Mycoplasma pneumoniae is a common cause of respiratory infections, which in some cases leads to extrapulmonary complications, including mucocutaneous eruptions. The authors describe a clinical case of a 5-year-old Polish boy presenting with symptoms consistent with the diagnostic criteria of Mycoplasma pneumoniae-induced rash and mucositis (MIRM), a relatively new clinical entity distinguished from the spectrum of erythema multiforme (EM), Stevens–Johnson syndrome (SJS), and toxic epidermal necrolysis (TEN). The patient was initially admitted to the acute admissions unit of the Children’s Health Centre in Poznan, Poland, due to prolonged fever, erosive-ulcerative lesions of the oral mucosa and lips, and slightly elevated, pink, rash-like skin eruptions. After 2 months, he was followed up in the University outpatient department. The treatment included systemic and local steroids (hydrocortisone, prednisolone), antibiotics (gentamicin), mucolytics (ambroxol), analgesics (paracetamol), and local antifungal agents (nystatin). This report discusses the clinical presentation, diagnostic procedures, differential diagnosis, and treatment. We recommend that pneumonia accompanied by mucocutaneous eruptions, especially in young patients, should raise clinical suspicion of MIRM. Differential diagnosis should include EM, SJS, TEN, and Kawasaki disease.

OBJECTIVES:Although antimicrobial resistance has not yet emerged as an overarching problem for Legionella pneumophila (L. pneumophila) infection, the description of clinical and environmental strains resistant to fluoroquinolones and macrolides is a cause of concern. This study aimed to investigate the antimicrobial susceptibility of L. pneumophila human isolates in Italy. METHODS:A total of 204 L. pneumophila clinical isolates were tested for sensitivity to 9 antibiotics using the broth microdilution assay (BMD). All isolates were typed by sequence-based typing, and Legionella pneumophila serogroup 1 (Lp1) isolates by monoclonal antibody subgrouping. Minimum inhibitory concentration (MIC) data were correlated with the possible source of infection and geographical distribution. The presence of the lpeAB efflux pump genes was also investigated. The genome sequences of a subpopulation of isolates showing reduced susceptibility to azithromycin were also analysed. RESULTS:The L. pneumophila isolates did not show significant resistance to the tested antibiotics, although a trend toward reduced sensitivity to azithromycin was observed in a subpopulation of 46 strains, most of which belonged to sequence type 1 (ST1), the second most widespread ST in Italy. An amplicon of the expected size overlapping the lpeAB genes was obtained only in the 46-subpopulation above mentioned. In 4 of the 46 isolates, sequencing analysis showed the occurrence of amino-acid substitutions already described in other strains. No further mutation was found. CONCLUSIONS:The presence of L. pneumophila strains with reduced susceptibility or resistance to azithromycin should be monitored to predict future trends and suggest to physicians a combined therapy with fluoroquinolones when a poor response to azithromycin is observed. © 2025 The Author(s). Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy.

This study investigated the detection of high-risk Human Papillomavirus (hrHPV) and seven other pathogens associated with sexually transmitted infections (STIs) in matched clinician-collected cervical samples and self-taken vaginal and urine specimens collected from 342 asymptomatic women referred to colposcopy to evaluate (i) the concordance in the molecular detection of investigated pathogen in three different sample types; (ii) the analytical sensitivity and specificity of STIs detection on self-samples; and (iii) the distribution of STIs in hrHPV-positive and hrHPV-negative women. Pathogens detection was performed using Anyplex™II HR and Anyplex™II STI-7e, respectively. Good/substantial agreement was observed between cervical and self-taken samples in detecting hrHPV (κ = 0.870 and κ = 0.773 for vaginal and urine). The agreement between cervical and self-taken samples for detecting STIs was found to be significant (κ = 0.779 and κ = 0.738 for vaginal and urine), with almost perfect agreement between urine and vaginal specimens (κ = 0.899). The positivity rate for all investigated STIs was found to be higher in hrHPV-positive compared to hrHPV-negative women. In conclusion, self-sampling proved to be a valid alternative to cervical samples to detect hrHPV and STIs, but further studies are required to evaluate the role of STI coinfections in cervical lesions development and progression.

