Multi-center evaluation of the Research Use Only NeuMoDx monkeypox virus (MPXV) fully automated real-time PCR assay.

The mpox outbreak, caused by monkeypox virus (MPXV), accelerated the development of molecular diagnostics. In this study, we detail the evaluation of the Research Use Only (RUO) NeuMoDx MPXV assay by multiple European and US sites. The assay was designed and developed by Qiagen for the NeuMoDx Molecular Systems. Primers and probes were tested for specificity and inclusivity in silico. The analytical sensitivity of the assay was determined by testing dilutions of synthetic and genomic MPXV DNA. A total of 296 clinical samples were tested by three sites; the Johns Hopkins University (US), UZ Gent (Belgium, Europe), and Hospital Universitario San Cecilio (Spain, Europe). The analytical sensitivity of the assay was 50 copies/mL for both clades I and II. The assay showed 100% in silico identity for 80 clade I and 99.98% in silico identity for 5,162 clade II genomes. Clade II primers and probes showed 100% in silico specificity; however, identity of at least one of the two sets of clade I primers and probes with variola, cowpox, camelpox, and vaccinia viruses was noticed. The clinical validation showed sensitivity of 99.21% [95% confidence interval (CI): 95.66-99.98%] and specificity of 96.64% (95% CI: 91.62-99.08%) for lesion swab samples. The NeuMoDx MPXV Test shows acceptable analytical and clinical performance. The assay improves the laboratory's workflow as it consolidates nucleic acid extraction, PCR, data analysis, and interpretation and can be interfaced. The Test Strip can differentiate clades I and II, which has important laboratory safety implications. IMPORTANCE: In this manuscript, we provide detailed in silico analysis and clinical evaluation of the assay using a large cohort of clinical samples across three academic centers in Europe and the United States. Because the assay differentiates MPXV clades I and II, this manuscript is timely due to the current need to rule out the regulated clade I by diagnostic clinical laboratories. In December 2023, and due to first report of cases of sexually transmitted clade I infections in the Democratic Republic of the Congo, when generic assays that do not differentiate the clades are used, samples are considered regulated. The assay meets the need of full automation and has a marked positive impact on the laboratory workflow.

Estimation of antimicrobial resistance of Mycoplasma genitalium, Belgium, 2022.

BackgroundAntimicrobial resistance (AMR) of Mycoplasma genitalium (MG) is a growing concern worldwide and surveillance is needed. In Belgium, samples are sent to the National Reference Centre of Sexually Transmitted Infections (NRC-STI) on a voluntary basis and representative or robust national AMR data are lacking.AimWe aimed to estimate the occurrence of resistant MG in Belgium.MethodsBetween July and November 2022, frozen remnants of MG-positive samples from 21 Belgian laboratories were analysed at the NRC-STI. Macrolide and fluoroquinolone resistance-associated mutations (RAMs) were assessed using Sanger sequencing of the 23SrRNA and parC gene. Differences in resistance patterns were correlated with surveillance methodology, socio-demographic and behavioural variables via Fisher's exact test and logistic regression analysis.ResultsOf the 244 MG-positive samples received, 232 could be sequenced for macrolide and fluoroquinolone RAMs. Over half of the sequenced samples (55.2%) were resistant to macrolides. All sequenced samples from men who have sex with men (MSM) (24/24) were macrolide-resistant. Fluoroquinolone RAMs were found in 25.9% of the samples and occurrence did not differ between socio-demographic and sexual behaviour characteristics.ConclusionAlthough limited in sample size, our data suggest no additional benefit of testing MG retrieved from MSM for macrolide resistance in Belgium, when making treatment decisions. The lower occurrence of macrolide resistance in other population groups, combined with emergence of fluoroquinolone RAMs support macrolide-resistance testing in these groups. Continued surveillance of resistance in MG in different population groups will be crucial to confirm our findings and to guide national testing and treatment strategies.

Human papillomavirus negative high grade cervical lesions and cancers: Suggested guidance for HPV testing quality assurance.

