Validating the ORACollect for the detection of cytomegalovirus

Targeted screening for Cytomegalovirus (CMV) in Deaf and Hard of Hearing (DHH) children is now internationally recommended. With newborn genomic screening for DHH children a future possibility, the commercially-available human genomic DNA collection kit (ORACollect, Oragene OCR-100) could enable one single sample to screen for CMV and genetic causes of deafness at scale with minimal additional costs. Our pilot study validated ORACollect against Copan FLOQswabs^® (gold standard clinical procedure) for detecting CMV using 15 sets of saliva samples from 14 infants/children, comparing CMV PCR results using different testing protocols. ORACollect stored at room temperature had high sensitivity (up to 89%), specificity (up to 80%) and percent agreement (up to 86%) in detecting CMV DNA compared to FLOQswabs^®. This suggests that ORACollect is an appropriate alternative to FLOQswabs^® for collecting viral CMV DNA for PCR testing, independent of the DNA extraction approach. This could be revolutionary in facilitating dual genomic and viral screening in newborns and would enable CMV screening in non-tertiary hospital settings where laboratory facilities are not available.

Congenital Cytomegalovirus (CMV) is the most common infectious cause of child hearing loss [1] and developmental disabilities, such as cerebral palsy [2]. Early targeted screening of CMV in newborn saliva is now recommended internationally [3]. CMV DNA is traditionally isolated from saliva collected using Copan FLOQswabs^® and analysed by Polymerase Chain Reaction (PCR) [4]. This process provides accurate clinical results but is difficult to implement in non-tertiary settings without infrastructure for immediate sample processing. While dedicated preservative systems for viral DNA are commercially available, these are distinct from systems designed for the isolation and genotyping of human genomic DNA. With the future possibility of newborn genomic screening, the ORACollect (DNA Genotek, Oragene OCR-100), normally designed to collect human genomic DNA and which can be stored at room temperature for up to two months for later testing [5], could be streamlined for screening CMV at scale with minimal additional costs and no additional collection processes. However, whether ORAcollect can detect viral CMV DNA is unknown. In this pilot study at the Royal Children’s Hospital (RCH), Victoria, Australia, we aimed to determine the sensitivity, specificity, percent agreement and Cohen’s kappa of CMV DNA detection using saliva collected by the ORACollect, compared to the gold standard FLOQswabs^®.

The RCH Electronic Medical Records (EPIC) and the Department of Microbiology’s Laboratory Information System (Medipath) databases were audited during July-August 2021 to identify newborns/children under 18 years old who had either: 1) tested positive for CMV on blood/urine/saliva, or 2) attended an RCH clinic for an appointment. This approach aimed to recruit newborns/children with saliva samples that were likely positive or likely negative for CMV. Families of eligible newborns/children were approached via their treating clinicians who obtained parental consent to be contacted by the study team. The study team contacted interested families to describe the study and obtain consent. Participating parents signed a consent form, and saliva was collected from their child (by the research assistant or the parent following verbal/written guidance) by rubbing each of the four swabs over their child’s gums against each side of their mouth 10 times, without touching the teeth [6].

ORAcollect swabs were stored at room temperature, and FLOQswabs^® at -30 ℃, until analysis. Before PCR analysis, saliva samples underwent DNA extraction using commercially available kits for either viral (Roche MagNA Pure 96 DNA and viral NA small volume kit) or human genomic (QIAsymphony SP with integrated assay) DNA extraction. This allowed assessment of the impact of the DNA isolation approach on CMV detection sensitivity and specificity. One FLOQswab^® was kept in storage for future use. All PCR tests were completed by the RCH Microbiology Laboratory using a certified clinical CMV diagnostic pipeline. Sensitivity is reported as the proportion of samples positive for CMV on FLOQswabs^® that were also positive on ORACollect and specificity as the proportion of samples negative for CMV on FLOQswabs^® that were also negative on ORACollect. Percent agreement between FLOQswab^® and ORACollect results are reported alongside Cohen’s kappa statistic to quantify the interrater agreement between CMV PCR test results from different saliva collection kits. Sensitivity, specificity, agreement and Cohen’s kappa statistic are reported for both viral and human DNA extraction starting material.

