Respiratory syncytial virus (RSV) is a leading cause of pediatric respiratory infections. This systematic review and meta-analysis aims to estimate the in-hospital positivity among children in Europe post-COVID-19 and prior to the implementation of the recent RSV prophylactic strategies. A systematic search was conducted across three databases: PubMed, Scopus and Web of Science from 01/01/2021-31/12/2025. Studies enrolling children aged 0-18 years who were hospitalized with respiratory infection and detected positive for RSV were eligible for inclusion. A random-effect model was applied and heterogeneity was assessed using I2 statistics. Thirty-eight studies met the inclusion criteria, encompasing 66,065 children. The overall pooled RSV in-hospital positivity was 48% (95%CI: 41%-56%). Positivity estimates per year were: 53% (95%CI:49%-56%) for 2021-2022, 49% (95%CI:36%-62%) for 2022-2023, and 62% (95%CI:51%-73%) for 2023-2024 (p = 0.308). RSV positivity in studies with infants (<12-months-old) was 69% vs 30% in studies including older children (0-18 years). Among RSV-positive patients, co-infections were detected in 17%, respiratory support was required in 62% and ICU/NICU admission in 11%. RSV-B predominated over RSV-A during the study period. A substantial burden of RSV-related hospitalizations was detected, especially in infants < 12-months-old; however, the overall pooled positivity estimate should be interpreted with caution. These findings provide an important pre-immunization baseline for evaluating the impact of maternal RSV vaccination and monoclonal antibodies and support the need for continued RSV surveillance across Europe.
BACKGROUND:Rotavirus vaccine has greatly decreased rotavirus illness since being introduced in 2006 in the United States. Post vaccine introduction, enzyme immunoassay (EIA) rotavirus testing false positivity rates rose, and reverse transcription-polymerase chain reaction (RT-PCR) testing became prominent. RT-PCR testing is highly sensitive and can detect low quantities of viral shedding that could be clinically irrelevant. OBJECTIVES:We compared RT-PCR cycle threshold (Ct) values to EIA tests to bridge EIA and RT-PCR testing results. METHODS:Stool specimens from 2325 children with acute gastroenteritis (AGE) and 544 healthy controls (HCs) enrolled in the New Vaccine Surveillance Network during 2011-16 were tested for rotavirus via both EIA and semiquantitative RT-PCR (qRT-PCR) to compare rotavirus vaccine effectiveness (VE) and calculate Youden's index (sensitivity + specificity - 1) for various Ct cutoffs. RESULTS:The Youden's index indicated a Ct value < 29 was optimal for comparing EIA and qRT-PCR rotavirus testing; Ct value cutoffs ranging from < 27 to < 29 produced comparable sensitivity, specificity, and rotavirus VE estimates. Ct values were lower among AGE children than HCs. Rotavirus VE ranged from 85.5% among participants positive via both EIA and qRT-PCR (Ct < 40) to 59.4% among participants with a Ct value ≥ 29. CONCLUSIONS:A qRT-PCR Ct value cutoff of < 27 to < 29 was most comparable to EIA for rotavirus VE estimates. The Youden's index indicated a Ct value < 29 was the best single cutoff for rotavirus test positivity to provide an accurate estimation of clinically relevant rotavirus cases.
Cytomegalovirus (CMV) infection after kidney transplantation is associated with poorer patient and graft outcomes. Nearly half of CMV seronegative kidney transplant recipients (KTR) with a CMV seropositive donor (D+R-) experience CMV infection. We hypothesized that the anti-CMV immunoglobulin G (IgG) titer of CMV seropositive donors is a risk factor for CMV infection in D+R- kidney transplantations, since higher titers may reflect more intense prior CMV viremia or impaired cell-mediated CMV control in donors. Anti-CMV IgG titers were measured using an enzymatic immunoassay. CMV DNAemia was defined as a positive CMV polymerase chain reaction (PCR), seroconversion as a positive anti-CMV IgG in a previously seronegative recipient. Cox regression analysis was performed to evaluate the risk of CMV infection. A total of 111 D+R- KTR with a living donor from the TransplantLines Study were included at 6 months post-transplantation. The mean age was 49.6 ± 15.0 years and 32 (29%) were female. Overall, 31 (28%) developed CMV DNAemia and 35 (32%) seroconverted. Recipients with donors with an anti-CMV IgG titer above 250 AU/mL (n = 19; 17%) had a significantly higher risk of CMV DNAemia (HR=2.49, 95% CI (1.15–5.42), p = 0.02) and CMV seroconversion (HR=3.07 (1.49–6.32), p = 0.002). High donor anti-CMV IgG titer is associated with an increased risk of CMV infection in CMV seronegative KTR. These findings support the hypothesis that a high anti-CMV IgG titer reflects a higher CMV tissue load, a poorer cell-mediated CMV response, or both. Donor anti-CMV IgG titer might be a useful parameter to guide individualized CMV prophylaxis and surveillance strategies.