BACKGROUND: Some high-grade cervical lesions and cervical cancers (HSIL+) test negative for human papillomavirus (HPV). The HPV-negative fraction varies between 0.03 % and 15 % between different laboratories. Monitoring and extended re-analysis of HPV-negative HSIL+ could thus be helpful to monitor performance of HPV testing services. We aimed to a) provide a real-life example of a quality assurance (QA) program based on re-analysis of HPV-negative HSIL+ and b) develop international guidance for QA of HPV testing services based on standardized identification of apparently HPV-negative HSIL+ and extended re-analysis, either by the primary laboratory or by a national HPV reference laboratory (NRL). METHODS: There were 116 initially HPV-negative cervical specimens (31 histopathology specimens and 85 liquid-based cytology samples) sent to the Swedish HPV Reference Laboratory for re-testing. Based on the results, an international QA guidance was developed through an iterative consensus process. RESULT: Standard PCR testing detected HPV in 55.2 % (64/116) of initially "HPV-negative" samples. Whole genome sequencing of PCR-negative samples identified HPV in an additional 7 samples (overall 61.2 % HPV positivity). Reasons for failure to detect HPV in an HSIL+ lesion are listed and guidance to identify cases for extended re-testing, including which information should be included when referring samples to an NRL are presented. CONCLUSION: Monitoring the proportion of and reasons for failure to detect HPV in HSIL+ will help support high performance and quality improvement of HPV testing services. We encourage implementation of QA strategies based on re-analysis of "HPV negative" HSIL+ samples.

Effect of screening for Neisseria gonorrhoeae and Chlamydia trachomatis on incidence of these infections in men who have sex with men and transgender women taking HIV pre-exposure prophylaxis (the Gonoscreen study): results from a randomised, multicentre, controlled trial.

BACKGROUND: Guidelines recommend screening for Neisseria gonorrhoeae and Chlamydia trachomatis at three anatomical sites (urethra, anus, and pharynx) every 3 months (3 × 3) in men who have sex with men (MSM) and transgender women taking HIV pre-exposure prophylaxis (PrEP). We present the first randomised controlled trial to compare the effect of screening versus non-screening for N gonorrhoeae and C trachomatis on the incidence of these infections in MSM and transgender women taking PrEP. METHODS: A multicentre, randomised, controlled trial of 3 × 3 screening for N gonorrhoeae and C trachomatis versus non-screening was done among MSM and transgender women taking PrEP in five HIV reference centers in Belgium. Participants attended the PrEP clinics quarterly for 12 months. N gonorrhoeae and C trachomatis was tested at each visit in both arms, but results were not provided to the non-screening arm, if asymptomatic. The primary outcome was incidence rate of N gonorrhoeae and C trachomatis infections in each arm, assessed in the per-protocol population. Non-inferiority of the non-screening arm was proven if the upper limit of the 95% CI of the incidence rate ratio (IRR) was lower than 1·25. This trial is registered with ClinicalTrials.gov, NCT04269434, and is completed. FINDINGS: Between Sept 21, 2020, and June 4, 2021, 506 participants were randomly assigned to the 3 × 3 screening arm and 508 to the non-screening arm. The overall incidence rate of N gonorrhoeae and C trachomatis was 0·155 cases per 100 person-days (95% CI 0·128-0·186) in the 3 × 3 screening arm and 0·205 (95% CI 0·171-0·246) in the non-screening arm. The incidence rate was significantly higher in the non-screening arm (IRR 1·318, 95% CI 1·068-1·627). Participants in the non-screening arm had a higher incidence of C trachomatis infections and symptomatic C trachomatis infections. There were no significant differences in N gonorrhoeae infections. Participants in the non-screening arm consumed significantly fewer antimicrobial drugs. No serious adverse events were reported. INTERPRETATION: We failed to show that non-screening for N gonorrhoeae and C trachomatis is non-inferior to 3 × 3 screening in MSM and transgender women taking PrEP in Belgium. However, screening was associated with higher antibiotic consumption and had no effect on the incidence of N gonorrhoeae. Further research is needed to assess the benefits and harms of N gonorrhoeae and C trachomatis screening in this population. FUNDING: Belgian Health Care Knowledge Centre.

Risk Factors for Natural Hearing Evolution in Newborns With Congenital Cytomegalovirus Infection.

