Background HIV-1 drug resistance (HIVDR) genotyping commonly requires PCR amplification before sequencing, with agarose gel electrophoresis used to assess amplification success. We investigated whether PCR products without visible gel bands could nevertheless support Nanopore sequencing and HIVDR profiling. Methods Thirteen protease/reverse transcriptase (PR/RT) products that had previously generated successful Sanger sequences subsequently produced no visible band following repeat PCR. These products were sequenced using Oxford Nanopore and consensus sequences and HIVDR profiles were compared with matched Sanger HIVDR profiles. Findings Nanopore sequencing generated HIV-1 PR/RT sequence from all 13 products. Across 286 drug–sample comparisons, Nanopore and Sanger HIVdb classifications agreed on susceptible versus resistant status in 74.5% (213/286), with exact agreement across the five HIVdb resistance levels in 68.5% (196/286). Discordance was directional where Nanopore detected only 53% (64/120) of Sanger-defined resistance yet rarely called resistance where Sanger did not (specificity 90%, 149/166), so errors were predominantly undercalls (56 versus 17 overcalls). Median Nanopore read quality was positively associated with Nanopore–Sanger consensus sequence identity (r = 0.67, p = 0.012), whereas read number was not significantly associated (r = 0.47, p = 0.11). Conclusion PCR products without visible agarose gel bands can contain sufficient HIV-1 material for Nanopore sequencing, but sequence recovery does not necessarily ensure reliable HIVDR profiling. Resistance was frequently undercalled in poorly performing samples, potentially reflecting differences in sequence quality, amplification and consensus generation. Nanopore sequencing may provide a rescue strategy for selected low-yield PCR products, but larger studies are needed to establish quality criteria for reliable resistance interpretation.
Background Congenital cytomegalovirus (cCMV) is typically diagnosed using nucleic acid amplification testing on saliva followed by urine confirmation if positive. Major challenges in saliva screening include low positive predictive value (PPV) and throughput constraints. We validated the cobas CMV assay on the cobas 8800 high-throughput real-time polymerase chain reaction (PCR) platform as a lab-developed test for newborn saliva swabs and urine, and evaluated its clinical performance characteristics for cCMV screening. Methods From April 2023 to January 2026, saliva swabs and urine from infants ≤ 21 days were tested in a cCMV screening program. Qualitative results were reported, with viral loads recorded offline. Urine PCR served as the reference standard to classify saliva results. Results CMV PCR was positive in 50/6559 (0.76%) saliva swabs meeting study inclusion criteria, with 29 (58%) confirmed by urine. Among 28 saliva-negative infants who had urine testing, all were negative. Median saliva viral load was significantly lower in false-positives than true-positives (below lower limit of quantitation at <35 IU/mL vs. 4.09 × 106 IU/mL, p < 0.001). Using urine PCR as the gold standard, saliva PCR performance varied by reporting threshold (from detected at any value up to 100 IU/mL) with sensitivity 93.1–100%, specificity 57.1–100%, PPV 58.0–100%, and negative predictive value (NPV) 96.1–100%. Conclusion The cobas CMV assay as validated in our laboratory demonstrated acceptable clinical performance characteristics for saliva-based cCMV screening. Clinical specificity and PPV are further improved through application of a viral load threshold for positivity reporting or an “equivocal” range.
Background The diagnostic value of upper respiratory tract (URT) viral testing as a surrogate for lower respiratory tract (LRT) viral detection in mechanically ventilated patients remains uncertain. We evaluated URT/LRT concordance in intubated intensive care unit (ICU) patients and identified factors associated with non-concordance. Methods We retrospectively studied adult and pediatric ICU patients receiving invasive mechanical ventilation who underwent paired URT and LRT respiratory viral polymerase chain reaction (PCR) testing between January 2014 and January 2025. Concordance was defined as detection of at least one identical virus in both specimens or negative results in both. Multivariable logistic regression identified factors associated with concordance. A simplified bedside prediction score was developed to predict non-concordance. Results Among 112 patients, 70 (62.5%) had concordant results. Specific positive agreement varied by virus: human metapneumovirus (88.9%), respiratory syncytial virus (80.0%), rhinovirus/enterovirus (75.0%), influenza virus (68.4%), adenovirus (34.8%), and human bocavirus (0.0%). A longer symptom-to-URT testing interval (adjusted odds ratio [aOR], 0.74 per day; 95% confidence interval [CI], 0.62–0.87) and chronic lung disease (aOR, 0.22; 95% CI, 0.06–0.80) were associated with lower concordance, whereas positive end-expiratory pressure > 5 cmH₂O was associated with higher concordance (aOR, 2.85; 95% CI, 1.07–7.59). The bedside score showed good discrimination for non-concordance (area under the receiver operating characteristic curve, 0.818). Conclusions URT/LRT concordance was moderate and varied by symptom timing, clinical factors, and viral pathogen. LRT testing may provide additional diagnostic value in patients at high risk of non-concordance. The bedside score may help identify these patients but requires further internal and external validation.
Objectives Rotavirus is a major cause of gastroenteritis in children worldwide. Denmark has no routine rotavirus vaccination programme. We aimed to describe national trends in rotavirus epidemiology and genotype distribution from 2014 to 2024, including changes during and after the COVID-19 pandemic. Methods Data on all diagnostic rotavirus analyses performed in Denmark between 2014 and 2024 were obtained from the Danish Microbiology Database. A subset of rotavirus-positive samples was submitted to the national reference laboratory for genotyping and linked with epidemiological data. Results A total of 18,246 rotavirus episodes were identified, and 1800 samples were selected for genotyping. The number of episodes declined during the COVID-19 restrictions in 2020–2021, followed by a substantial increase in 2022. The median age of infected patients increased from 20 to 27 months before the pandemic to 32 months in 2022. Test positivity returned to pre-pandemic levels in 2023–2024, whereas incidence remained elevated. Genotype distribution shifted over time, with G1P[8] dominating in 2014–2015, G3P[8] in 2016–2017 and 2022–2024, and G9P[8] in 2019–2021. Reinfections were uncommon. Conclusions Rotavirus epidemiology in Denmark changed substantially during and after the COVID-19 pandemic, with changes in disease incidence, age distribution, and circulating genotypes. Continued surveillance remains important to monitor epidemiological trends.
