Risk of healing impairment following tooth extraction in patients administered with antiresorptive and non-antiresorptive polypharmacy.

INTRODUCTION: Lack of evidence existed related to the essential role by which anticancer medications alone or in combination with other polypharmacy would be accountable for wound healing impairment post-dental extraction. The following study was conducted to assess the influence of antiresorptive (AR) and non-antiresorptive (non-AR) drugs and other patient-related risk factors on wound healing status following tooth extraction. MATERIAL AND METHODS: A total of 353 patients (age range: 40-90 years, average age: 67.4 years, clinical and radiological follow-up) were recruited. All the patients were divided into three groups, which included, patients used polypharmacy with non-AR drugs, polypharmacy with a combination of AR + non-AR drugs, and the control group. Based on time of healing, the outcome was defined as, normal healing, delayed healing, and Medication-related osteonecrosis of the jaw (MRONJ). The polypharmacy score was categorized depending on the sum of the number of administered medications. RESULTS: The odds of delayed healing were significantly higher in 80+ years old patients (OR=6.98, 95 %CI:2.45-19.88, p = < 0.001) administered with AR+ non-AR drugs (OR=14.68, 95 %CI:4.67-46.14, p = < 0.001), having a major polypharmacy score (OR= 15.37, 95 %CI:4.83-48.91, p = < 0.001). On the contrary, patient administered with non-AR drugs (OR=11.52, 95 %CI: 4.45-29.83, p = < 0.001) with hyper polypharmacy (OR=58.86, 95 %CI:25.03-138.40, p = < 0.001) were significantly more likely to develop MRONJ. Smoking and extraction sites showed no significant impact on wound healing impairment. DISCUSSION: Wound healing status in patients administered with both non-AR and AR+ non-AR polypharmacy was significantly impaired following tooth extraction. Other risk factors, such as increased age and high polypharmacy scoring, also significantly contributed towards the occurrence of delayed healing and MRONJ.

Glucose and redox metabolism in meiotically blocked in vitro grown mouse antral follicles.

PURPOSE: Glucose and redox metabolism characterization in mouse antral follicles with meiotically blocked oocytes, after in vitro follicle culture (IFC) from the early secondary stage. METHODS: Following IFC (10 days), oocytes, corresponding cumulus (CC), and granulosa cells (GC) were collected from antral follicles: (i) on day 9-immature, germinal vesicle (GV) stage; (ii) on day 10, after hCG/EGF stimulation-mature, metaphase II (MII) stage and meiotically blocked (MB) immature GV stage. The metabolic profiles of all samples (GV, MII, and MB) were compared by measuring changes in metabolites involved in glycolysis, tricarboxylic acid (TCA) cycle, pentose phosphate pathway (PPP), and redox activity via enzymatic spectrophotometric assays in each cell type. RESULTS: Within MB follicles, GCs drive higher levels of glycolysis and lactic acid fermentation (LAF) while oocytes exert more PPP activity. MB-oocytes had significantly larger diameters compared to day 9 GVs. MB follicles revealed limited metabolic changes in the somatic compartment compared to their GV counterparts (before stimulation). MB-CCs showed increased aconitase and glucose-6-phosphate dehydrogenase activities with lower malate levels comparted to GV-CCs. MB and MII in vitro grown follicles displayed comparable metabolic profiles, suggesting culture induces metabolic exhaustion regardless of the maturation stage. CONCLUSIONS: Current results suggest that in addition to impaired nuclear maturation, metabolic disruption is present in MB follicles. MB follicles either compensate with high levels of TCA cycle and PPP activities in CCs, or are unable to drive proper levels of aerobic metabolism, which might be due to the current culture conditions.

The impact of CBCT-head tilting on 3D condylar segmentation reproducibility.

