Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Nyu Medical Center on 02/08/15 For personal use only.
Key Paper Evaluation
Diagnosis and monitoring of cytomegalovirus infection by the quantification of viral load in dried blood spots samples Expert Rev. Mol. Diagn. 14(2), 139–142 (2014)
Giulia Piccirilli‡, Angela Chiereghin‡, Liliana Gabrielli* and Tiziana Lazzarotto Operative Unit of Clinical Microbiology, St. Orsola-Malpighi General Hospital, University of Bologna, Via Massarenti 9, 40138 Bologna, Italy *Author for correspondence: Tel.: +39 051 6364645 Fax: +39 051 307397 [email protected] ‡
Authors contributed equally
Evaluation of: Limaye AP, Santo Hayes TK, Huang ML, et al. Quantitation of cytomegalovirus DNA load in dried blood spots correlates well with plasma viral load. J. Clin. Microbiol. 51, 2360–2364 (2013). Cytomegalovirus (CMV) represents the major infectious cause of birth defects, as well as an important pathogen for immune-compromised individuals. Several studies described the use of dried blood spots (DBS) for the detection of CMV DNA for late diagnosis of congenital CMV infection in cases of strong clinical suspicion. In the article under evaluation, Limaye et al. perform for the first time the quantification of CMV in pairs of finger-stick DBS and plasma samples collected from transplant patients. The work concluded that finger-stick DBS could be an alternative sample type for quantification of CMV load that correlates well with plasma levels. Prospective trials to evaluate the use of DBS for monitoring CMV load in transplant recipients will be required. KEYWORDS: congenital infection • cytomegalovirus • dried blood spots • transplant patient • viral load
This article evaluates a paper recently published by Limaye et al., which suggests that an assay to accurately quantitate cytomegalovirus (CMV) load in finger-stick-collected dried blood spots (DBS) could potentially be useful for analyzing patient self-collected specimens [1]. DBS assay may well possibly be a useful tool for serial detection of CMV viral load in patients who do not have convenient access to phlebotomy facilities or who are unwilling to undergo venipuncture. CMV causes severe morbidity and mortality in congenitally infected newborns and immune-compromised patients, most notably transplant recipients and HIV-infected people. Specifically, congenital CMV infection is the most common infectious cause of mental retardation and deafness. Following symptomatic congenital CMV infection, progressive hearing loss develops in up to 60% of cases and mental retardation in 35–50% of patients [2]. informahealthcare.com
10.1586/14737159.2014.883278
In solid organ transplant recipients, CMV infection causes both direct and indirect effects including allograft rejection, decreased graft and patient survival and predisposition to opportunistic infections and malignancies [3]. In immune-compromised patients, diagnostic methods for CMV infection and CMV-associated diseases include detection and quantification of viral nucleic acids using molecular methods. Quantification of CMV DNA has correlated disease and infection severity with the degree of viral replication [4]. Different compartments of the blood have been used in the diagnosis of CMV infection. In the article by Limaye et al., CMV DNA load was evaluated in paired venipuncturecollected plasma samples and finger-stick DBS from solid organ transplant patients, using a previously validated quantitative PCR assay. In order to compare the CMV levels measured on blood card samples and on plasma
2014 Informa UK Ltd
ISSN 1473-7159
139
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Nyu Medical Center on 02/08/15 For personal use only.
Key Paper Evaluation
Piccirilli, Chiereghin, Gabrielli & Lazzarotto
samples instead of whole blood, DBS CMV results were normalized to copies of CMV per 1 ml of plasma using specific formulas, considering that in blood, approximately 60% of the volume is plasma. Which of the various blood compartments is optimal for CMV DNA detection has been the subject of several studies [5–7]. Some authors reported that there was a good correlation between plasma and whole blood viral loads. However, the absolute value for the two viral loads was different: in most patients whole blood viral loads were approximately 1 log higher than plasma. Moreover, the higher sensitivity of whole blood was observed in detecting low-level viral loads [7]. Summary of methods & results
In the analysis of Limaye et al., 106 prospectively collected pairs of finger-stick DBS and plasma samples were processed using a validated quantitative real-time TaqMan CMV PCR to assess CMV viral load. The commercial Qiagen 96 blood kit (Qiagen Inc.) was used to extract DNA from 200 ml plasma samples according to the manufacturer’s instructions. For DBS specimens, DNA was extracted from four 3-mm diameter paper puncher resuspended into 100 ml of autoclave-sterilized 5% Chelex (Bio-Rad Inc.) and incubated at 95˚C for 30 min. To compare the CMV levels measured on plasma and on DBS samples, CMV results were normalized to copies of CMV per 1 ml of plasma using specific formulas and then conversion to IU/ml was performed. Statistical analysis was performed using linear regression, logistic, probit and complementary log-log regression analyses. The DBS levels showed a linear correlation with plasma levels throughout the measuring range of the assay (slope of the regression line = 1.0; r = 0.92). Twenty-five of the 106 (23.6%) sample pairs were positive by plasma PCR (with low viral load: median range of 320 copies/ml), but negative by DBS assay. In 76/81 (94%) of paired DBS and plasma samples, the difference in CMV load was
