Influence of different extraction methods and PCR techniques on the sensitivity of HCMV-DNA detection in dried blood spot (DBS) filter cards

doi:10.1016/j.jcv.2010.04.011

Journal of Clinical Virology

Volume 48, Issue 4, August 2010, Pages 278-281

https://doi.org/10.1016/j.jcv.2010.04.011 Get rights and content

Abstract

Background

Infection with human cytomegalovirus (HCMV) is the most common congenital virus infection, affecting about 0.5–2% of newborns. Using DBS on Guthrie cards, it is possible to discriminate congenital from postnatal HCMV-infection. However, a recent European trial revealed serious problems in detection of low HCMV-DNA levels from DBS-filter-cards (Barbi et al., 2008).⁷

Objectives

Evaluation of the most sensitive combination of sample size, DNA extraction method and PCR system for the detection of low copy numbers of HCMV-DNA from DBS-filter-cards.

Study design

We compared three different manual extraction methods for the detection of HCMV-DNA out of DBS: the QIAmp-blood-Mini-Kit, a heat-extraction-method and traditional phenol–chloroform extraction. Additionally, we tested an automated nucleic acid extraction system (NucliSense EasyMag/Biomerieux). Different punch-sizes of DBS spiked with defined HCMV AD169-DNA copy numbers were analyzed. For detection, we used a quantitative in-house-LightCycler-PCR targeting the gB-region using the hybridisation-probe-format. We compared the sensitivity of the real-time-PCR with IE1Ex4-targeted nested-PCR.

Results

The highest sensitivity with 200 copies HCMV DNA/ml was achieved using the phenol–chloroform method in combination with the nested-PCR and 6 mm, 3 × 3 mm punches or the whole DBS. The QIAmp-blood-Mini-Kit also showed a very high sensitivity by using the whole DBS and the nested-PCR.

Conclusion

These results may have strong implications for retrospective diagnosis of congenital HCMV (cHCMV) infection, since a defined combination of the area of punch, the extraction method, and PCR method determine the probability of detection of viral DNA from DBS according to a logistic model.

Introduction

Congenital HCMV (cHCMV)-infection is the most frequent vertically transmitted viral infection in humans. Although 90% of congenitally infected infants are asymptomatic at birth, about 10% show severe clinical outcome with neurological damages, hearing loss, visual impairment and mental retardation.¹ For retrospective diagnostic reasons and in the context of epidemiological studies² the analysis of DBS-filter-cards is very important. The detection methods of HCMV-DNA out of DBS are very heterogeneous with respect to DNA extraction.3, 4, 5, 6 Unfortunately, there is no international standard protocol for the detection of HCMV-DNA out of DBS. Recent external-quality-assessment studies on HCMV-detection from DBS revealed a very poor detection limit with 8.8 × 10⁴ copies/ml. These results showed an urgent need for improvement of the various HCMV-detection-methods from DBS.⁷ including optimization of minimal punch sizes for successful viral DNA detection.

Section snippets

Methods

Native HCMV-seronegative EDTA-blood was spiked with commercially available AD169-DNA (tebu-bio) in a range of 10⁵–10¹ copies/50 μl and dropped on filter-cards (V_F = 50 μl). After drying, different areas of the DBS-filter-cards were collected: 1 × 6 mm punch (area of punch 28.3 mm²), 1 × 3 mm punch (area of punch 7.1 mm²), 2 × 3 mm punches (area of both attached punches 14.1 mm²), 3 × 3 mm punches (area of all attached punches 21.1 mm²) and the whole area of the DBS (area of punch 126.7 mm²). Four different

Results

Using one individual 3 mm punch of the DBS (area of punch 7.1 mm²), the detection limit of the nPCR using phenol–chloroform-extraction corresponded to 20,000 HCMV DNA copies/ml of native spiked EDTA-blood. Using the QIAmp-blood-DNA-Mini-Kit for extraction the sensitivity decreased to 4,000,000 copies/ml while using the NucliSenseEasyMag the sensitivity increased again to 200,000 copies/ml. With the heat-extraction-method in combination with the nPCR and one 3 mm punch the detection-limit was above