OBJECTIVES: To investigate whether variations in head positioning may influence the reproducibility of cone-beam CT (CBCT) three-dimensional (3D) segmented models of the mandibular condyle. METHODS: Five fresh frozen cadaver heads were scanned in four different positions: reference position (RP) and a set of three tilted alternative head positions (AP) in anteroposterior direction (AP1: 2 cm anterior translation, AP2: 5° pitch rotation, AP3: 10° pitch rotation). Surface models of mandibular condyles were constructed and compared with the condylar reference position using voxel-based registration. Descriptive statistics and a linear mixed-effects model were performed to compare condylar volumetric differences and root mean square (RMS) distance between surfaces of AP vs RP. RESULTS: The mean differences in condylar volumes of AP vs RP were 14.1 mm³ (95% CI [-79.3, 107.4]) for AP1, 1.0 mm³ (95% CI [-87.2, 89.2]) for AP2 and 0.1 mm³ (95% CI [-88.3, 88.4]) for AP3. Mean and absolute volumetric differences did not exceed earlier reported intraoperator differences of 30 mm³. The RMS distance values obtained per group were 0.12 mm (95% CI [0.05,0.20]) for AP1, 0.17 mm (95% CI [0.10, 0.22]) for AP2 and 0.17 mm for AP3 (95% CI [0.10,0.22]). The confidence intervals (CI) for RMS distance remained far below the threshold for clinical acceptability (0.5 mm). CONCLUSIONS: Within the limits of the present study, it is suggested that tilted head positions may affect the reproducibility of 3D condylar segmentation, thereby influencing outcome in repeated CBCT scanning. Nevertheless, observed differences are unlikely to have a meaningful impact on clinical patient diagnosis and management.

Classification of "Near-patient" and "Point-of-Care" SARS-CoV-2 Nucleic Acid Amplification Test Systems and a first approach to evaluate their analytical independence of operator activities.

BACKGROUND: European legislation defines as "near-patient testing" (NPT) what is popularly and in other legislations specified as "point-of-care testing" (POCT). Systems intended for NPT/POCT use must be characterized by independence from operator activities during the analytic procedure. However, tools for evaluating this are lacking. We hypothesized that the variability of measurement results obtained from identical samples with a larger number of identical devices by different operators, expressed as the method-specific reproducibility of measurement results reported in External Quality Assessment (EQA) schemes, is an indicator for this characteristic. MATERIALS AND METHODS: Legal frameworks in the EU, the USA and Australia were evaluated about their requirements for NPT/POCT. EQA reproducibility of seven SARS-CoV-2-NAAT systems, all but one designated as "POCT", was calculated from variabilities in Ct values obtained from the respective device types in three different EQA schemes for virus genome detection. RESULTS: A matrix for characterizing test systems based on their technical complexity and the required operator competence was derived from requirements of the European In Vitro Diagnostic Regulation (IVDR) 2017/746. Good EQA reproducibility of the measurement results of the test systems investigated implies that different users in different locations have no recognizable influence on their measurement results. CONCLUSION: The fundamental suitability of test systems for NPT/POCT use according to IVDR can be easily verified using the evaluation matrix presented. EQA reproducibility is a specific characteristic indicating independence from operator activities of NPT/POCT assays. EQA reproducibility of other systems than those investigated here remains to be determined.

Analytical aspects of the antinuclear antibody test by HEp-2 indirect immunofluorescence: EFLM report on an international survey.