Key Paper Evaluation
Diagnosis and monitoring of cytomegalovirus infection by the quantification of viral load in dried blood spots samples Expert Rev. Mol. Diagn. 14(2), 139–142 (2014)
Giulia Piccirilli‡, Angela Chiereghin‡, Liliana Gabrielli* and Tiziana Lazzarotto Operative Unit of Clinical Microbiology, St. Orsola-Malpighi General Hospital, University of Bologna, Via Massarenti 9, 40138 Bologna, Italy *Author for correspondence: Tel.: +39 051 6364645 Fax: +39 051 307397 [email protected] ‡
Authors contributed equally
Evaluation of: Limaye AP, Santo Hayes TK, Huang ML, et al. Quantitation of cytomegalovirus DNA load in dried blood spots correlates well with plasma viral load. J. Clin. Microbiol. 51, 2360–2364 (2013). Cytomegalovirus (CMV) represents the major infectious cause of birth defects, as well as an important pathogen for immune-compromised individuals. Several studies described the use of dried blood spots (DBS) for the detection of CMV DNA for late diagnosis of congenital CMV infection in cases of strong clinical suspicion. In the article under evaluation, Limaye et al. perform for the first time the quantification of CMV in pairs of finger-stick DBS and plasma samples collected from transplant patients. The work concluded that finger-stick DBS could be an alternative sample type for quantification of CMV load that correlates well with plasma levels. Prospective trials to evaluate the use of DBS for monitoring CMV load in transplant recipients will be required. KEYWORDS: congenital infection • cytomegalovirus • dried blood spots • transplant patient • viral load
This article evaluates a paper recently published by Limaye et al., which suggests that an assay to accurately quantitate cytomegalovirus (CMV) load in finger-stick-collected dried blood spots (DBS) could potentially be useful for analyzing patient self-collected specimens [1]. DBS assay may well possibly be a useful tool for serial detection of CMV viral load in patients who do not have convenient access to phlebotomy facilities or who are unwilling to undergo venipuncture. CMV causes severe morbidity and mortality in congenitally infected newborns and immune-compromised patients, most notably transplant recipients and HIV-infected people. Specifically, congenital CMV infection is the most common infectious cause of mental retardation and deafness. Following symptomatic congenital CMV infection, progressive hearing loss develops in up to 60% of cases and mental retardation in 35–50% of patients [2]. informahealthcare.com
10.1586/14737159.2014.883278
In solid organ transplant recipients, CMV infection causes both direct and indirect effects including allograft rejection, decreased graft and patient survival and predisposition to opportunistic infections and malignancies [3]. In immune-compromised patients, diagnostic methods for CMV infection and CMV-associated diseases include detection and quantification of viral nucleic acids using molecular methods. Quantification of CMV DNA has correlated disease and infection severity with the degree of viral replication [4]. Different compartments of the blood have been used in the diagnosis of CMV infection. In the article by Limaye et al., CMV DNA load was evaluated in paired venipuncturecollected plasma samples and finger-stick DBS from solid organ transplant patients, using a previously validated quantitative PCR assay. In order to compare the CMV levels measured on blood card samples and on plasma
2014 Informa UK Ltd
ISSN 1473-7159
139
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Nyu Medical Center on 02/08/15 For personal use only.
Key Paper Evaluation
Piccirilli, Chiereghin, Gabrielli & Lazzarotto
samples instead of whole blood, DBS CMV results were normalized to copies of CMV per 1 ml of plasma using specific formulas, considering that in blood, approximately 60% of the volume is plasma. Which of the various blood compartments is optimal for CMV DNA detection has been the subject of several studies [5–7]. Some authors reported that there was a good correlation between plasma and whole blood viral loads. However, the absolute value for the two viral loads was different: in most patients whole blood viral loads were approximately 1 log higher than plasma. Moreover, the higher sensitivity of whole blood was observed in detecting low-level viral loads [7]. Summary of methods & results
In the analysis of Limaye et al., 106 prospectively collected pairs of finger-stick DBS and plasma samples were processed using a validated quantitative real-time TaqMan CMV PCR to assess CMV viral load. The commercial Qiagen 96 blood kit (Qiagen Inc.) was used to extract DNA from 200 ml plasma samples according to the manufacturer’s instructions. For DBS specimens, DNA was extracted from four 3-mm diameter paper puncher resuspended into 100 ml of autoclave-sterilized 5% Chelex (Bio-Rad Inc.) and incubated at 95˚C for 30 min. To compare the CMV levels measured on plasma and on DBS samples, CMV results were normalized to copies of CMV per 1 ml of plasma using specific formulas and then conversion to IU/ml was performed. Statistical analysis was performed using linear regression, logistic, probit and complementary log-log regression analyses. The DBS levels showed a linear correlation with plasma levels throughout the measuring range of the assay (slope of the regression line = 1.0; r = 0.92). Twenty-five of the 106 (23.6%) sample pairs were positive by plasma PCR (with low viral load: median range of 320 copies/ml), but negative by DBS assay. In 76/81 (94%) of paired DBS and plasma samples, the difference in CMV load was