In HPV-primary screening, sample quality significantly influences test accuracy. Unlike cytology-based screening, no consensus guidelines presently exist for sample quality assessment in HPV testing. This study aims to evaluate the impact of sample cellularity on HPV testing. A total of 37 592 liquid-based cytology (LBC) samples from women undergoing HPV-primary screening (aged 30-64, median 48; IQR: 40-56 years) were analyzed using Cobas 4800 HPV Test (Roche). Sample adequacy was assessed by the assay's β-globin internal control and by an independent quantitative cellularity assessment (OncoPredict HPV, Hiantis). HPV positivity rates (PR) were stratified according to β-globin Ct values. Among the analyzed samples, 50.0%, 47.1%, 2.3%, and 0.6% had β-globin Ct values of ≤ 28, > 28 to ≤ 32, > 32 to ≤ 34, and > 34, respectively. Overall HPV-PR was 7.7% (2891/37 592). PR reached 9.7% in samples with β-globin ≤ 28 Ct (1820/18 801), decreasing markedly to 1.4% for β-globin > 34 Ct (3/214), (p < 0.001). Quantitative analysis showed that Cobas 4800 β-globin Ct = 34 corresponds to approximately 1.5 × 10^3 nucleated cells/reaction. A subset of 195 HPV-negative samples with β-globin Ct ≥ 34 was evaluated by liquid based cytology (LBC): 19% had inadequate cellularity according to LBC guidelines, 8% were ≥ ASC-US and 73% NILMs. 65% of adequate LBC showed cellular atrophy. These findings emphasize the importance of assessing cellularity in HPV-screening to avoid potentially false-negative results due to inadequate samples. Future research should focus on establishing standardized cellularity thresholds to improve screening accuracy.

Wastewater-based epidemiology has emerged as a complementary tool for the monitoring of COVID-19 pandemic waves and for the circulation of viral variants. The selection, standardization, and dynamics of different SARS-CoV-2 RNA targets in wastewater requires further investigation. In the present study, 106 wastewater samples were collected over a 24-month period from the wastewater treatment plant of Sondrio, north Italy, and were analyzed for the presence of SARS-CoV-2 RNA through the quantification of ORF1b, N1, and N3 gene targets via one-step real-time qPCR. In general, the three RNA targets demonstrated different performances and dynamics over the studied time period, underlying the usefulness of multiple viral targets in the surveillance of SARS-CoV-2 in wastewater. During the first 12 months, the quantification of the selected SARS-CoV-2 viral targets also correlated with the reported clinical cases in the same geographical area; however, from the overall data analysis this did not appear to significantly anticipate the epidemic waves. In conclusion, this study further supports the use of wastewater surveillance as a real time indicator of the human circulation of SARS-CoV-2. Moreover, the use of multiple viral gene targets has been shown to improve the reliability of SARS-CoV-2 surveillance in wastewater over time.

Wastewater-based environmental surveillance enables the monitoring of SARS-CoV-2 dynamics within populations, offering critical epidemiological insights. Numerous workflows for tracking SARS-CoV-2 have been developed globally, underscoring the need for interlaboratory comparisons to ensure data consistency and comparability. An inter-calibration test was conducted among laboratories within the network monitoring SARS-CoV-2 in wastewater samples across the Lombardy region (Italy). The test aimed to evaluate data reliability and identify potential sources of variability using robust statistical approaches. Three wastewater samples were analyzed in parallel by four laboratories using identical pre-analytical (PEG-8000-based centrifugation) and analytical processes (qPCR targeting N1/N3 and Orf-1ab). A two-way ANOVA framework within Generalized Linear Models was applied, and multiple pairwise comparisons among laboratories were performed using the Bonferroni post hoc test. The statistical analysis revealed that the primary source of variability in the results was associated with the analytical phase. This variability was likely influenced by differences in the standard curves used by the laboratories to quantify SARS-CoV-2 concentrations, as well as the size of the wastewater treatment plants. The findings of this study highlight the importance of interlaboratory testing in verifying the consistency of analytical determinations and in identifying the key sources of variation.