OBJECTIVES: Detection of antinuclear antibodies (ANA) by indirect immunofluorescence assay using HEp-2 cells (HEp-2 IFA) is used to screen for various autoimmune diseases. HEp-2 IFA suffers from variability, which hampers harmonization. METHODS: A questionnaire was developed to collect information on HEp-2 IFA methodology, computer-assisted diagnosis (CAD) systems, training, inter-observer variability, quality assessment, reagent lot change control, and method verification. The questionnaire was distributed to laboratories by Sciensano (Belgium), national EASI groups (Italy, Croatia, Portugal, Estonia, Greece) and ICAP (worldwide). Answers were obtained by 414 laboratories. The results were analysed in the framework of the recent EFLM/EASI/ICAP ANA recommendations (companion paper). RESULTS: Laboratories used either HEp-2, HEp-2000, or HEp-20-10 cells and most laboratories (80%) applied the same screening dilution for children and adults. The conjugate used varied between laboratories [IgG-specific (in 57% of laboratories) vs. polyvalent]. Sixty-nine percent of CAD users reviewed the automatic nuclear pattern and 53% of CAD users did not fully exploit the fluorescence intensity for quality assurance. Internal quality control was performed by 96% of the laboratories, in 52% of the laboratories only with strongly positive samples. Interobserver variation was controlled by 79% of the laboratories. Limited lot-to-lot evaluation was performed by 68% of the laboratories. Method verification was done by 80% of the respondents. CONCLUSIONS: Even though many laboratories embrace high-quality HEp-2 IFA, substantial differences in how HEp-2 IFA is performed and controlled remain. Acting according to the EFLM/EASI/ICAP ANA recommendations can improve the global performance and quality of HEp-2 IFA and nurture harmonization.

Local radiographic risk factors for MRONJ in osteoporotic patients undergoing tooth extraction.

OBJECTIVE: To identify local radiographic risk factors for Medication-Related Osteonecrosis of the Jaws (MRONJ) in osteoporotic patients treated with antiresorptive drugs (ARD) and undergoing tooth extraction. MATERIAL AND METHODS: Patients were included in this retrospective, longitudinal, case-control study, if having at least one administration of ARD, underwent tooth extraction(s), and had pre- and post-operative panoramic radiographs. Additionally, a matched control group was selected. Three calibrated, blinded, and independent observers assessed each tooth extraction site. Statistical analysis compared control against study group, and within the latter, sites MRONJ+ and MRONJ-. RESULTS: In total, 120 patients (99 females/21 males) with 354 tooth extractions were included, from which nine patients (7.5%) and eleven tooth extraction sites (3.1%) developed MRONJ. When comparing control with study group, the latter showed significantly more thickened lamina dura, persistence of the alveolar socket, heterogeneous bone patterns, and sequestrum formation. In the study group, MRONJ developed significantly more in males (19%, p = 0.049), smokers (25%, p = 0.008), in the mandible (82%, p = 0.027), when identifying a radiolucent or sclerotic trabecular pattern (p = 0.004) or when extracting teeth with furcation involvement (p < 0.001), root remnants (p = 0.017), or unrestored caries lesions (p = 0.005). CONCLUSIONS: Tooth extraction sites showing radiographic signs of chronic dental infection are prone to MRONJ.

Retrospective cohort assessment of survival and complications of zygomatic implants in atrophic maxillae.

OBJECTIVES: To report on zygomatic implant (ZI) survival rate and associated complications through a longitudinal retrospective cohort assessment. MATERIAL AND METHODS: A total of 940 ZIs (rough: 781, machined: 159; immediate loading: 454, delayed loading: 486) and 451 standard implants (rough: 195, machined: 256; immediate loading: 58, delayed loading: 393) were placed in 302 adult patients with atrophic maxilla from December 1998 till September 2020. Following data collection reported complications were grouped based on their origin as infectious/ non-infectious biological and mechanical. Statistical analysis was performed to identify risk factors and preceding complications leading to implant loss (P < 0.05). RESULTS: The survival rate of ZI was found to be 89.9% and the average time between implant placement and an eventual loss was 4.8 years. The mean ZI follow-up period was 7.9 ± 4.9 years. Amongst the infectious biological complications, sinusitis was the most reported (n = 138) occurring at a mean follow-up time-point of 4.5 years, whereas infraorbital nerve hypoesthesia occurred more frequently in the non-infectious biological category (n = 8, meantime: 0.3 years). The prosthetic screw fracture was the most reported complication of mechanical origin (n = 29, meantime: 4 years). Furthermore, sinusitis, standard implant loss, zygomatic/peri-zygomatic region infection, and oroantral communication were significantly associated with ZI loss. CONCLUSIONS: ZI placement offered a high survival rate for the rehabilitation of severely atrophied maxilla with most losses occurring within the first 5 years at follow-up. The most frequently observed complication was sinusitis which tends to develop several years following implant placement.

