Laboratory diagnosis of congenital CMV infection in newborns: Impact of pre-analytic factors

Kohmer, Niko; Nagel, A.; Berger, Annemarie; Enders, Martin; Hamprecht, Klaus; Korn, Klaus; Kortenbusch, Marhild; Überla, Klaus; Rabenau, Holger F.

doi:10.1016/j.jcv.2019.03.017

Cited by 9 publications

(11 citation statements)

References 12 publications

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“…Summarized, our study confirmed reliable detection of different pathogens by real-time PCR after sample storage and transport on Whatman papers and flocked swabs at ambient temperature in a tropical environment. Such reliable detection has been suggested before for bacteria, protozoa and even RNA viruses in previous assessments (Uttayamakul et al 2005;Rajendram et al 2006;Menassa et al 2010;Jones et al 2012;Petrov et al 2014;Koontz et al 2015;Lalani et al 2015;Randriamparany et al 2016;Silbert et al 2017;Zainabadi et al 2017;Nag et al 2018;Natarajan et al 2018;Kohmer et al 2019;Panda et al 2019;Walker et al 2019). Of note, Cohen's kappa for the comparison of the standard extraction with the assessed alternative nucleic acid extraction approaches ranged from fair (0Á21) to substantial (0Á76) (Table 2).…”

Section: Resultssupporting

confidence: 60%

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Study of enteric pathogens among children in the tropics and effects of prolonged storage of stool samples

Schotte

Hoffmann

Schwarz

et al. 2021

Letters Applied Microbiology

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The study was performed to compare real‐time PCR after nucleic acid extraction directly from stool samples as well as from samples stored and transported on Whatman papers or flocked swabs at ambient temperature in the tropics. In addition, the possible suitability for a clear determination of likely aetiological relevance of PCR‐based pathogen detections based on cycle threshold (Ct) values was assessed. From 632 Tanzanian children <5 years of age with and without gastrointestinal symptoms, 466 samples were subjected to nucleic acid extraction and real‐time PCR for gastrointestinal viral, bacterial and protozoan pathogens. Equal or even higher frequencies of pathogen detections from Whatman papers or flocked swabs were achieved compared with nucleic acid extraction directly from stool samples. Comparison of the Ct values showed no significant difference according to the nucleic acid extraction strategy. Also, the Ct values did not allow a decision whether a detected pathogen was associated with gastrointestinal symptoms.

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Section: Resultssupporting

confidence: 60%

“…However, different types of swabs can stabilize pathogen nucleic acids with varying efficacy, leading to varying degrees of diagnostic reliability, as was shown for gastroenteric pathogens (Silbert et al 2017;Walker et al 2019) and viruses such as African swine fever virus (Petrov et al 2014) or cytomegalovirus (Kohmer et al 2019).…”

Section: Introductionmentioning

confidence: 99%

Study of enteric pathogens among children in the tropics and effects of prolonged storage of stool samples

Schotte

Hoffmann

Schwarz

et al. 2021

Letters Applied Microbiology

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“…(DOCX) S2 Table. Influence of storage conditions on CMV DNA recovery. The flocked swabs of the eNAT™ kit (Copan Italia, Bresca, Italy; order number: 608CS01R), were immersed in a virus suspension with rotating movements for 4-6 seconds and subsequently transferred into 1 ml of eNAT™ medium or 1 ml virus transport medium (Sigma-Virocult, Medical Wire & Equipment, Corsham, Wiltshore, UK) as described by Kohmer et al [13]. After a storage time of eight days at room temperature reflecting a reasonable time interval between sampling and laboratory testing in centralized screening approaches, in a part of the samples DNA was extracted from 200μl and eluted in 60μl as described in the Material and Methods section prior CMV PCR.…”

Section: Discussionmentioning

confidence: 99%

“…Saliva samples. Consistent with a previous study, which evaluated the performance of different swabbing materials and transport time with regard to the efficacy of CMV DNA recovery [13], buccal swabs were obtained with the eNAT™ kit consisting of 1 ml eNAT™ transport and preservation medium in 12x80mm screw cap tubes and a regular FLOQSwab™ (Copan, Brescia, Italy, order number eNAT™ kit: 608CS01R). Swabs were immediately immersed in the eNAT™ medium, stored at 4˚C, and shipped weekly from the screening clinics at ambient temperature to the Institute of Clinical and Molecular Virology at the University Hospital Erlangen, Germany (central study laboratory) for CMV DNA analyses.…”