BACKGROUND:Human papillomavirus (HPV) testing is the primary method for cervical cancer screening, but reliable detection depends on adequate sample cellularity. Cycle threshold (Ct) values for the assay's internal control (IC), such as β-globin, are commonly used as proxies for adequacy, yet standardized Ct cut-offs are lacking. We aimed to contribute evidence-based thresholds for sample adequacy using real-world data. METHODS:We analyzed 237,853 clinician-collected and self-collected samples tested with the BD Onclarity™ HPV Assay between 2022 and 2024. β-globin Ct values were assessed by HPV status to evaluate adequacy. Histologically confirmed CIN2+ outcomes were linked via the National Cervical Screening Registry to assess clinical performance. RESULTS:Among 110,482 clinician-taken samples, 73.63 % (81,350) were HPV negative; 74.32 % (60,457) of these had β-globin Ct ≤28, and only 1.28 % exceeded Ct 32.1. In 127,390 self-collected samples, 83.47 % (106,329) were HPV negative; 99.66 % (105,967) had Ct ≤28 and only 0.06 % exceeded Ct 32.1. HPV positivity declined gradually beyond Ct 26 and more markedly above Ct 28. CIN2+ cases (n = 5546) were rarely HPV negative (n = 73), and these showed low β-globin Ct values, indicating adequate cellularity. Self-collected samples had significantly lower Ct values than clinician-taken ones (median 21.5 vs. 26.5; p < 2.2e-16), likely due to lower resuspension volume. CONCLUSIONS:Both clinician- and self-collected samples showed adequate cellularity, with potentially false negative HPV results from low cellular content appearing rare. Observed patterns suggest Ct <26 as optimal and Ct <28 as a minimum for program-level quality assurance with the BD Onclarity™ HPV Assay.

The introduction of self-sampling in cervical cancer screening has raised the importance of HPV test validation on self-collected samples. This study aimed to evaluate the clinical accuracy of the OncoPredict HPV Screening (SCR) assay on self-collected vaginal and first-void urine (FVU) samples, relative to cervical specimens, using the VALHUDES Framework. FVU and vaginal self-samples followed by a clinician-collected cervical brushing were collected from 500 women referred to colposcopy and tested using OncoPredict HPV SCR assay. The assay demonstrated clinical sensitivity to detect cervical intraepithelial neoplasia grade 2 or worse (≥ CIN2) similar to cervical samples in FVU (ratio: 0.95, [95% CI: 0.88-1.02]) and vaginal self-samples (ratio: 0.96 [95% CI: 0.90-1.02]). The clinical specificity for < CIN2 was lower in vaginal (ratio: 0.90 [95% CI: 0.84-0.96]) but not in FVU samples (ratio: 1.03 [95% CI: 0.96-1.12) when compared to cervical samples. However, the relative specificity improved following cut-off optimization (ratio: 0.94, 95% CI: [0.88-1.01]). Moderate to excellent agreement in HPV detection between self-collected and cervical samples was demonstrated (Kappa values: 0.53-1.00). To conclude, OncoPredict HPV SCR assay demonstrated similar accuracy on FVU and cervical samples. On vaginal compared to cervical samples sensitivity was similar with a lower specificity, which improved with cut-off optimization.