Postoperative complications following orthognathic surgery in patients with rheumatic diseases: A 2-year follow-up study.

OBJECTIVE: The purpose of this study was to describe the complications following orthognathic surgery in patients with rheumatic diseases and to evaluate rheumatic disease as a possible risk factor. METHODS: A retrospective cohort study was conducted during a 6-year period. The sample consisted of rheumatic and healthy patients who underwent orthognathic surgery. The outcome variables included infection, relapse, respiratory complications, hemorrhage, neurosensory disturbances, temporomandibular joint complications, and removal of osteosynthesis material. Bivariate analysis and logistic regression were applied to identify rheumatic disease as an independent risk factor for complications after orthognathic surgery. RESULTS: Twenty patients were identified as having rheumatic diseases (male: 2; female: 18; mean age: 37.8 ± 13.6 years), and 278 patients were systemically healthy (male: 105; female: 173; mean age: 25.8 ± 11.8 years). The most frequent complications in rheumatic and healthy patients were delayed recovery from neurosensory disturbance (55% and 33%), removal of osteosynthesis material (45% and 26%), and infection (35% and 7%). Following adjustment for possible confounders, rheumatic disease showed a significant association with infection (OR = 4.191, p = 0.016). CONCLUSION: Patients with rheumatic diseases are at a higher risk of postoperative infection following orthognathic surgery compared to healthy patients.

Radiographic predictors for MRONJ in oncologic patients undergoing tooth extraction.

Tooth extraction is a risk factor for the development of osteonecrosis of the jaw following treatment with antiresorptive drugs (ARDs), but not all extraction sites develop this pathology. Therefore, we aimed to identify local radiographic predictors of Medication-Related Osteonecrosis of the Jaw (MRONJ) in panoramic images of oncologic patients undergoing tooth extraction. Based on a retrospective longitudinal cohort study design, patients were included if undergoing one or more tooth extraction, with at least one administration of ARDs, and presence of pre- and post-operative panoramic radiographs. After data collection, blinded and independent observations were performed. Eleven distinct imaging-related parameters were assessed preoperatively and five postoperatively, at each extraction site. A case-control and subgroup analysis assessing MRONJ development was performed. Significance level is set to 0.05 (5%). A total of 77 oncologic patients were selected, undergoing 218 tooth extractions, from which 63 teeth (29%) in 39 patients (51%) developed MRONJ. Results showed that patients developed significantly more MRONJ with longer ARD treatment (p = 0.057), teeth with absent and incomplete endodontic fillings with caries, widened periodontal ligament space and/or periapical lesions (p = 0.005), and sclerotic and heterogenous bone patterns (p = 0.005). In conclusion, tooth extraction sites presenting with infections and bone sclerosis are at higher risk to develop MRONJ.

Characterization of carbohydrate metabolism in in vivo- and in vitro-grown and matured mouse antral follicles†.

Establishing an ideal human follicle culture system for oncofertility patients relies mainly on animal models since donor tissue is scarce and often of suboptimal quality. The in vitro system developed in our laboratory supports the growth of prepubertal mouse secondary follicles up to mature oocytes. Given the importance of glucose in preparing the oocyte for proper maturation, a baseline characterization of follicle metabolism both in the culture system and in vivo was carried out. Markers of glucose-related pathways (glycolysis, tricarboxylic acid [TCA] cycle, pentose phosphate pathway [PPP], polyol pathway, and hexosamine biosynthetic pathway), as well as the antioxidant capacity, were measured in the different follicle cell types by both enzymatic activities (spectrophotometric detection) and gene expression (qPCR). This study confirmed that in vivo the somatic cells, mainly granulosa, exhibit intense glycolytic activity, while oocytes perform PPP. Throughout the final maturation step, oocytes in vivo and in vitro showed steady levels for all the key enzymes and metabolites. On the other hand, ovulation triggers a boost of pyruvate and lactate uptake in cumulus cells in vivo, consumes reduced nicotinamide adenine dinucleotide phosphate, and increases TCA cycle and small molecules antioxidant capacity activities, while in vitro, the metabolic upregulation in all the studied pathways is limited. This altered metabolic pattern might be a consequence of cell exhaustion because of culture conditions, impeding cumulus cells to fulfill their role in providing proper support for acquiring oocyte competence.

