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

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.

Performance evaluation of fully automated cobas® 6800 CMV PCR for the detection and quantification of cytomegalovirus DNA in neonatal urine and saliva, and adult urine, saliva, and vaginal secretion.

Laboratory testing for cytomegalovirus (CMV) in bodily fluids is essential to manage congenital and prenatal CMV infection. The rapid and fully automated cobas® CMV PCR is approved only for the testing of plasma in transplant patients. To evaluate the performance of the cobas® CMV to detect and quantify CMV DNA in neonatal and adult female urine, saliva, and vaginal secretion, the limit of detection (LoD), limit of quantification (LoQ), imprecision, linearity, PCR efficiency, bias, analytical specificity, cross-reactivity, and cross-contamination of the cobas® CMV for urine, saliva, and vaginal secretion was determined. The performance of the assay was evaluated prospectively with two laboratory-developed PCR assays using neonatal and adult urine, saliva swabs, and vaginal swabs. The LoD and LoQ were 31 and 100 IU/mL, respectively, for urine, and 81 and 100 IU/mL, respectively, for vaginal secretion. The LoD and LoQ for saliva were the same (200 IU/mL). The cobas® CMV was precise (coefficient of variation ≤10%), linear (R2 ≥ 0.995), and efficient (1.07 and 1.09) between 100 and 250,000 IU/mL for the sample types. The bias and analytical specificity was <±0.30 log10 IU/mL and 100%, respectively. Cross-reactivity with non-CMV pathogens was not detected. Cross-contamination rate was 0.28%. The diagnostic accuracy, sensitivity, and specificity of the cobas® CMV for neonatal urine and saliva were ≥95.0%, ≥93.3%, and ≥90.4%, respectively. The overall percent agreement for adult urine, saliva, and vaginal secretion was 86.6%, 94.5%, and 89.4%, respectively. Taken together, the cobas® CMV demonstrated acceptable analytical and diagnostic performance, and is suitable for routine diagnostic laboratory investigation of CMV infection in neonates and adults.

Performance evaluation of the Viasure PCR assay for the diagnosis of monkeypox: A multicentre study.

BACKGROUND: Monkeypox virus (MPXV) is the causative agent of the 2022 monkeypox global outbreak. Rapid detection of MPXV infection is essential to inform patient management and public health response. Currently, there is a lack of established real-time PCR assays to support a rapid diagnosis of monkeypox. OBJECTIVES: To evaluate the performance characteristics of the Viasure MPXV PCR assay in three London teaching hospitals. STUDY DESIGN: Prospectively collected paired patient swabs from matched or unmatched anatomical sites were evaluated by the reference laboratory and Viasure MPXV PCR assays. A subset of samples were also tested for HSV, VZV, and/or Treponema pallidum DNA. RESULTS: 217 paired samples were evaluated. 91.2% of the paired swabs generated concordant results whilst 8.8% generated discordant results. The accuracy, diagnostic sensitivity, diagnostic specificity, positive predictive value, negative predictive value, likelihood ratio positive, and likelihood ratio negative of the Viasure PCR assay across the hospitals were 93.2 - 96.3%, 90.0 - 100%, 88.2 - 100%, 94.9 - 100%, 87.9 - 100%, 8.50 - 14.41, and 0.00 - 0.10 respectively. MPXV co-infections with HSV were detected in two patients. Five patients were negative for monkeypox but positive for herpes or chickenpox. CONCLUSIONS: The Viasure MPXV PCR assay demonstrated excellent performance characteristics, was easy to use, and is fit for routine diagnostic purpose. Where implemented, the assay would allow rapid and accurate laboratory diagnosis of MPXV infections and support a timely management of monkeypox. To reduce the risk of false negative detections, vesicular lesions from any anatomical site should be preferentially and optimally sampled.

Screening for cytomegalovirus shedding in vagina and saliva: Significant differences between biological fluids, swab types and storage durations in DNA recovery.

BACKGROUND: Cytomegalovirus (CMV) shedding in genital and oral secretions during pregnancy is associated with adverse outcomes. Sample collection methods between studies are not uniform and currently there are limited data on the impact of biological fluids, swab types and storage durations on the detection of CMV DNA. OBJECTIVES: To evaluate the absorption efficiency and CMV DNA recovery of various commercially available swabs in vaginal and saliva fluids. STUDY DESIGN: The absorption volume of different swab types was evaluated. The recovery of CMV DNA over time from vaginal and saliva fluids, and vaginal and saliva swabs, was evaluated by PCR measurements of samples spiked with CMV standard. RESULTS: Absorption efficiency of swabs varied significantly. The duration of storage did not affect CMV DNA recovery from vaginal fluid or swabs, but did significantly affect CMV DNA recovery from neat saliva fluid and saliva swabs stored dry or in viral transport media (VTM). Flocked swab/eNAT media recovered the highest amount of CMV DNA from both fluids. In saliva, flocked swab/eNAT media and polyester swab/cobas media demonstrated a higher CMV DNA recovery than foam swab/VTM. 25% of dry saliva foam swabs were falsely negative for CMV DNA. CONCLUSIONS: Recovery of CMV DNA is dependent on sample type and swab type used. Flocked swab/eNAT media and polyester swab/cobas media appear acceptable, though flocked swab/eNAT media was superior demonstrating the best recovery of CMV DNA. For saliva, foam swabs stored dry or in VTM was shown to be inferior to flocked or polyester swabs.

Verification of the Roche cobas® 6800 PCR 200 µl and 500 µl protocols for the quantification of HIV-1 RNA, HBV DNA and HCV RNA and evaluation with COBAS® Ampliprep/COBAS® TaqMan® assays.

PURPOSE: The analytical performance of the cobas 6800 HIV-1, HBV and HCV assays was verified and evaluated to the COBAS Ampliprep/COBAS TaqMan assays. METHODOLOGY: The precision, limit of detection (LoD), limit of quantification (LoQ) and linearity were verified using pooled residual clinical samples. The analytical specificity was verified with negative plasma. HIV-1 analytical reactivity was verified with WHO reference preparations. Accuracy was verified using EQA plasma panels. Evaluation of the equipment was performed using prospectively collected clinical whole blood samples. RESULTS: Excellent precision was demonstrated using both testing protocols (coefficient of variation ≤15 %). The LoDs using the 500 and 200 µl protocols were 20 and 50 cp ml-1 for HIV-1, 10 and 20 IU ml-1 for HBV and 15 and 40 IU ml-1 for HCV, respectively. The LoQs were 40 and 100 cp ml-1 for HIV-1, 20 and 25 IU ml-1 for HBV and 30 and 80 IU ml-1 for HCV, respectively. Assays demonstrated good linearity (R2 ≥0.96). The analytical specificity of the assays was 100 %. There was excellent agreement between the cobas 6800 and CAP/CTM assays (kappa>0.94). The sensitivity, specificity, positive predictive value and negative predictive value for each of the assays were ≥99 %. The cobas HIV-1 and HCV mean quantifications were 0.03 log10 cp ml-1 and 0.17 log10 IU ml-1 higher than the CAP/CTM. The cobas HBV mean quantification was 0.17 log10 IU ml-1 lower than the CAP/CTM. Subtype/genotype specific differences were not observed. CONCLUSION: Cobas 6800 equipment and assays demonstrated excellent performance and correlated well with CAP/CTM assay results.