Discussion

Recent results of testing the clinical sensitivity of HCMV-DNA-PCR methods with Guthrie cards from confirmed congenitally infected neonates revealed, that the combination of easyMAG-DNA extraction with a conventional PCR-protocol showed only a sensitivity of 45%.¹⁵ In our hands, the most sensitive combination of HCMV-extraction-method and HCMV-amplification-system for the detection of HCMV-DNA out of experimentally spiked DBS consisted of a conventional nPCR using primers of the

Conflict of interest

No competing interests.

Acknowledgements

This work was supported by the Robert-Koch-Institute, Berlin in context of the clinical trial “Epidemiology of congenital HCMV-Infection in Germany”.

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2023, Journal of Clinical Virology
Cytomegalovirus (CMV) is a leading cause of congenital infections. Dried blood spots (DBS) collected in the first week of life (Guthrie cards) have been used in the diagnosis of CMV infection outside the three-week window period following birth. The present work summarizes the results of a 15-year observational study in which DBS from 1388 children were used for a late diagnosis of congenital CMV infection.
Three groups of children were studied: (i) symptomatic (with symptoms at birth or late sequelae) (N = 779); (ii) born to mothers with serological profile of primary CMV infection (N = 75); (iii) without any information (N = 534). A highly sensitive method of DNA extraction (heat-induced) from the DBS was used. CMV DNA was detected by a nested PCR.
In total CMV DNA was detected in 7.5% (104/1388) of children. Symptomatic children showed a low rate of CMV DNA detection (6.7%) than children born to mothers with serological profile of primary CMV infection (13.3%) (p = 0.034). Sensorial hearing loss and encephalopathy were the two clinical manifestations with the highest CMV detection rate (18.3% and 11.1%, respectively). Children whose mothers had a confirmed primary infection showed a higher rate of CMV detection (35.3%) when compared with children whose mothers had a not confirmed primary infection (6.9%) (p = 0.007).
The present work emphasises the importance of testing DBS in symptomatic children even a long time after symptoms onset and in children born to mothers with serologic diagnosis of maternal primary CMV infection when they miss the diagnosis during the three-week window following birth.
Prospective multicenter comparison of urine culture with PCR on dried blood spots using 2 different extraction and PCR methods in neonates suspected for congenital cytomegalovirus infection
2020, Diagnostic Microbiology and Infectious Disease
To evaluate the potential of dried blood spots (DBS) as a congenital cytomegalovirus (cCMV) testing specimen, the laboratory diagnostic accuracy of polymerase chain reaction (PCR) on DBS was compared to viral urine cultures from neonates suspected for cCMV. Two different extraction methods (EasyMAG, bioMérieux versus Qiagen) and 2 real-time PCR protocols (in-house versus Argene) were compared. We were able to collect both DBS and urine samples in 6 Belgian neonatal units from 276 neonates suspected for cCMV registered in CMVREG (an online neonatal registry system). Forty-eight neonates (17.4%) were positive by viral culture in urine. Laboratory diagnostic accuracy parameters of DBS-PCR were both extraction method and PCR protocol dependent. Not all DBS-CMV-PCR methods successfully detected urine-culture–positive neonates born after first-trimester seroconversions. Interestingly, however, all urine-culture–positive neonates having clinical signs of cCMV did consistently score positive.
Evaluation of rapid and sensitive DNA extraction methods for detection of cytomegalovirus in dried blood spots
2019, Journal of Virological Methods
Citation Excerpt :