Analytical performance of eight enzymatic assays for ethanol in serum evaluated by data from the Belgian external quality assessment scheme.

OBJECTIVES: Fast and reliable ethanol assays analysis are used in a clinical context for patients suspected of ethanol intoxication. Mostly, automated systems using an enzymatic reaction based on ethanol dehydrogenase are used. The manuscript focusses on the evaluation of the performance of these assays. METHODS: Data included 30 serum samples used in the Belgian EQA scheme from 2019 to 2021 and concentrations ranged from 0.13 to 3.70 g/L. A regression line between target concentrations and reported values was calculated to evaluate outliers, bias, variability and measurement uncertainty. RESULTS: A total of 1,611 results were taken into account. Bias was the highest for Alinity c over the whole concentration range and the lowest for Vitros for low concentrations and Cobas 8000 using the c702 module for high concentrations. The Architect and Cobas c501/c502 systems showed the lowest variability over the whole concentration range. Highest variability was observed for Cobas 8000 using the 702 module, Thermo Scientific and Alinity c. Cobas 8000 using the c702 module showed the highest measurement uncertainty for lower concentrations. For higher concentrations, Alinity c, Thermo Scientific and Vitros were the methods with the highest measurement uncertainty. CONCLUSIONS: The bias of the enzymatic techniques is nearly negligible for all methods except Alinity c. Variability differs strongly between measurement procedures. This study shows that the Alinity c has a worse measurement uncertainty than other systems for concentrations above 0.5 g/L. Overall, we found the differences in measurement uncertainty to be mainly influenced by the differences in variability.

Mechanical properties and cellular content of leukocyte- and platelet-rich fibrin membranes of patients on antithrombotic drugs.

INTRODUCTION: The aim of this study was to examine the potential influence of antithrombotics on leukocyte- and platelet-rich fibrin (L-PRF) membranes. METHODS: Tensile tests and cell counts were performed with L-PRF membranes originating from patients on anticoagulants and antiplatelets versus patients not taking antithrombotics. RESULTS: For the tensile tests, 13 control patients, 12 on anticoagulants, and 10 on antiplatelets donated blood. Compared to controls, membranes from anticoagulated donors were weaker (strength 0.57 ± 0.24 MPa vs. 0.80 ± 0.27 MPa, p = .03) and could not be stretched as far (1.8 ± 0.3 vs. 2.1 ± 0.3 times the initial length, p = .01). For the cell counting, 23 control patients, 16 on anticoagulants, and 16 on antiplatelets donated blood. The percentage of platelets was ±50% in the three groups. The percentage of leukocytes was lower in the anticoagulant group compared with controls (69 ± 10% vs. 78 ± 8%, p = .04). However, because of the unknown error of method, it is questionable whether the statistical significance is meaningful. There was no difference between membranes from the control group and the group on antiplatelets. CONCLUSION: Our results indicate that L-PRF membranes originating from patients on anticoagulants are weaker, stretch less far, and contain less leukocytes than L-PRF membranes of patients not taking these drugs.

Improving Youden plots by including analytical performance specifications.

Historically, Youden plots are made of a scatter plot of the results of two EQA samples with an elliptical confidence area around them. Because Youden plots compare individual laboratory results with respect to other laboratories, they intrinsically evaluate with state of the art. In the field of medical laboratory science, analytical performance specifications have been proposed and various EQA providers show these limits on Youden plots by horizontal and vertical lines near the limits of total error. This study shows the weaknesses of these limits of total error and proposes a new approach for drawing acceptance areas on Youden plots. The classical Youden plot is extended with two acceptance areas: A first acceptance area evaluates the reported results with respect to maximum allowed variability, a second acceptance area evaluates the results with respect to maximum allowed variability and bias. The ellipses are calculated using quantiles of the Chi-square and noncentral Chi-square distribution. Results show that the new approach allows evaluating results from individual laboratories and from measurement procedures by comparing the position and overlap of the confidence area and the acceptance areas. It is demonstrated that his approach controls the type I error better than the rectangular limits.