All processes were approved by the RCH Human Research and Ethics Department prior to research commencing (HREC: 77000).

During July-August 2021, the families of 14 newborns/children, of whom 2 had recently been diagnosed with congenital CMV and 9 had recently tested positive for CMV, were approached. Fourteen participants (age range 1 month to 17 years) provided 15 sets of saliva samples. Twenty percent (3/15) of the saliva samples collected were from infants with congenital CMV diagnosed or confirmed at Melbourne’s RCH. CMV PCR results are summarised in Table 1, with further detail including CMV Ct values available in Supplementary Table 1. After DNA extraction, DNA was amplified on all ORAcollect and all FLOQswabs^® except for the sample for Participant 4. Participant 4’s FLOQswab^® internal control (beta-globin) was negative following initial DNA extraction and follow-up re-extraction. The Laboratory concluded the result of this specimen could not be interpreted due to inadequate specimen. Therefore, Participant 4 was excluded from sensitivity, specificity, percent agreement, and Cohen’s kappa statistic analysis. Of the remaining participants, 9 had CMV DNA detected and 5 had no CMV DNA detected.

ORACollect combined with viral DNA extraction successfully detected CMV using PCR with 78% sensitivity (7/9 CMV positive on ORACollect), 80% specificity (4/5 CMV negative on ORACollect) and 79% agreement between PCR results from FLOQswab^® and ORACollect-derived samples. Cohen’s kappa indicated moderate agreement in PCR results (k = 0.55 ). ORACollect combined with human DNA extraction successfully detected CMV using PCR with 89% sensitivity (8/9 CMV positive on ORACollect), 80% specificity (4/5 CMV negative on ORACollect) and 86% agreement on PCR results from FLOQswab^® and ORACollect-derived samples. Cohen’s kappa indicated substantial agreement in PCR results (k = 0.69).

This pilot study is the first to demonstrate the utility of ORACollect, designed for the isolation of human genomic DNA, for the detection of CMV in saliva. DNA isolated from ORACollect stored at room temperature have high sensitivity (up to 89%), specificity (up to 80%), and percent agreement (up to 86%) in detecting CMV DNA when compared to FLOQswabs^® which were stored at –30 ℃ following collection. We collected multiple real-life samples from newborns and children. For ORACollect, we performed both viral DNA extraction and human genomic DNA extraction prior to PCR.

There were some limitations. We were only able to collect 3 samples (20% of samples) from infants diagnosed with congenital CMV. However, the majority of samples (12 of 15, 80%) were collected when participants were aged two years or less. It is possible that collecting multiple saliva samples may have led to insufficient DNA or saliva in the FLOQswabs^® collected after ORACollect in our protocol. This is likely to have been the case for Participant 4 where no DNA was amplified from the FLOQswab^®.

Despite these considerations, results from this pilot study suggest that ORACollect may be an appropriate alternative to clinically-utilised FLOQswabs^® for collecting viral CMV DNA for CMV PCR testing, independent of the DNA extraction approach. These results should be confirmed in a larger sample prior to the ORACollect being used clinically.

The use of ORACollect could have immense utility for large-scale screening in newborns with hearing loss, as a single sample, collected for genomic testing, could also be utilised for CMV testing. The potential to use ORACollect to test for CMV also expands the settings in which CMV screening may be applicable, with two months of room temperature storage appropriate for ORAcollect after sample collection, enabling non-refrigerated transport to a PCR-enabled laboratory.

The authors thank study participants and their families for contributing their time to this research.

Support statement

This pilot study did not receive any funding. Research at the Murdoch Children’s Research Institute is supported by the Victorian Government’s Operational Infrastructure Support Programme. A/Prof Sung was supported by a Melbourne Children’s Clinician Scientist Fellowship 2021. A/Prof Sung and Dr. Gillespie were funded by a National Health and Medical Research Council (NHMRC). Clinical Trials and Cohort Studies grant (2006491). Prof Wake was supported by an NHMRC Principal Research Fellowship (1160906). 

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