Microorganisms, microbiomes, and their products (e.g., enzymes, metabolites, antibiotics, etc.) are key players in the functioning of both natural and anthropized Earth ecosystems; they can be exploited for both research purposes and biotechnological applications, including fighting the big challenges of our era, such as climate change. Culture collections (CCs) and microbial Biological Resource Centres (mBRCs) are repositories of microorganisms that investigate and safeguard biodiversity and facilitate the scientific and industrial communities’ access to microbial strains and related know-how by providing external users with skills and services. Considering this, CCs and mBRCs are pivotal institutions for the valorisation of microorganisms, the safeguarding of life, and the fostering of excellent bioscience. The aim of this review is to present the state-of-the-art of Italian CCs and mBRCs, highlighting strengths, weaknesses, threats, and opportunities. Italy is, indeed, a hotspot of microbial biodiversity with a high rate of endemism and incredible potential, not only for the food and beverage sector (i.e., “Made in Italy” products), where microorganisms can have a beneficial or a spoiling function, but also to guarantee environmental sustainability and foster the bioeconomy through the design of new bioprocesses and products. However, weaknesses, such as the lack of management rules in accordance with international quality standards, are also analysed and ways of overcoming them are discussed. In this context, an overview is given of the Joint Research Unit MIRRI-IT and the European-funded SUS-MIRRI.IT project, which aims to improve the management and sustainability of Italian microbial collections, and serves as a starting point for an innovative revolution in the context of CCs and mBRCs worldwide.

While HC2 and GP5+/6+ PCR-EIA were pivotal in test validation of new HPV assays, they represent the first generation of comparator tests based upon technologies that are not in widespread use anymore. In the current guideline, criteria for second-generation comparator tests are presented that include more detailed resolution of HPV genotypes. Second-generation comparator tests should preferentially target only the 12 genotypes classified as carcinogenic (IARC-group I), and show consistent non-inferior sensitivity for CIN2+ and CIN3+ and specificity for ≤CIN1 compared to one of the first-generations comparators, in at least three validation studies using benchmarks of 0.95 for relative sensitivity and 0.98 for relative specificity. Validation should take into account used storage media and other sample handling procedures. Meta-analyses were conducted to identify the assays that fulfill these stringent criteria. Four tests fulfilled the new criteria: (1) RealTime High-Risk HPV Test (Abbott), (2) Cobas-4800 HPV test (Roche Molecular System), (3) Onclarity HPV Assay (BD Diagnostics), and (4) Anyplex II HPV HR Detection (Seegene), each evaluated in three to six studies. Whereas the four assays target 14 carcinogenic genotypes, the first two identify separately HPV16 and 18, the third assay identifies five types separately and the fourth identifies all the types separately.

BACKGROUND:The VALHUDES initiative was established to assess the clinical accuracy of HPV assays to detect cervical precancers using urine and vaginal self-samples compared to cervical clinician-collected samples. Here, the clinical performance of OncoPredict HPV Quantitative Typing (QT) assay (OncoPredict QT) was evaluated. METHODS:490 women referred to colposcopy self-collected a urine and a vaginal specimen using Colli-Pee and FLOQSwab, respectively. Subsequently, a colposcopy was performed, and a cervical sample was collected with Cervex-Brush, followed by biopsy if clinically indicated. Vaginal samples were transported dry and resuspended in 5 mL of eNAT medium, whilst cervical brushings were immediately transferred in 20 mL ThinPrep. RESULTS:The clinical sensitivity of OncoPredict HPV QT testing for CIN2+ in urine and vaginal self-samples was similar to cervical samples (ratios of 0.99 [95 % CI 0.94-1.05] and 1.00 [95 % CI 0.96-1.04]), respectively, when manufacturer's cut-offs were applied. The specificity for <CIN2 on both self-samples was lower than on cervical samples (urine/cervical ratio = 0.91 [95 % CI 0.84-0.98]; vaginal/cervical ratio = 0.90 [95 % CI 0.84-0.98]). Cut-off optimisation improved specificity without compromising sensitivity. Median viral load values adjusted for cellularity were significantly higher in cervical samples compared to urine or vaginal self-samples, in general for all 12 high-risk HPV and in particular for HPV16, 18, 31, 33, 35, 45, 51, 58 (p < 0.05). No difference was observed in median viral loads between urine and vaginal samples. CONCLUSION:Following cut-off optimisation OncoPredict HPV QT assay demonstrated similar accuracy on self-collected versus cervical samples.

