DBS PCR were 91.7% (95% CI, 61.5%-99.8%) and 99.8% (95% CI, 99.6%-99.9%), respectively. CONCLUSION:Among newborns, CMV testing with DBS real-time PCR compared with saliva rapid culture had low sensitivity, limiting its value as a screening test.

SUBMITTER: Boppana SB

PROVIDER: S-EPMC2997517 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Dried blood spot real-time polymerase chain reaction assays to screen newborns for congenital cytomegalovirus infection.

Boppana Suresh B SB Ross Shannon A SA Novak Zdenek Z Shimamura Masako M Tolan Robert W RW Palmer April L AL Ahmed Amina A Michaels Marian G MG Sánchez Pablo J PJ Bernstein David I DI Britt William J WJ Fowler Karen B KB

JAMA 20100401 14

<h4>Context</h4>Reliable methods to screen newborns for congenital cytomegalovirus (CMV) infection are needed for identification of infants at increased risk of hearing loss. Since dried blood spots (DBS) are routinely collected for metabolic screening from all newborns in the United States, there has been interest in using DBS polymerase chain reaction (PCR)-based methods for newborn CMV screening.<h4>Objective</h4>To determine the diagnostic accuracy of DBS real-time PCR assays for newborn CMV ...[more]

PMID: 20388893

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Project description:BackgroundCongenital cytomegalovirus (CMV) infection is an important cause of hearing loss, and most infants at risk for CMV-associated hearing loss are not identified early in life because of failure to test for the infection. The standard assay for newborn CMV screening is rapid culture performed on saliva specimens obtained at birth, but this assay cannot be automated. Two alternatives--real-time polymerase-chain-reaction (PCR)-based testing of a liquid-saliva or dried-saliva specimen obtained at birth--have been developed.MethodsIn our prospective, multicenter screening study of newborns, we compared real-time PCR assays of liquid-saliva and dried-saliva specimens with rapid culture of saliva specimens obtained at birth.ResultsA total of 177 of 34,989 infants (0.5%; 95% confidence interval [CI], 0.4 to 0.6) were positive for CMV, according to at least one of the three methods. Of 17,662 newborns screened with the use of the liquid-saliva PCR assay, 17,569 were negative for CMV, and the remaining 85 infants (0.5%; 95% CI, 0.4 to 0.6) had positive results on both culture and PCR assay. The sensitivity and specificity of the liquid-saliva PCR assay were 100% (95% CI, 95.8 to 100) and 99.9% (95% CI, 99.9 to 100), respectively, and the positive and negative predictive values were 91.4% (95% CI, 83.8 to 96.2) and 100% (95% CI, 99.9 to 100), respectively. Of 17,327 newborns screened by means of the dried-saliva PCR assay, 74 were positive for CMV, whereas 76 (0.4%; 95% CI, 0.3 to 0.5) were found to be CMV-positive on rapid culture. Sensitivity and specificity of the dried-saliva PCR assay were 97.4% (95% CI, 90.8 to 99.7) and 99.9% (95% CI, 99.9 to 100), respectively. The positive and negative predictive values were 90.2% (95% CI, 81.7 to 95.7) and 99.9% (95% CI, 99.9 to 100), respectively.ConclusionsReal-time PCR assays of both liquid- and dried-saliva specimens showed high sensitivity and specificity for detecting CMV infection and should be considered potential screening tools for CMV in newborns. (Funded by the National Institute on Deafness and Other Communication Disorders.).

Project description:BackgroundReal-time polymerase chain reaction (PCR) is a sensitive and specific method for diagnosing schistosomiasis. However, this method should be performed in a laboratory, usually located distant from the sample collection site. Therefore, it is important to have fast sampling preservation methods, which allow simple transport prior to DNA extraction and amplification. The aim of this study was to verify if blood samples applied to filter paper are suitable for analysis of Schistosoma mansoni DNA by real-time PCR.MethodsA cross-sectional study was conducted among 100 study participants aged 17 to 70 years in a fishing village on the southern shore of Lake Victoria, Tanzania. Serum samples and ethylenediaminetetraacetic acid (EDTA)-anticoagulated whole blood for preparation of dried blood spots (DBS) were collected to test for Schistosoma mansoni infection by real-time PCR. A combined diagnostic reference of positive results of serum-based real-time PCR and the Kato-Katz (KK) method was used for analysis. Sensitivity and negative predictive value (NPV) were calculated. The Wilcoxon signed-rank test was chosen to compare the mean cycle threshold (Ct) values from serum and DBS.ResultsAccording to the reference, 92.5% S. mansoni positive samples were determined. The serum-based real-time PCR performed excellently with 95.4% sensitivity, whereas the DBS-based real-time PCR showed a low sensitivity (45.4%). The Ct-values were significantly higher in DBS (median: 37.3) than in serum samples (median: 27.5, P?<?0.001), reflecting a lower parasite-specific DNA load on the filter cards. With increasing egg counts, an increase in sensitivity was observed for all methods. The POC-CCA test and the serum-based real-time PCR showed a sensitivity of 100% for medium and severe infections. The DBS real-time PCR showed a sensitivity of only 85.7% even for severe infections.ConclusionsDBS-based real-time PCR did not provide good results in our study and therefore should not be recommended or must be tested concerning temperature of storage, storage duration, use of different filter papers and extraction methods before it is used in future studies. In contrast, our results showed that the POC-CCA test is a sensitive and precise test for detecting S. mansoni infections .

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Dried blood spot real-time polymerase chain reaction assays to screen newborns for congenital cytomegalovirus infection.

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Dried blood spot real-time polymerase chain reaction assays to screen newborns for congenital cytomegalovirus infection.

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