BACKGROUND:Forkhead box protein A1 (FOXA1) is linked to cancer development in different anatomical sites. We previously demonstrated that FOXA1 markedly increases high-risk human papillomavirus (HPV) transcription by directly binding to the long control region (LCR). Herein, we aimed to assess FOXA1 expression and its prognostic significance in cervical cancer (CC). METHODS:Patients with CC treated at the Instituto do Cancer do Estado de São Paulo (ICESP), Brazil, were included. FOXA1 levels were measured using immunohistochemistry (IHC). RESULTS:Overall, FOXA1 staining was observed in 85% (208/245) of the samples, primarily localized in both the nucleus and cytoplasm (71%). High FOXA1 staining was more frequently detected in squamous cell carcinoma (SCC) compared to adenocarcinomas (ADC) (p = 0.003). High FOXA1 staining was significantly associated with HPV-positive CC (p = 0.005). It was also associated with worse overall survival (OS) and disease-free survival (DFS) when considering all CC samples, as well as when restricting the analysis to SCC cases. Among HPV-positive cases, high FOXA1 staining was also significantly associated with poorer OS and DFS in women with SCC. Finally, high FOXA1 staining was associated with poor OS (HR 1.79; 95% CI: 1.14-2.79; p = 0.011) and poor DFS (HR: 1.64; 95% CI: 0.98-2.74; p = 0.056) among SCC cases in comparison to cases with low/moderate staining. No association was found between FOXA1 levels and prognosis in ADC. CONCLUSIONS:Our data suggest that FOXA1 may be involved in HPV-induced carcinogenesis and could serve as a prognostic biomarker in patients with cervical SCC.
BACKGROUND AND OBJECTIVES:Orthohantaviruses are negative-sense RNA viruses that persistently infect rodents and can cause severe human disease. Orthohantavirus diversity complicates comprehensive surveillance strategies and diagnostic tools. Currently, serological assays and nested PCR with degenerate primers are used for hantavirus diagnosis and rodent surveillance, but these assays have long turnaround-times. Here, a broadly reactive Orthohantavirus real-time PCR assay is presented that can be incorporated into public health and research laboratories across the Americas. STUDY DESIGN:Starting from an existing rRT-PCR primer-probe set originally developed for the detection of Sin Nombre virus, a pan-America Orthohantavirus assay (HantAmeriQ) was developed that can be used for both human and rodent surveillance. RESULTS:Using synthetic RNA, the HantAmeriQ assay detected all New World Orthohantaviruses known to cause human disease at a limit of detection between 25 (Blue River virus) and 28,000 (Bayou virus) copies/reaction. Genetically diverse hantaviruses from North America (including Sin Nombre, New York, Monongahela, Blue River, and Bayou viruses) and Central/South America (including Catacamas, Carrizal, Choclo, Maciel, and Andes viruses) were detected within similar limits. The HantAmeriQ assay performed as well as or better than a commercially available assay for the detection of North American, but not South American hantaviruses and detected diverse hantaviruses from rodent and human specimens. CONCLUSIONS:Here, the HantAmeriQ assay detected New World Orthohantaviruses in both rodent and human samples from across the United States, and hantaviruses from Central and South America. The assay demonstrates strong potential for diagnostic use in North American clinical laboratories and for surveillance and research applications in rodent-centered field studies.
BACKGROUND:The non-pathogenic Torque Teno Virus (TTV) is currently being investigated as a functional biomarker for the degree of immunosuppression. A reliable assay for quantification of TTV is extremely important. The primary objective of this study was to assess the reliability and agreement between two quantitative methods, an in-house PCR assay and the commercial TTV R-GENE, for the detection of TTV viral loads. The secondary objectives included testing of the in-house assay by performing external quality assessment (EQA) and assessing the TTV prevalence in 101 Danish blood donors. METHODS:Parallel TTV viral load quantification by both PCR assays was conducted in samples from heart transplant recipients, kidney transplant recipients, and patients with atopic dermatitis. Bland-Altman statistics and Intraclass Correlation Coefficient (ICC) were implemented for the evaluation of agreement and reliability between assays. Additionally, 12 EQA samples and samples from 101 blood donors were analyzed applying the in-house assay. RESULTS:Qualitative comparison revealed a high concordance (80.6%) between assays. Bland-Altman statistics demonstrated a mean difference of 0.72 Log10 copies/mL. with the highest viral loads obtained with the in-house assay. Reliability, defined with an ICC of 0.82, was considered good. All EQA samples were acceptable. The prevalence of TTV in Danish blood donors was 60.4%. CONCLUSION:This study demonstrated qualitative concordance, and moderate agreement and reliability between an in-house assay and the commercial TTV R-GENE kit. The in-house assay obtained acceptable results within the EQA, suggesting that the in-house assay is suitable for quantification of TTV viral loads in clinical practice.