ABSTRACTThis study assessed the accuracy of high-risk human papillomavirus testing of BD Onclarity HPV (Onclarity) assay on vaginal self-collected FLOQSwab versus cervical samples to ensure similar accuracy to detect cervical intraepithelial neoplasia. Testing was performed on two automated platforms, BD Viper LT and BD COR, to evaluate the effect of machine and using two vaginal self-samples to analyze the influence of collection, transport, and freezing-unfreezing on the results. A cervical sample and two self-samples were collected from 300 women. The first collected vaginal and the cervical sample were tested on BD Viper LT, and the second swab was frozen and subsequently tested on both automated systems. Test results on vaginal and cervical specimens were considered the index and comparator, respectively; colposcopy and histology were reference standards. Relative sensitivity for ≥CIN2 on vaginal samples analyzed versus the cervical sample was 1.01 (0.97–1.06), 1.01 (0.97–1.06), and 1.00 (0.95–1.05), for the first, second self-collected sample tested on BD VIPER LT, and second self-collected sample tested on BD COR, respectively. Relative specificity was 0.83 (0.73–0.94), 0.76 (0.67–0.87), and 0.82 (0.73–0.92) using the three different workflows. Cut-off optimization for human papillomavirus (HPV) positivity defined at Ct ≤38.3 for HPV16, ≤ 34.2 for HPV18, and ≤31.5 for all other types showed an increased relative specificity with similar sensitivity. No significant difference was observed between self-samples tested with the two platforms and between first- and second-collected swabs. Onclarity assay on FLOQSwab using both platforms showed similar sensitivity but lower specificity to detect ≥CIN2 compared to cervical samples. By cut-off optimization, non-inferior specificity could be reached.IMPORTANCEHuman papillomavirus (HPV) testing on self-collected vaginal samples has been shown to improve women’s participation to cervical cancer screening programs, particularly in regions with limited access to health care. Nevertheless, the introduction of self-sampling in cervical cancer screening programs requires prior clinical validation of the HPV assay in combination with a self-sample collection device, including also the laboratory workflow and automation required for high-throughput testing in screening. In this study, the performance of BD Onclarity HPV on FLOQSwab-collected vaginal self-samples has been compared to clinician-taken liquid-based cytology samples, to detect high-grade cervical intraepithelial neoplasia using two high-throughput platforms, BD Viper LT and BD COR. The study findings have shown a similar performance of BD Onclarity on testing self-collected samples, confirming the validation of the proposed pre-analytical and analytical protocols for their use in cervical cancer screening programs based on self-collected vaginal samples.

The introduction of Human Papillomavirus (HPV) testing in cervical cancer screening enhanced the opportunity to introduce self-collection as an innovative approach to improve coverage rates. Validation and standardization of the pre-analytical and analytical procedures are crucial for the quality assurance of HPV tests on self-collected samples.This study evaluated the analytical performance and the stability of self-collected vaginal samples resuspended in 5 mL of two non-alcohol-based media, eNat® and MSwab® compared to a professionally collected cervical sample, resuspended in 20 mL ThinPrep®, for the detection of high-risk HPV (hrHPV). The impact of the suspension volumes on analytical performance was also evaluated (2 and 5 ml).A good analytical concordance in hrHPV detection in cervical and vaginal self-collected swabs suspended in 5 ml of both non-alcohol-based media was demonstrated (eNat®: 91.2%, k=0.821; MSwab®: 91.4%; k= 0.798). A similar analytical performance was found for samples resuspended in 2 mL (eNat®: 92.9%, k=0.811; MSwab®: 92.9%, k=0.811) compared to cervical samples.Good nucleic acid stability was demonstrated for vaginal samples stored at 22°C and 37°C for up to 4 weeks.Results of this preliminary study support the introduction of these media for vaginal self-sampling-based prevention programs. Nevertheless, further research is necessary to evaluate clinical accuracy in larger settings.