Importance: Congenital cytomegalovirus (cCMV) is the major cause of congenital nonhereditary sensorineural hearing loss in children. Currently, criteria to identify infants at increased risk for unfavorable hearing outcome are lacking. Objective: To identify risk factors associated with cCMV-related hearing improvement, hearing deterioration, and late-onset hearing loss. Design, Setting, and Participants: This multicenter cohort study included patients from 6 secondary and tertiary hospitals enrolled in the Flemish CMV registry (Belgium). Newborns with untreated cCMV infection with at least 4-year audiological follow-up were included. Patients who presented with other possible causes of sensorineural hearing loss were excluded. Data were collected for 15 years (January 1, 2007, to February 7, 2022) and analyzed from September 26, 2022, to January 16, 2023. Main Outcomes and Measures: Primary outcome was hearing evolution (per-ear analysis; described as stable hearing, improvement, or deterioration). The association of gestational characteristics, clinical findings, timing of seroconversion, viral load, and hearing status at birth with hearing evolution was investigated using effect sizes (Cramer V, odds ratio [OR], or Hedges g). Results: Of the 387 children, 205 of 385 with nonmissing data were male (53.2%), 113 (29.2%) had a symptomatic infection, and 274 (70.8%) had an asymptomatic infection. Every child was 4 years or older at final hearing evaluation. A total of 701 of 774 ears (90%) showed stable hearing (normal hearing or stable hearing loss since birth) over time. Late-onset hearing loss (normal hearing at birth followed by hearing loss) was present in 43 of 683 ears (6.3%). Among children with hearing loss present at birth, 24 of 34 ears (70.6%) had hearing deterioration, and 6 of 91 ears (6.6%) had hearing improvement. Prematurity was associated with a higher chance of hearing improvement (OR, 12.80; 95% CI, 2.03-80.68). Late-onset hearing loss was more prevalent in a first trimester infection (OR, 10.10; 95% CI, 2.90-34.48). None of the 104 ears of children with a third trimester seroconversion developed late-onset hearing loss. Conclusions and Relevance: Findings of this cohort study support that ongoing audiological follow-up for untreated children with congenital hearing loss is important, as the majority of patients had hearing deterioration. The timing of seroconversion was associated with the risk of developing late-onset hearing loss. These insights can aid in parental counseling, patient stratification, and follow-up. Future research should focus on the effect of treatment, the influence of determined risk factors, and the study of eventual new risk factors in patients at high risk to develop hearing loss.

Longevity of the humoral and cellular responses after SARS-CoV-2 booster vaccinations in immunocompromised patients.

We assessed the humoral and cellular immune responses after two booster mRNA vaccine administrations [BNT162b2 (Pfizer-BioNTech vaccine)] in cohorts of immunocompromised patients (n = 199) and healthy controls (HC) (n = 54). All patients living with HIV (PLWH) and chronic kidney disease (CKD) patients and almost all (98.2%) of the primary immunodeficiency (PID) patients had measurable antibodies 3 and 6 months after administration of the third and fourth vaccine dose, comparable to the HCs. In contrast, only 53.3% and 83.3% of the multiple sclerosis (MS) and rheumatologic patients, respectively, developed a humoral immune response. Cellular immune response was observed in all PLWH after administration of four vaccine doses. In addition, cellular immune response was positive in 89.6%, 97.8%, 73.3% and 96.9% of the PID, MS, rheumatologic and CKD patients, respectively. Unlike the other groups, only the MS patients had a significantly higher cellular immune response compared to the HC group. Administration of additional vaccine doses results in retained or increased humoral and cellular immune response in patients with acquired or inherited immune disorders.

Evaluation of the applicability of internal controls on self-collected samples for high-risk human papillomavirus is needed.

BACKGROUND: Self-collection of cervical samples to detect high-risk human papillomavirus (hr-HPV) is a trending topic in primary cervical cancer screening. This study evaluates the applicability of a self-sampling device to routine molecular procedures for hr-HPV detection. METHODS: In a primary health care facility in Kinshasa, Congo, 187 self-collected samples (Evalyn Brush) were gathered and sent to Ghent University Hospital (UZ Ghent) and Algemeen Medisch Labo (AML) in Belgium where routine tests for hr-HPV were applied (Abbott RealTime hr-HPV and qPCR (E6/E7), respectively). Sample type effect was evaluated by comparing the internal control (IC) between the self-collected samples and routine, clinician-taken samples randomly selected from the UZ Ghent archive. RESULTS: In UZ Ghent an error was encountered in 9.1% (17/187) of self-collected samples due to a lack of IC signal. The hr-HPV prevalence in the remaining 170 samples was 18,8%. Comparing IC results between the self-collected and clinician-collected groups, a significant difference (p < 0,001) was found, with higher IC signals in the clinician-collected group. In AML, an error was encountered in 17.6% (33/187) of samples, including 16/17 of the UZ Ghent. The remaining sample with IC error gave a negative result in AML. Among the 154 samples without IC error at AML, a correlation of 90% was seen between both laboratories with a 77% negativity rate. CONCLUSION: Testing the self-collected specimens by 2 routine hr-HPV tests gave a high IC error rate (9.1-17.6%). A possible solution would be to differentiate cut-offs for IC values depending on sample type, as currently used cut-offs are set for clinician-taken samples.

Evaluation of a smartphone-operated point-of-care device using loop-mediated isothermal amplification technology for rapid and remote detection of SARS-CoV-2.