BACKGROUND:T-cell responses are important for controlling viral infections, but underutilized in routine diagnostics. In recent years, whole-blood interferon-gamma release assays (IGRAs) have emerged as a simple and reliable method for detecting virus-specific T-cells, making them well suited for clinical use. Monitoring of Epstein-Barr virus (EBV) infection is clinically important in transplant recipients and EBV-associated conditions such as Multiple Sclerosis (MS). However, serological assessment may be confounded by disease-modifying treatments, making T-cell-based assays a robust approach for determining EBV-specific immune status. OBJECTIVE:To develop a whole-blood IGRA for the detection of EBV-specific T-cells and to apply it in a clinical context i.e. in patients with MS (pwMS). METHODS:Blood samples from 50 healthy individuals (HI) and 20 pwMS were stimulated with EBNA1 and one self-designed pool (EIHM). IFNγ levels were measured via ELISA and compared to anti-VCA IgG serostatus. RESULTS:The IGRA reliably detected EBV-specific T-cell responses in HI with prior EBV infection using the EBNA1 and EIHM pools. Anti-VCA IgG-positive HI showed significantly higher IFNγ level than EBV-seronegative HI (p < 0.0001), and these responses correlated with anti-VCA IgG levels (EBNA1: p < 0.0001, rs = 0.54; EIHM: p < 0.0001, rs = 0.72). In pwMS, EBV-specific T-cell responses were detectable before therapy and tended to be higher than in HI. After anti-CD20 treatment, EBV-specific IFNγ responses declined significantly (EBNA1: p = 0.0020; EIHM: p = 0.0078). CONCLUSION:The EBV IGRA reliably detects EBV-specific T-cells in seropositive individuals and enables monitoring of cellular immunity in clinical settings.
BACKGROUND:Hepatitis E virus (HEV) infection is the leading cause of acute viral hepatitis globally. OBJECTIVES:We compared the performance of the two fully-automated anti-HEV IgM and IgG assays available: The LIAISON XL murex assay (DiaSorin) and the newly introduced Elecsys® Anti-HEV IgG and Elecsys Anti-HEV IgM assays (Roche Diagnostics). STUDY DESIGN:The study included n = 1808 samples tested across three testing sites. Overall percentage agreement (OPA), relative sensitivity, and specificity were calculated using a multi-comparator approach with CE-IVD-marked assays (recomWell, Wantai, and Vidas) as reference. RESULTS:OPA between Murex and Elecsys assays was 97.9% (95%-CI: 97.1-98.5%) and 93.9% (95%-CI: 92.6-95.0) for the detection of anti-HEV IgM and IgG, respectively, and kappa (κ) was κ= 0.94 (95%-CI: 0.92-0.96) and κ= 0.86 (95%-CI: 0.85-0.90). Using clinical samples from HEV RNA-positive (n = 216), acute (n = 338), and resolved infections (n = 156), relative sensitivity was determined as > 95.7% for the Murex and > 98.6% for the Elecsys assay. Relative specificity was > 98.8% for the Murex and > 89.7% for the Elecsys assays, as determined using samples from asymptomatic blood donors (n = 500), routine diagnostic samples with clinical suspicion of viral hepatitis (n = 498), and pregnant women (n = 100). Of n = 47 anti-HEV IgG Elecsys-only reactive samples, 95.7% (n = 45/47) were confirmed by neutralization, increasing relative specificity to 98.8%. CONCLUSION:Both assays demonstrated high agreement across comprehensive clinical cohorts. Notably, the Elecsys Anti-HEV IgG assay showed higher sensitivity for anti-HEV-IgG detection, which may have implications for immunity estimates and seroprevalence studies. Overall, both assays appear well suited to meet the growing demand for HEV diagnostics.