Background: Reliable Human Papillomavirus (HPV) testing and genotyping are essential for quality assurance in HPV-based primary screening, disease management and for monitoring the impact of HPV vaccination. The clinical validation of HPV molecular diagnostic assays has significantly contributed to these objectives; however, little emphasis has been placed on assuring sample quality. This study aimed to evaluate the accuracy of sample cellularity assessment using the C-C Motif Chemokine Receptor 5 (CCR5) gene target as a marker of sample adequacy in molecular diagnostics. Methods: Jurkat cell line samples were counted using both a Thoma cell-counting chamber and Fluorescence-Activated Cell Sorting (FACS). Jurkat cell line samples at three different concentrations were subsequently evaluated using the OncoPredict HPV Quality Control (QC) real-time PCR assay, employing CCR5 for molecular cellularity quantification. Results: The cellularity values obtained were comparable across the three different methods for all dilutions of the cell line tested. Conclusions: The results obtained from this study show that CCR5 represents a promising molecular marker for the accurate quantification of sample cellularity, confirming its use as a reliable sample adequacy control, thus reducing the risk of “false-negative” results.

HPV51 belongs to group I high-risk HPV (hrHPV) genotypes with a high prevalence of infection in numerous epidemiological studies, although its occurrence varies across geographical regions. To better understand the presence of variants and genetic variation of HPV51, this study aimed to perform a phylogenetic analysis of the E6, E7 and L1 regions to detect the hrHPV variants circulating in women referred to colposcopy in two different Italian regions (Lombardy and Sardinia). Cervical samples were tested using Anyplex™ II HPV HR Detection. HPV51 positive samples were further analysed using previously described PCR protocols to obtain sequences of E6, E7 and L1. A positivity rate for HPV51 of 7.4% (26/351) and 13.6% (11/81) was found in Lombardy and Sardinia, respectively. The majority (34 out of 35) of the analyzed sequences clustered into the A lineage and only one sequence was grouped into B lineage. L1 capsid gene showed the presence of different mutations but none under positive selective pressure. Three nucleotide polymorphisms T6311G, A6312T and G6313A related to the two aminoacidic mutations V264G and G265S were found among all analysed sequences. This study contributes to implement data regarding the molecular epidemiology of HPV but additional analysis on a larger number of sequences is necessary to better understand the role of HPV variants in cervical lesions development.