During the SARS-CoV-2 pandemic, rapid and sensitive detection of SARS-CoV-2 has been of high importance for outbreak control. Reverse transcriptase polymerase chain reaction (RT-PCR) is the current gold standard, however, the procedures require an equipped laboratory setting and personnel, which have been regularly overburdened during the pandemic. This often resulted in long waiting times for patients. In contrast, reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) is a simple, cost-efficient, and fast procedure, allowing for rapid and remote detection of SARS-CoV-2. In the current study, we performed a clinical evaluation of a new point-of-care test system based on LAMP-technology for SARS-CoV-2 detection, providing a result within 25 min (1copy™ COVID-19 MDx Kit Professional system). We tested 112 paired nasopharyngeal swabs, collected in the COVID-19 Ghent University Hospital test center, using the 1copy™ COVID-19 MDx Kit Professional system, and RT-PCR as the reference method. The test system was found to have a clinical sensitivity of 93.24% (69/74) (95% confidence interval [CI]: 84.93%-97.77%) and specificity of 97.37% (37/38) (95% CI: 86.19%-99.93%). Due to its easy smartphone operation and ready-to-use reagents, it ought to be easily applied in for instance general practices, pharmacies, nursing homes, schools, and companies. This would facilitate an efficient SARS-CoV-2 outbreak control and quarantine policy, as diagnosis can occur sooner in a near-patient setting.

Intra- and interlaboratory reproducibility of the RIATOL qPCR HPV genotyping assay.

The implementation of cervical screening based on human papillomavirus (HPV) continues to progress rapidly across countries. Evidence has shown that assays detecting high-risk human papillomavirus (hrHPV) deoxyribonucleic acid (DNA) are more effective than cytology-based screening. Validation of new hrHPV DNA assays requires both noninferior clinical accuracy compared to a standard comparator for cervical precancer and good reproducibility. This study builds upon previous diagnostic accuracy assessments of the RIATOL HPV genotyping qPCR assay and aims to evaluate the international validation criteria for reproducibility. The intra- and interreproducibility of the RIATOL-qPCR assay were assessed using 550 remnant cervical cell material from the cytology archive of the National Reference Center for HPV in Belgium. Specimens were collected in the context of cervical cancer screening and tested in two different laboratories. The international reproducibility criteria include the lower bound of 95% confidence interval of the intra- and interlaboratory agreement regarding the detection of hrHPV DNA exceeding 87% with kappa ≥0.50. The RIATOL-qPCR assay demonstrated excellent intralaboratory reproducibility, achieving an overall agreement of 98.2 (95% CI 96.6-99.1%) and a kappa of 0.96. Interlaboratory testing showed an overall agreement of 98.5 (95% CI 97.1-99.4%) with a kappa of 0.97. The RIATOL-qPCR assay fulfills the third criterion for HPV test reproducibility requirement for use in cervical cancer screening.

Preanalytical variables influencing the interpretation and reporting of biological tests on blood samples of living and deceased donors for human body materials.

With the present paper, the Working Group on Cells, Tissues and Organs and other experts of the Superior Health Council of Belgium aimed to provide stakeholders in material of human origin with advice on critical aspects of serological and nucleic acid test (NAT) testing, to improve virological safety of cell- and tissue and organ donation. The current paper focusses on a number of preanalytical variables which can be critical for any medical biology examination: (1) sampling related variables (type of samples, collection of the samples, volume of the sample, choice of specific tubes, identification of tubes), (2) variables related to transport, storage and processing of blood samples (transport, centrifugation and haemolysis, storage before and after centrifugation, use of serum versus plasma), (3) variables related to dilution (haemodilution, pooling of samples), and (4) test dependent variables (available tests and validation). Depending on the type of donor (deceased donor (heart-beating or non-heart beating) versus living donor (allogeneic, related, autologous), and the type of donated human material (cells, tissue or organs) additional factors can play a role: pre- and post-mortem sampling, conditions of sampling (e.g. morgue), haemodilution, possibility of retesting.

No Significant Association between 25-OH Vitamin D Status and SARS-CoV-2 Antibody Response after COVID-19 Vaccination in Nursing Home Residents and Staff.

Vitamin D is an essential nutrient for various physiological functions, including immunity. While it has been suggested that higher vitamin D levels/supplementation are associated with a better immune response to COVID-19 vaccination, conflicting data exist. Therefore, we aimed to investigate the association between vitamin D (25-hydroxyvitamin D) deficiency/supplementation, and SARS-CoV-2 antibody responses post-vaccination in nursing home residents (NHRs) and staff (NHS). Blood samples were collected from 115 NHRs and 254 NHS at baseline and 14 days after primary course BNT162b2 vaccination. Baseline samples were assessed for serum 25-hydroxyvitamin D levels, while follow-up samples were analyzed for spike protein S1 receptor-binding domain (S1RBD) IgG antibody concentrations and 50% pseudoneutralization titers. Vitamin D supplementation status was obtained from NHRs medical records. We compared immune responses between (severe) vitamin D-deficient and -sufficient NHRs/NHS and between supplemented and non-supplemented NHRs, stratified for history of SARS-CoV-2 infection and participant type. No significant differences in either binding or neutralizing COVID-19 vaccine antibody response were found between groups. The prevalence of vitamin D deficiency (<20 ng/mL) was 45% (95% CI: 36-54%) among NHRs and 60% (95% CI: 54-66%) among NHS. Although we showed that vitamin D status may not be related to a better COVID-19 vaccine antibody response, addressing the high prevalence of vitamin D deficiency in the nursing home population remains important.