Moreover, sensitivity of CMV detection in DBS is strongly correlated with the DNA extraction method used (de Vries et al., 2009; Koontz et al., 2015; Vauloup-Fellous et al., 2007; Gohring et al., 2010). In addition to the widely used thermal shock method (Yamamoto et al., 2001; de Vries et al., 2009; Koontz et al., 2015; Gohring et al., 2010; Shibata et al., 1994; Barbi et al., 1996; Kharrazi et al., 2010), other extraction methods such as silica-based columns (Leruez-Ville et al., 2011; Walter et al., 2008; de Vries et al., 2009; Koontz et al., 2015; Vauloup-Fellous et al., 2007; Gohring et al., 2010), magnetic bead technologies (Boppana and Ross, 2010; de Vries et al., 2009; Koontz et al., 2015; Gohring et al., 2010), and phenol-chloroform (Leruez-Ville et al., 2011; Johansson et al., 1997; Vauloup-Fellous et al., 2007; Gohring et al., 2010) have been used. We have previously shown that thermal shock performs well compared to column and bead-based extraction methods (Koontz et al., 2015).
Dried blood spots (DBS), collected universally from newborns in the U.S., could be used as a matrix for the detection of cytomegalovirus (CMV) infection in infants. However, sensitivity to detect CMV in DBS as compared to saliva and urine is variable across studies largely due to the DNA extraction method. Thermal shock, a widely used DNA extraction method, is highly sensitive for the detection of CMV in DBS, however, the processing time required is not practical for high-throughput testing.
To determine if rapid and cost-effective DNA extraction methods amenable to newborn screening (NBS) could achieve the same sensitivity as the thermal shock method.
DBS were prepared from CMV positive blood samples from 20 organ transplant recipients. Three DNA extraction methods were compared for relative yield and sensitivity of detection of CMV DNA: thermal shock, KOH Tris buffer, and DNA Extract All. CMV DNA was detected by real-time quantitative polymerase chain reaction (qPCR).
The KOH Tris and DNA Extract All methods gave higher yields and sensitivity of CMV detection in DBS than thermal shock, which were significantly greater when viral loads were ≤ 10,000 copies/ml blood. Both methods gave faster turnaround times than thermal shock and would be better suited for NBS.
The choice of DNA extraction method greatly influences the ability to detect low levels of CMV DNA in DBS. Moreover, development of highly sensitive yet rapid methods for CMV detection could help facilitate future newborn screening of CMV in DBS.
Discrepancy in impact of maternal milk on vertical transmission between Hepatitis B virus and Human cytomegalovirus
2015, International Journal of Infectious Diseases
This study aims to elucidate the role of breastfeeding on vertical transmission of HCMV and HBV and to investigate the difference in perinatal transmission via breast milk between HBV and HCMV.
This detailed study monitored the kinetics of viral DNA load in maternal milk for both HBV and HCMV, demonstrated the rate of transmission to infants, and compared HBV infection rate with that of HCMV.
There was no difference in overall DNAlactia+ between HBV (23.86%) and HCMV (29.54%, P=0.140) for seropositive mothers, while HBsAg prevalence (0.75%) was significantly lower than HCMV IgG+ (27.44%, P<0.001) for the breast-fed babies. Between breast-fed babies of seropositive mothers and those of seronegative mothers, HBV infection rate had no difference (HBsAg+: 0.75% vs 0%, P=0.538; DNAemia+: 0.38% vs 0%, P=0.664), but HCMV infection rate of the former was significantly higher than that of the latter (IgG+: 27.07% vs 18.00%, P=0.045; DNAemia+: 15.79% vs 4.00%, P=0.027).
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Evaluation of DNA extraction methods for the detection of Cytomegalovirus in dried blood spots
2015, Journal of Clinical Virology
Citation Excerpt :
Concordant with our study, the Investigator manual and thermal shock methods showed superior performance for detection of CMV. A third DBS methods comparison by Gohring [19] included four methods and showed the Qiagen Mini kit performed much better than thermal shock (referred to as heat). However the Gohring study used AD169-spiked blood instead of naturally infected blood, and their thermal shock (heat) method did not include the important pre-incubation of DBS at either 4 °C overnight [10] or at room temperature for 2 h.
Dried blood spots (DBS) are collected universally from newborns and may be valuable for the diagnosis of congenital Cytomegalovirus (CMV) infection. The reported analytical sensitivity for DBS testing compared to urine or saliva varies greatly across CMV studies. The purpose of this study was to directly compare the performance of various DNA extraction methods for identification of CMV in DBS including those used most often in CMV studies.