ABSTRACTThe coronavirus disease 2019 (COVID-19) pandemic demonstrated the need for accurate diagnostic testing for the early detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although the pandemic has ended, accurate assays are still needed to monitor viral spread at national levels and beyond through population and wastewater surveillance. To enhance early detection, SARS-CoV-2 assays should have high diagnostic accuracy and should be validated to assure accurate results. Three distinct SARS-CoV-2 assays were evaluated with clinical samples using the VALCOR (VALidation of SARS-CORona Virus-2 assays) framework, with the TaqPath COVID-19 assay (ThermoFisher Scientific, USA) as a comparator. We evaluated clinical sensitivity, specificity, limit of detection (LOD), and overall concordance between comparator and three index Allplex SARS-CoV-2 assays (Seegene, South Korea): Allplex-SC2, Allplex-SC2Fast (Fast PCR), and Allplex-SC2FabR (SARS-CoV-2/FluA/FluB/respiratory syncytial virus). Analytical performance and LOD of index assays were assessed using a dilution series of three synthetic SARS-CoV-2 sequence reference materials (RMs). Ninety SARS-CoV-2 positives and 90 SARS-CoV-2 negatives were tested. All Allplex assays had 100.0% sensitivity (95%CI = 95.9%–100.0%). Allplex-SC2 and Allplex-SC2Fast assays had 97.8% specificity (95%CI = 92.3%–99.7%) and 98.9% overall concordance [κ = 0.978 (95%CI = 0.947–1.000)]. Allplex-SC2FabR assay showed 100.0% specificity (95%CI = 95.9%–100.0%) and 100.0% overall concordance [κ = 1.000 (95%CI = 1.000–1.000)]. LOD assessment of index assays revealed detection down to 2.61 × 102 copies/mL in clinical samples, while the analytical LOD was 9.00 × 102 copies/mL. In conclusion, the evaluation of the three Seegene Allplex SARS-CoV-2 assays showed high sensitivity and specificity and an overall good assay concordance with the comparator. The assays showed low analytical LOD using RM and even a slightly lower LOD in clinical samples. Non-overlapping target gene sequences between SARS-CoV-2 assays and RMs emphasize the need for aligning targeted sequences of diagnostic assays and RMs.IMPORTANCEThe coronavirus disease 2019 pandemic has a significant impact on global public health, economies, and societies. As shown through the first phases of the pandemic, accurate and timely diagnosis is crucial for disease control, prevention, and monitoring. Though the pandemic phase of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has concluded, diagnostic assays remain in demand to monitor SARS-CoV-2 at the individual patient level, regionally, and nationally, as well as to remain an infectious disease preparedness instrument to monitor any new SARS-CoV-2 dissemination across borders using population and wastewater surveillance. The anticipation by WHO and central health care policy entities such as the Center for Disease Control, EMA, and multiple national health authorities is that SARS-CoV-2 will reside as an endemic respiratory disease for years to come. The key strategic consideration is hence shifting from combating a pandemic situation with a high number of patients to instead allowing precise diagnostics of suspected patients with the intention of correct management in a low-prevalence setting.

Background: Epigenetic biomarkers in cancer have emerged as promising tools for early detection, prognosis, and treatment response prediction. In cervical cells, hypermethylation of the host and viral HPV-genome increases with the severity of lesions, providing a useful biomarker in the triage of hr-HPV-positive women and during treatment. The present study focuses on evaluating the clinical performance of the FAM19A4/miR124-2 methylation test in a population-based cervical screening program. Methods: Previously collected cervical samples, after bisulfite-converted DNA, were analyzed by PreCursor-M+ kit (distributed by Fujirebio Europe), for DNA methylation. The sensitivity, specificity, and negative/positive predictive values of DNA methylation were compared to histology, colposcopy, the HPV-DNA test, and cytology results. Results: Among the 61-sample set, the specificity of methylation vs. positive histology (≥CIN2) and colposcopy (≥G2) were 87% and 90%, whereas the sensitivity was 50% and 33.3%, respectively. The combination of methylation analysis with standard methods increases diagnostic accuracy. Conclusions: Overall, we found a good specificity of DNA methylation in comparison to currently used techniques. Further larger studies could support the use of FAM19A4/miR124-2 as reliable biomarkers in the prevention of cervical cancer as triage in the screening protocol.

High-risk Human Papillomavirus (hrHPV) persistent infection is acknowledged as the primary cause of cervical cancer. Coinfection with other sexually transmitted infections (STIs) may be associated with a higher risk of cervical lesion progression. This study investigated the detection of hrHPV and 7 other STIs in matched clinician-collected cervical samples and self-taken vaginal and urine specimens of 345 asymptomatic women referred to colposcopy for abnormal cervical cytology. The association of sexually transmitted coinfections with cervical dysplasia was evaluated. Detection of hrHPV and 7 other sexually transmitted pathogens was carried out using Anyplex™II HR and Anyplex™II STI-7e, respectively. The positivity of hrHPV infections in cervical, vaginal and urine samples were respectively 67.0%, 71.3% and 68.1%, while STIs were detected in 47.9%, 57.9% and 56.4%. A good analytical agreement was observed between cervical and self-taken samples for hrHPV and STIs detection. STIs positivity rate was found to be higher in hrHPV-positive compared to hrHPV-negative women, but no association was found between STIs coinfections and the grade of cervical lesions. In conclusion, self-collected specimens proved to be a valid non-invasive alternative to cervical samples to detect hrHPV and STIs. Longitudinal studies are required to evaluate the role of STIs coinfections in lesions progression.