BACKGROUND:Dried blood spot (DBS) testing enables hepatitis C virus (HCV) testing in hard‑to‑reach patient populations but typically lacks full genotype and drug resistance information. This study evaluates a commercially available hybrid‑capture whole‑genome sequencing (WGS) workflow for HCV from DBS to determine suitability for clinical diagnostic and surveillance purposes. METHODS:We analysed 147 DBS and 24 plasma samples known to be HCV‑RNA positive using the QIAGEN QIAseq xHYB HepC panel. Whole genome sequences were generated using an in-house custom designed iterative bioinformatic pipeline. We assessed coverage characteristics, genotyping accuracy, resistance‑associated mutation (RAM) detection, and analytical sensitivity to determine panel performance. RESULTS:WGS success rates were high for both DBS (95%) and plasma (96%). Genome enrichment was uniform across major HCV genotypes, with no systematic dropouts. Genotyping showed 100% concordance with reference laboratory results including for novel genotype 1 subtypes. RAMs were detected in 27% of samples assessed, with full concordance with reference laboratory results in those with known resistance profiles. Analytical sensitivity was calculated at < 7301 IU/ml for WGS with slightly poorer performance when assessing the NS5a gene in isolation. CONCLUSION:This protocol reliably generates high‑quality HCV genomes from DBS, enabling accurate genotype, subtype, and resistance profiling. This approach overcomes limitations of venous sampling in hard-to-reach populations and could support decentralised diagnostics and enhancing genomic surveillance in populations underserved by traditional healthcare pathways.
BACKGROUND:Graft rejection and opportunistic infections are among the main causes of graft dysfunction and loss in renal transplant recipients. Torque Teno Virus (TTV) is a highly prevalent, non-pathogenic virus that has been postulated as an immunosuppression biomarker in renal transplant patients. This study aimed to develop predictive models for these adverse events based on TTV viremia. METHODS:From 130 patients, 871 plasmas were tested for TTV viral load. Patients' follow-up was documented up to 3 years post-transplantation. To develop predictive logistic regression models, ROC curve analyses were performed. The basal model analyzed only TTV viral loads while the enhanced model analyzed TTV viral load and patients' variables. RESULTS:TTV prevalence by real time PCR was 81.2% pre-transplantation and 97.6% at month 3. The global rejection rate -clinical and subclinical rejection- was 36.6% and infections rate was 55.4%. The basal models showed areas under the curve (AUC) of 0.595 and 0.662 for rejection and infection prediction, respectively. In the enhanced models, AUCs increased to 0.781 and 0.765, respectively. Sensitivity and specificity with the enhanced models were 61.1% and 81.8% for graft rejection prediction; and 81.8% and 68.7% for infection prediction. NPV were 97.7% and 94.3% respectively. Internal validation of the enhanced models yielded comparable AUCs. CONCLUSIONS:Enhanced models showed high probabilities to predict graft rejection and opportunistic infections in renal transplant patients. Incorporating these tools in the clinical setting can better identify patients at risk of developing adverse events. All statistical parameters improved with the enhanced model, achieving a very high NPV. Internal validation confirmed the robustness of these results.
BACKGROUND:After the COVID-19 pandemic, enterovirus circulation rebounded, but enteroviral meningitis remained low in South Korea, suggesting that viral activity alone may not reflect disease burden. METHODS:We analyzed hospital-based and national surveillance data from 2015 to 2025 across the pre-pandemic period (2015-2019), pandemic period (2020-2021), and post-pandemic period (2022-2025). A Kitagawa-type decomposition was applied to quantify the relative contributions of changes in circulating type distribution and type-specific risk. RESULTS:In the post-pandemic period, despite partial recovery in enterovirus activity (IRR 0.379-0.637), meningitis incidence remained markedly low (IRR 0.060-0.077). The proportion of enteroviral meningitis among aseptic meningitis declined from 72.3% to 24.4%. This discordance was accompanied by a shift in circulating types, with echoviruses declining from 40.3% to 1.3% and coxsackievirus A increasing from 34.2% to 93.8% (P < 0.001). The overall decline in meningitis (-60.7 %age points) was explained by changes in type distribution (-27.9 %age points) and type-specific risk (-32.8 %age points), with echoviruses contributing the largest share (58.8%). CONCLUSIONS:In South Korea, post-pandemic enterovirus epidemiology showed a discordance between overall enterovirus incidence and meningitis burden. This pattern was driven by shifts in circulating type distribution and type-specific risk, indicating that meningitis burden cannot be inferred from overall enterovirus incidence alone.