Comparison between the Roche Cobas 4800 Human Papillomavirus (HPV), Abbott RealTime High-Risk HPV, Seegene Anyplex II HPV28, and Novel Seegene Allplex HPV28 Assays for High-Risk HPV Detection and Genotyping in Mocked Self-Samples.

Infection with high-risk human papillomavirus (hrHPV) is well recognized as the main cause of cervical cancer. The recently developed Seegene Allplex HPV28 assay is a novel quantitative PCR (qPCR) assay designed to separately detect and quantify 28 distinct HPV genotypes in a fully automated and user-friendly manner. This study evaluated and compared the performance of this new assay with the performance of the Roche Cobas 4800, the Abbott RealTime high-risk HPV, and the Seegene Anyplex II HPV28 assays. A total of 114 mocked self-samples, i.e., semicervical samples collected by gynecologists using the Viba-Brush, were analyzed with all four HPV assays. Agreement in terms of detecting and genotyping HPV was assessed by the mean of the Cohen's kappa (κ) coefficient. Results of all four HPV assays agreed in 85.9% of the cases when using the Abbott RealTime manufacturer's recommended quantification cycle (Cq) cutoff for positivity (<32.00) and 91.2% when using an adapted range (32.00 to 36.00). An intercomparison of the included assays demonstrated an overall agreement ranging from 85.9 to 100.0% (κ = 0.42 to 1.00) when using the manufacturer's guidelines and 92.9 to 100.0% (κ = 0.60 to 1.00) with the adapted range. For all assays, highly significant, strongly positive Pearson correlations were shown between the Cq values of positive test results. This study thereby shows high concordance between results of the included HPV assays on mocked self-samples. Based on these findings, we imply that the novel Allplex HPV28 assay demonstrates a comparable performance to those of available qPCR HPV assays, potentially providing opportunities for the simplification and standardization of future large-scale testing. IMPORTANCE This study proves that the novel Allplex HPV28 assay has a good diagnostic performance in comparison with the well-known, validated, and frequently used Roche Cobas 4800, Abbott RealTime, and Anyplex II HPV28 assays. According to our experience, the novel Allplex HPV28 assay had a user-friendly and automated workflow with short hands-on time, had an open platform which facilitates the use of add-on assays, and provided quick and easy-to-interpret results. Together with its ability to detect and quantify 28 HPV genotypes, the Allplex HPV28 assay could therefore potentially provide opportunities for the simplification and standardization of future diagnostic testing programs.

SARS-CoV-2 infection and COVID19 vaccination across eight immune-mediated inflammatory disorders: A prospective, real-life Belgian cohort study - the BELCOMID study.

Background: The risks and impact of COVID19 disease and vaccination in patients with Immune Mediated Inflammatory Diseases (IMID) remain incompletely understood. IMID patients and particularly patients receiving immunosuppressive treatment were excluded from the original, registrational phase-3 COVID19 vaccination efficacy and safety trials. Real-world observational data can help to fill this gap in knowledge. The BELCOMID study aims to explore the interaction between IMIDs, immune-modulating treatment modalities and SARS-CoV-2 infection and vaccination in a real-life patient cohort. Methods: A multidisciplinary, prospective, observational cohort study was set up. Consecutive patients with IMIDs of the gut, joints and skin followed at two high-volume referral centers were invited. Both patients under conventional treatment or targeted immune modulating therapies were included. Patient data and serological samples were collected at 3 predefined periods (before COVID19 vaccination, before booster vaccination, after booster vaccination). Primary endpoints were positive PCR-test and SARS-CoV-2 serology reflecting previous SARS-CoV-2 infection or vaccination. Associations with IMID treatment modality and IMID disease activity were assessed. Results of the first two inclusion periods (before booster vaccination) are reported. Results: At the first inclusion period data was assessed of 2165 IMID-patients before COVID19 vaccination. At the second inclusion period, data of 2065 patients was collected of whom 1547 had received complete baseline COVID19 vaccination and 222 were partially vaccinated. SARS-CoV-2 infection rate remained low in both groups. No significant increase in IMID flare-up rate was noted in patients with prior SARS-CoV-2 infection. Multiple logistic regression analyses did not show a significant influence of IMID-treatment modality or IMID activity on SARS-CoV-2 infection risk (based on PCR positivity or N-serology). Patients treated with conventional immunomodulators, systemic steroids, and patients on advanced therapies such as biologics or small molecules, had reduced S-antibody seroconversion. S-antibody response was also lower in patients without prior SARS-CoV-2 infection and in active smokers. A subset of patients (4.1%) had no S- nor N-antibody seroconversion following complete baseline vaccination. Conclusion: The BELCOMID study results confirm the benign course of COVID19 infection and vaccination in a large real-life IMID-population. However, our results underscore the need for repeated vaccination and smoking cessation in patients with IMIDs treated with immune-modulating therapies or systemic steroids during the pandemic.