Section: Sample Collectionmentioning

confidence: 99%

“…The manufacturer warrants a 100% DNA stabilization at room temperature or 4˚C for four weeks. In a study exploring the influence of pre-analytic factors, this approach has revealed the best CMV DNA recovery efficiency compared to a viral transport medium with or without prior DNA extraction ( [13], S2 Table).…”

Section: Test Procedures Of Ccmv Screening and Confirmationmentioning

confidence: 99%

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Characterization of a universal screening approach for congenital CMV infection based on a highly-sensitive, quantitative, multiplex real-time PCR assay

et al. 2020

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The majority of congenital cytomegalovirus (cCMV) infections are asymptomatic at birth and therefore not diagnosed. Approximately 10-15% of these infants develop late-onset hearing loss and other developmental disorders. Implementation of a universal screening approach at birth may allow early initiation of symptomatic interventions due to a closer follow-up of infants at risk and offers the opportunity to consider treatment of late-onset disease. Realtime PCR assays for the detection of CMV DNA in buccal swab samples demonstrated feasibility and good clinical sensitivity in comparison to a rapid culture screening assay. Because most cCMV infections remain asymptomatic, a universal screening assay that stratifies CMV infected infants according to low and high risk of late-onset cCMV disease could limit the parental anxiety and reduce follow-up costs. We therefore developed and characterized a screening algorithm based on a highly-sensitive quantitative real-time PCR assay that is compatible with centralized testing of samples from universal screening and allows to determine CMV DNA load of saliva samples either as International Units (IU)/ml saliva or IU/10 5 cell equivalents. 18 of 34 saliva samples of newborns that tested positively by the screening algorithm were confirmed by detection of CMV DNA in blood and/or urine samples obtained during the first weeks of life. All screening samples that could not be confirmed had viral loads of <2.3x10 5 IU/ml saliva (median: 6.8x10 3) or 1.3x10 5 IU/10 5 cell equivalents (median: 4.0x10 2). The viral load of screening samples with confirmed cCMV infection ranged from 7.5x10 2 to 8.2x10 9 IU/ml saliva (median: 9.3x10 7) or 1.5x10 2 to 5.6x10 10 IU/10 5 cell equivalents (median: 3.5x10 6). Clinical follow-up of these newborns with confirmed cCMV infection should reveal whether the risk of late-onset cCMV disease correlates with CMV DNA load in early life saliva samples and whether a cutoff can

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Detecting human cytomegalovirus in urine, vagina and saliva: Impact of biological fluids and storage durations and temperatures on CMV DNA recovery

Tan,

Carrington,

Pope

2023

Journal of Medical Virology

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Sample collection, transport and storage conditions vary in the human cytomegalovirus (CMV) shedding literature. Currently, limited data exist on the impact of biological fluids and pre‐analytical sample handling on the detection of CMV DNA. To evaluate CMV DNA recovery from urine, vaginal fluid and saliva stored in different conditions, adult urine, vaginal and saliva fluids and swabs, stored with or without selected nucleic acid preservation media at various durations and temperatures, was compared by polymerase chain reaction (PCR) quantitation of spiked samples and self‐collected urine (n = 45) and vaginal swabs (n = 58) from CMV seropositive pregnant women. There was a time‐dependent reduction in CMV DNA recovery from urine, urine diluted in phosphate‐buffered saline, and saliva stored at 2−8°C, but not from urine preserved in cobas® PCR transport media (CPM) (urine/CPM). For vaginal fluid, a reduction in recovery was evident after 7 days storage at 2−8°C. CMV DNA recovery over 91 days was similar between −80°C and −20°C storage for urine and vaginal swabs preserved in CPM, and saliva swabs preserved in eNAT® PCR transport media. A statistically significant change in CMV DNA recovery after 25 months storage (median) at −80°C was not observed for self‐collected urine/CPM and vaginal swab/CPM from pregnant women. Taken together, recovery of CMV DNA is dependent on fluid type and storage conditions. To improve the validity and reliability of detection at different storage durations and temperatures, the use of nucleic acid preserving transport media at the point of collection for urine, vaginal fluid and saliva may be essential.

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Laboratory diagnosis of congenital CMV infection in newborns: Impact of pre-analytic factors

Cited by 9 publications

References 12 publications

Study of enteric pathogens among children in the tropics and effects of prolonged storage of stool samples

Study of enteric pathogens among children in the tropics and effects of prolonged storage of stool samples

Characterization of a universal screening approach for congenital CMV infection based on a highly-sensitive, quantitative, multiplex real-time PCR assay

Detecting human cytomegalovirus in urine, vagina and saliva: Impact of biological fluids and storage durations and temperatures on CMV DNA recovery

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