Abstract Background The COVID-19 pandemic highlighted the importance of diagnostic testing against curbing the spread of SARS-CoV-2. The urgent need and scale for diagnostic tools resulted in manufacturers of SARS-CoV-2 assays receiving emergency authorization that lacked robust analytical or clinical evaluation. As it is highly likely that testing for SARS-CoV-2 will continue to play a central role in public health, the performance characteristics of assays should be evaluated to ensure reliable diagnostic outcomes are achieved. Methods VALCOR or “VALidation of SARS-CORona Virus-2 assays” is a study protocol designed to set up a framework for test validation of SARS-CoV-2 virus assays. Using clinical samples collated from VALCOR, the performance of Aptima SARS-CoV-2 assay was assessed against a standard comparator assay. Diagnostic test parameters such as sensitivity, specificity and overall per cent agreement were calculated for the clinical performance of Aptima SARS-CoV-2 assay. Results A total of 180 clinical samples were tested with an addition of 40 diluted clinical specimens to determine the limit of detection. When compared to the standard comparator assay Aptima had a sensitivity of 100.0% [95% CI 95.9–100.0] and specificity of 96.7% [95% CI 90.8–99.3]. The overall percent agreement was 98.3% with an excellent Cohen’s coefficient of κ = 0.967 [95% CI 0.929–1.000]. For the limit of detection, Aptima was able to detect all of the diluted clinical samples. Conclusion In conclusion. validation of Aptima SARS-CoV-2 assay using clinical samples collated through the VALCOR protocol showed excellent test performance. Additionally, Aptima demonstrated high analytical sensitivity by detecting all diluted clinical samples corresponding to a low limit of detection.

Background: Given the diagnostic accuracy of HPV-DNA tests in terms of self-collected samples, in order to implement self-sampling in cervical screening programs, the standardization of the pre-analytical phase, including decisions concerning the choice of medium, the volume of elution, and storage conditions, are necessary, in addition to understanding the potential factors involved in acceptability by women. On this basis, we carried out a cross-sectional study to assess (i) the stability of dry vaginal self-collected samples stored at room temperature for up to 4 weeks after elution in 2 mL of eNat® (Copan) medium, and (ii) the acceptability of self-collection in enrolled women. Methods: 185 women were enrolled in the LILT (Italian League Against Tumors) regional project. A self-sampling kit, including a dry FLOQSwab® (Copan), instructions for use, and a satisfaction questionnaire, were supplied for each woman and sent by mail to the laboratory. The HPV-DNA test was carried out using the Anyplex™ II HPV HR (Seegene) kit. To evaluate the specimen’s stability, 185 dry vaginal swabs were eluted in eNat®, a lyses-based molecular medium and tested for HPV detection at two different time points (<6 days and 1 month after elution). The Cohen’s Kappa coefficients and McNemar test were used to assess the agreement of HPV-DNA at different times. Results: We found high agreement in terms of HPV-DNA results among the samples tested at two different time points (Cohen K = 0.98; p < 0.0001). Moreover, most of the women found it easy to use self-collection devices and the pictorial instructions clear to understand. Approximately half of the enrolled women declared preferring self-sampling to clinician-collected methods. Conclusion: Our results display the high reliability and accuracy of HPV-DNA tests using dry vaginal self-collection FLOQSwabs® devices eluted in 2 mL of molecular medium. The analysis of the questionnaire showed a high acceptability of self-collection among women, although a high percentage preferred standard collection devices. Overall, our preliminary results support the adoption of self-collection in screening programs, even though further analyses should be performed to optimize and standardize protocols for HPV tests on self-samples, and educational campaigns are needed to adequately inform and increase responsiveness in a target population.