Quality assurance in human papillomavirus testing for primary cervical screening.

The recommendation for cervical screening is that it should be based on human papillomavirus (HPV) molecular testing. For all screening programs, attention to quality assurance is required to fully realize the benefits. Internationally recognized quality assurance recommendations for HPV-based screening are needed that are ideally applicable for a variety of settings, including in low- and middle-income countries. We summarize the main points of quality assurance for HPV screening, with a focus on the selection, implementation, and use of an HPV screening test, quality assurance systems (including internal quality control and external quality assessment), and staff competence. While we recognize that it might not be possible to fulfill all points in all settings, an awareness of the issues is essential.

Immunologic Monitoring after Allogeneic Stem Cell Transplantation: T-SPOT.CMV and QuantiFERON-CMV, Are They the Same?

Despite prophylactic and preemptive strategies, cytomegalovirus (CMV) reactivation and disease remains major concerns after allogeneic hematopoietic stem cell transplantation (allo-HSCT). In recent years, immunologic monitoring using CMV commercially available IFN-γ release assays (IGRAs) has gained interest to better risk-stratify immunocompromised patients or to guide prophylactic therapy. CMV-IGRA can quantify CMV cell-mediated immunity by measuring the IFN-γ that is released by CD4+ and CD8+ T lymphocytes in the presence of CMV antigens. However, the 2 most widely used CMV-IGRAs, T-SPOT.CMV and QuantiFERON-CMV, had not yet been compared in the setting of an allo-HSCT. In the present study, we performed a method comparison between T-SPOT.CMV and QuantiFERON-CMV at 28 days and 100 days post-allo-HSCT, and to assess predictive values of both tests for CMV reactivation. Twenty-seven patients were included in a bicentric prospective trial. Samples were obtained on days +28 and +100 post-allo-HSCT, and patients' clinical information was collected up to day +270 post-HSCT. Comparisons of methods were performed using Cohen's κ. On day +28 (n = 26) post-allo-HSCT, T-SPOT.CMV yielded 3 positive test results and QuantiFERON-CMV yielded 2 positive results. On day +100 (n = 24), T-SPOT.CMV produced 7 positive test results, and QuantiFERON-CMV produced 9. One discordant result was obtained at day +28 (n = 26), and 6 discordant results were obtained at day +100 (n = 24). Method comparison showed a strong agreement on day +28 (κ = .780; 95% confidence interval [CI], .366 to 1.000) but only a moderate agreement on day +100 (κ = .442; 95% CI, .070 to .814) and in pooled data from both time points (κ = .578; 95% CI, .300-.856). Four clinically significant CMV infections (CS-CMVi) were observed, all occurring after discontinuation of letermovir prophylaxis. None of those 4 patients had a positive result with either test at day +100 (or day +28). Thus, the negative predictive value (NPV) and sensitivity were very high, at 100% for both tests measured at day +100. Positive predictive values (PPVs) and specificity were considerably lower at day +100 (T-SPOT.CMV: PPV, 23.5%; specificity, 35%; QuantiFERON-CMV: PPV, 26.7%; specificity, 45%). T-SPOT.CMV and QuantiFERON-CMV had only moderate agreement (at day +100) after allo-HSCT. Although these IGRAs are very promising, as shown by their very high NPVs for protection against CS-CMVi, they are not interchangeable. Future research should stipulate which IGRA was used, and future guidelines preferably should be assay-specific. As QuantiFERON-CMV still lacks a large post-allo-HSCT validation study, the moderate agreement with T-SPOT.CMV poses a significant hurdle in the routine implementation of this test.

Risk Factors for Hearing Loss at Birth in Newborns With Congenital Cytomegalovirus Infection.