Whatman^® Grade 903 filter paper cards were spotted with blood samples from 25 organ transplant recipients who had confirmed CMV viremia. Six DNA extraction methods were compared for relative yield of viral and cellular DNA: 2 manual solution-based methods (Gentra Puregene, thermal shock), 2 manual silica column-based methods (QIAamp DNA Mini, QIAamp DNA Investigator), and 2 automated methods (M48 MagAttract Mini, QIAcube Investigator). DBS extractions were performed in triplicate followed by real-time quantitative PCR (qPCR).
For extraction of both viral and cellular DNA, two methods (QIAamp DNA Investigator and thermal shock) consistently gave the highest yields, and two methods (M48 MagAttract Mini and QIAamp DNA Mini) consistently gave the lowest yields. There was an average 3-fold difference in DNA yield between the highest and lowest yield methods.
The choice of DNA extraction method is a major factor in the ability to detect low levels of CMV in DBS and can largely account for the wide range of DBS sensitivities reported in studies to date.
Common childhood viral infections
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Citation Excerpt :
In a congenital CMV-suspect infant older than 21 days, dried blood spots collected in the first few days of life for newborn metabolic screening can be tested by CMV PCR. Despite not having perfect sensitivity, PCR on dried blood spots can detect low levels of CMV, confirming a diagnosis of congenital CMV that may be otherwise impossible to make.62 No treatment is indicated for immunocompetent individuals with acute CMV infection because the expected course is recovery.
Infections caused by viruses are universal during childhood and adolescence. Clinicians will regularly care for children and adolescents who present with infections caused by a wide number of viral pathogens. These infections have varied presentations. Many infections may have clinical presentations that are specific to the infecting virus but present differently, based on the age and immunocompetence of the patient. Some children are directly impacted early in their lives when maternal disease results in an in utero infection (cytomegalovirus, rubella virus, or parvovirus B19). Other viruses may infect children in a predictable pattern as they grow older (rhinovirus or influenza virus). Fortunately, many viral infections frequently encountered in the past are no longer extant due to widespread immunization efforts. Recognition of these vaccine-preventable infections is important because outbreaks of some of these diseases (mumps or measles) continue to occur in the United States. Vigilance in vaccine programs against these viral agents can prevent their re-emergence. In addition, an increasing number of viral infections (herpes simplex virus, influenza virus, varicella zoster virus, or cytomegalovirus) can now be successfully treated with antiviral medications. Most viral infections in children result in self-limited illness and are treated symptomatically and infected children experience full recovery. This review will address the epidemiology, clinical presentation, diagnosis, treatment, and prevention of viral infections commonly encountered by the clinician.

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Short communicationInfluence of different extraction methods and PCR techniques on the sensitivity of HCMV-DNA detection in dried blood spot (DBS) filter cards

Abstract

Background

Objectives

Study design

Results

Conclusion

Introduction

Section snippets

Methods

Results

Discussion

Conflict of interest

Acknowledgements

J Clin Virol

J Clin Virol

J Virol Methods

J Virol Methods

J Clin Virol

Clin Diagn Virol

Congenital cytomegalovirus infection: outcome and diagnosis

Pediatr Infect Dis

Diagnosis of congenital HCMV infection via dried blood spots

Rev Med Virol

Detection of human cytomegalovirus DNA in dried newborn blood filter paper

J Virol Methods

Short communication
Influence of different extraction methods and PCR techniques on the sensitivity of HCMV-DNA detection in dried blood spot (DBS) filter cards