BACKGROUND:Detection and quantification of hepatitis D virus RNA is essential for diagnosis and treatment of chronic hepatitis D virus (HDV) infection. This study evaluated the diagnostic performance of the Altostar HDV RT-PCR Kit 1.5 in real-world clinical samples with and without treatment with bulevirtide (BLV). METHODS:Plasma samples of 40 patients with chronic HDV infection were analyzed with the Altostar HDV RT-PCR Kit 1.5 and compared to the local standard, the RoboGene HDV RNA Quantification Kit 2.0, with additional analyses of corresponding serum and plasma samples and on-treatment samples during BLV therapy. RESULTS:HDV RNA levels in plasma samples quantified by both assays showed a significant positive correlation. All samples were quantifiable by Altostar 1.5, whereas two samples were undetectable by Robogene 2.0 During BLV therapy, mean baseline (BL) and follow-up (FU) HDV RNA levels were comparable between the two assays. There was a similar proportion of patients with virological response (Altostar 1.5: 75% [n = 15/20] vs. Robogene 2.0: 70% [n = 14/20]), whereas the proportion of samples with undetectable HDV RNA at FU was considerably higher in measurements with Robogene 1.5 (40% vs. 0%). CONCLUSIONS:HDV RNA levels quantified by the Altostar HDV RT-PCR Kit 1.5 were comparable to the local standard. The higher number of detectable samples may reflect increased sensitivity, occasional false-positive results with Altostar 1.5, or the impact of automated extraction in the Robogene workflow. Importantly, treatment response during BLV treatment was not influenced by the type of quantification assay.
INTRODUCTION:Laboratory developed tests (LDTs) are high-complexity tests developed by individual laboratories. LDTs have been critical for addressing clinical needs not available commercially. Automation facilitates workflow efficiency and expansion of testing menus. At the Johns Hopkins Medical Microbiology Laboratory, different automation solutions were evaluated for molecular LDTs to transition manual real-time PCR diagnostics to automated platforms. This study evaluated the performance of the Panther Fusion Open Access platform, with a focus on assays developed for viral infections of the central nervous system (CNS). METHODS:Molecular assays for HSV-1, HSV-2, VZV, enterovirus, and parechovirus were developed on the Panther Fusion platform using Open Access. Analytical studies included determination of sensitivity, specificity, inclusivity, onboard reagent stability, and agreement. Clinical evaluations were performed and compared to standard-of-care assays, including the BioFire Meningitis/Encephalitis panel and DiaSorin Simplexa HSV-1&2 and VZV Direct assays. RESULTS:Analytical sensitivities were comparable or better than standard-of-care tests. A blind panel of 297 unique samples demonstrated 100% agreement and reproducibility. Among 101 clinical samples tested retrospectively and prospectively, the developed viral assays showed 100% positive and 98.8% negative agreement with comparator assays. Patients with positive viral results were more frequently treated with antivirals rather than antibiotics, had final diagnoses of viral meningitis or encephalitis, and exhibited significantly higher cerebrospinal fluid (CSF) mononuclear cell counts, supporting the clinical validity of developed and comparator assays. CONCLUSION:Assays developed on the Panther Fusion Open Access platform demonstrate excellent analytical and clinical performance for CNS viral targets. The platform provides a highly efficient, fully automated open-access solution for LDTs in high-complexity laboratories.
The 2024 dengue outbreak in Brazil was accompanied by intense co-circulation of other arboviruses, complicating clinical diagnosis. In this retrospective study, serological evidence of unrecognized Alphavirus chikungunya (CHIKV) exposure was investigated among patients hospitalized with dengue-like illness during the 2024 outbreak in Minas Gerais, Brazil. A total of 1119 serum samples from 553 patients admitted to a referral hospital for infectious diseases between March and May 2024 were analyzed. Patients were initially managed under the clinical suspicion of dengue and had previously undergone molecular testing for arboviruses. Retrospective serological testing for CHIKV-specific IgM and IgG antibodies was performed using a commercial ELISA. CHIKV IgM antibodies were detected in 16.6% of patients, while IgG antibodies were identified in 8.3%. Notably, 31% of patients who tested negative by PCR for all investigated arboviruses tested positive for CHIKV IgM, indicating a substantial burden of unrecognized CHIKV exposure. Furthermore, 7.5% of patients who were PCR-positive for DENV were also CHIKV IgM-positive. Hematological analysis of CHIKV-positive (PCR and/or IgM) patients revealed marked thrombocytopenia, accompanied by mild to moderate leukopenia. These findings demonstrate that CHIKV exposure was frequently overlooked among hospitalized patients initially suspected of dengue, underscoring the limitations of molecular testing alone in late-presenting cases and highlighting the importance of combined molecular and serological diagnostic strategies during outbreaks.