Importance: With a prevalence between 0.2% and 6.1% of all live births, congenital cytomegalovirus (cCMV) infection is a major cause of congenital nonhereditary sensorineural hearing loss. Despite the large amount of research on cCMV-related hearing loss, it is still unclear which newborns are at risk of hearing loss. Objective: To identify independent risk factors for cCMV-related congenital hearing loss and predictors of hearing loss severity at birth. Design, Setting, and Participants: This cross-sectional study of newborns with cCMV infection used data included in the Flemish CMV registry that was collected from 6 secondary and tertiary hospitals in Flanders, Belgium, over 15 years (January 1, 2007, to February 7, 2022). Data were analyzed March 3 to October 19, 2022. Patients were included in the study after confirmed diagnosis of cCMV infection and known hearing status at birth. Patients who presented with other possible causes of sensorineural hearing loss were excluded. Main Outcomes and Measures: Primary outcome was hearing status at birth. Clinical, neurological, and laboratory findings along with the timing of seroconversion and blood viral load were separately considered as risk factors. Binary logistic regression was performed to identify independent risk factors for congenital hearing loss in newborns with cCMV. Effect sizes were measured using Hedges g, odds ratio, or Cramer V. Results: Of the 1033 newborns included in the study (553 of 1024 [54.0%] boys), 416 (40.3%) were diagnosed with symptomatic cCMV infection and 617 (59.7%) with asymptomatic cCMV infection. A total of 15.4% of the patients (n = 159) presented with congenital hearing loss; half of them (n = 80 [50.3%]) had isolated hearing loss. The regression model revealed 3 independent risk factors for congenital hearing loss: petechiae at birth (adjusted odds ratio [aOR], 6.7; 95% CI, 1.9-23.9), periventricular cysts on magnetic resonance imaging (MRI; aOR, 4.6; 95% CI, 1.5-14.1), and seroconversion in the first trimester (aOR, 3.1; 95% CI, 1.1-9.3). Lower viral loads were seen in patients with normal hearing compared with those with congenital hearing loss (median [IQR] viral load, 447.0 [39.3-2345.8] copies per milliliter of sample [copies/mL] vs 1349.5 [234.3-14 393.0] copies/mL; median difference, -397.0 [95% CI, -5058.0 to 174.0] copies/mL). Conclusions and Relevance: Findings of this cross-sectional study suggest that newborns with cCMV infection and petechiae at birth, periventricular cysts on MRI, or a seroconversion in the first trimester had a higher risk of congenital hearing loss. Clinicians may use these risk factors to counsel parents in the prenatal and postnatal periods about the risk of congenital hearing loss. Moreover, linking clinical features to hearing loss may provide new insights into the pathogenesis of cCMV-related hearing loss. The importance of viral load as a risk factor for congenital hearing loss remains unclear.

Diagnostic performance of the SARS-CoV-2 S1RBD IgG ELISA (ImmunoDiagnostics) for the quantitative detection of SARS-CoV-2 antibodies on dried blood spots.

Dried Blood Spots (DBS) are broadly used in SARS-CoV-2 surveillance studies, reporting either the presence or absence of SARS-CoV-2 antibodies. However, quantitative follow-up has become increasingly important to monitor humoral vaccine responses. Therefore, we aimed to evaluate the performance of DBS for the detection of anti-spike SARS-CoV-2 antibody concentrations using a commercially available assay, reporting in a standardised unitage (International Units/mL; IU/mL). To assess the sensitivity and specificity of the ImmunoDiagnostics ELISA on serum and DBS for SARS-CoV-2 antibody detection, we analysed 72 paired DBS and serum samples. The SARS-CoV-2 S1 IgG ELISA kit (EUROIMMUN) on serum was used as the reference method. We performed a statistical assessment to optimise the cut-off value for DBS and serum and assessed the correlation between DBS and serum antibody concentrations. We found that anti-spike SARS-CoV-2 antibody concentrations detected in DBS are highly correlated to those detected in paired serum (Pearson correlation 0.98; p-value < 0.0001), allowing to assess serum antibody concentration using DBS. The optimal cut-off for antibody detection on DBS was found to be 26 IU/mL, with 98.1% sensitivity and 100% specificity. For serum, the optimal cut-off was 14 IU/mL, with 100% sensitivity and 100% specificity. Therefore, we conclude that the ImmunoDiagnostics ELISA kit has optimal performance in the detection of SARS-CoV-2 antibodies on both DBS and serum. This makes DBS ideal for large-scale follow-up of humoral SARS-CoV-2 immune responses, as it is an easy but valuable sampling method for quantification of SARS-CoV-2 antibodies, compared to serum.

Efficacy and safety of camostat mesylate in early COVID-19 disease in an ambulatory setting: a randomized placebo-controlled phase II trial.

OBJECTIVES: This study aimed to assess the efficacy and safety of 300 mg camostat mesylate three times daily in a fasted state to treat early phase COVID-19 in an ambulatory setting. METHODS: We conducted a phase II randomized controlled trial in symptomatic (maximum 5 days) and asymptomatic patients with confirmed COVID-19 infection. Patients were randomly assigned in a 2:1 ratio to receive either camostat mesylate or a placebo. Outcomes included change in nasopharyngeal viral load, time to clinical improvement, the presence of neutralizing antibodies, and safety. RESULTS: Of 96 participants randomized between November 2020 and June 2021, analyses were performed on the data of 90 participants who completed treatment (N = 61 camostat mesylate, N = 29 placebo). The estimated mean change in cycle threshold between day 1 and day 5 between the camostat and placebo group was 1.183 (P = 0.511). The unadjusted hazard ratio for clinical improvement in the camostat group was 0.965 (95% confidence interval, 0.480-1.942, P = 0.921 by Cox regression). The percentage distribution of the 50% neutralizing antibody titer at day 28 visit and frequency of adverse events were similar between the two groups. CONCLUSION: Under this protocol, camostat mesylate was not found to be effective as an antiviral drug against SARS-CoV-2. TRIAL REGISTRATION: ClinicalTrials.gov NCT04625114; November 12, 2020.

Screening of chlamydia trachomatis in non-partner donors: situation of stored donations and proposal for periodic screening.

RESEARCH QUESTION: Lack of guidance on the frequency of Chlamydia trachomatis screening in non-partner donors has led to heterogeneous testing protocols. C. trachomatis was checked in sperm donations unscreened for C. trachomatis to determine the risk for C. trachomatis infection in recipients using historic sperm donations unscreened for C. trachomatis. A C. trachomatis screening protocol is proposed to harmonize C. trachomatis screening, for which a cost evaluation is provided. DESIGN: Retrospective study of sperm donations carried out between 2009 and 2019 from healthy non-partner donors for whom at least one straw was available. A straw was selected from the still available donations that had not been tested for C. trachomatis in urine at the time of donation. These sperm samples were screened for C. trachomatis by nucleic acid amplification (NAT). RESULTS: Forty donors were included in the analysis. The 210 analysed straws tested negative for C. trachomatis. A C. trachomatis screening protocol following the European Centre for Disease Prevention and Control (ECDC) protocol for other sexually transmitted diseases (STD), i.e. NAT C. trachomatis screening of donor eligibility and first and last donation, provided these occur within 90 days, is cost advantageous compared with screening of all samples (approximately 75% reduction). CONCLUSION: A negligible risk for C. trachomatis infection was found in recipients when using historical sperm samples stored at the sperm bank. C. trachomatis screening following the ECDC protocol for other STDs is supported as it significantly reduces workload and cost compared with screening all samples.

Evaluation of Humoral and Cellular Responses in SARS-CoV-2 mRNA Vaccinated Immunocompromised Patients.

Background: Immunocompromised patients are at increased risk of severe COVID-19 and impaired vaccine response. In this observational prospective study, we evaluated immunogenicity of the BNT162b2 mRNA vaccine in cohorts of primary or secondary immunocompromised patients. Methods: Five clinical groups of immunocompromised patients [primary immunodeficiency (PID) (n=57), people living with HIV (PLWH) (n=27), secondary immunocompromised patients with a broad variety of underlying rheumatologic (n=23) and homogeneous (multiple sclerosis) neurologic (n=53) conditions and chronic kidney disease (CKD) (n=39)] as well as a healthy control group (n=54) were included. Systemic humoral and cellular immune responses were evaluated by determination of anti-SARS-CoV-2 Spike antibodies using a TrimericS IgG assay (Diasorin) and through quantification of interferon gamma release in response to SARS-CoV-2 antigen with QuantiFERON SARS-CoV-2 assay (Qiagen), respectively. Responses were measured at pre-defined time-points after complete vaccination. Results: All healthy controls, PLWH and CKD-patients had detectable antibodies 10 to 14 days (T2) and 3 months (T3) after administration of the second vaccination. In contrast, only 94.5% of the PID, 50.0% of the rheumatologic and 48.0% of neurologic patients developed antibodies at T2 and only 89.1% of the PID, 52.4% of the rheumatologic and 50.0% of neurologic patients developed antibodies at T3. At T3 no significant differences in cellular response between the healthy control group and the PLWH and CKD groups were found, while proportions of reactive subjects were lower in PID and rheumatologic patients and higher in neurologic patients. Humoral and cellular immune responses significantly correlated in the healthy control, PID, PLWH groups for all 3 antigens. Conclusion: Patients with acquired or inherited immune disorders may show variable immune responses to vaccination with the BNT162b2 mRNA vaccine against SARS-CoV-2. Whether humoral, cellular or both immune responses are delayed depends on the patient group, therapy and individual risk factors. These data may guide the counselling of patients with immune disorders regarding vaccination of SARS-CoV-2.