Peri-zygomatic infection associated with zygomatic implants: A retrospective longitudinal cohort study.

OBJECTIVES: The main objective of this retrospective, longitudinal, cohort study was to describe the occurrence of peri-zygomatic infection (PZI) as a complication associated with zygomatic implant (ZI) placement in a period of 22 years. MATERIALS AND METHODS: A retrospective search was carried out in the department of oral and maxillofacial surgery of Saint John's hospital in Genk, Belgium. Patients that had a severely atrophic fully or partially edentulous maxilla, and at least one ZI placed, were included. RESULTS: A total of 302 eligible patients, underwent ZI surgery between 1998 and 2020. From a total of 940 ZI, 45 were associated with the development of PZI. PZI was located in the upper portion of the cheek in relation to the external corner of the eye, one or two centimeters under the lower lid. The total number of affected patients was 25 (8.3%), who had a mean age of 58.1 years. In this subset, PZI occurred in 15 cases on the right side, in eight cases on the left side, and in two cases bilaterally. Ultimately, 16 ZI were lost in the PZI site. The mean time since the implant placement to the diagnosis of PZI was 1.9 years (SD ±2.4) and to the ZI removal of 3.8 years (SD ±3.7). After implant removal, the PZI symptomatology dissipated in all patients. CONCLUSION: Peri-zygomatic infection should be informed to the patients as a possible complication after ZI placement. Once identified, it should be acknowledged as a risk factor for ZI failure.

External quality assessment providers' services appear to more impact the immunohaematology performance of laboratories than national regulatory and economic conditions.

OBJECTIVES: Medical laboratories may, at their own discretion, exceed but not undercut regulatory quality requirements. Available economic resources, however, may drive or hinder eagerness to exceed minimum requirements. Depending on the respective scopes of regulatory and economic framework conditions, differing levels of quality efforts to safeguard laboratory performance can be anticipated. However, this has not yet been investigated. METHODS: Immunohaematology external quality assessment (EQA) results collected by 26 EQA providers from their participant laboratories in 73 countries from 2004 to 2019 were evaluated. Error rates were aggregated in groups according to the respective national regulatory and economic framework conditions, to whether or not expert advice was provided in case of incorrect results, and the frequency of EQA samples. RESULTS: These representative data indicate no association between national regulatory (mandatory participation in EQA, monitoring of performance of individual laboratories by authorities, financial consequences of incorrect results) and economic (level of national income, share of national health expenditure) conditions to the quality performance of medical laboratories in immunohaematology. However, EQA providers' support for laboratories in the event of incorrect results appear to be associated with lower error rates, but a high EQA sample frequency with higher error rates. CONCLUSIONS: Further research into the impact of introducing or changing services of EQA providers is needed to confirm the results found in this first of its kind study.

A framework for evaluating the frequency of external quality assessment challenges.

BACKGROUND AND AIMS: No clear rules about the optimal frequency of organizing External Quality Assessment (EQA) rounds exist. More frequent challenges will facilitate faster responses and more reliable statistics. Adding extra samples leads to extra information, but the correlation between results from different samples reduces the extra information from additional samples. MATERIALS AND METHODS: Data were used for ALT and Albumin from the RCPAQAP EQA scheme. Every two weeks, laboratories analysed two samples. Correlation between results of different samples was calculated to determine the power of distinguishing poorly from well-performing laboratories. The power was compared to hypothetical cases of no correlation and one-sample-per-week to estimate the number of samples negated due to correlation. RESULTS: Correlation leads to negation of a number of samples, but not more than 50% of samples were negated. The number of negated samples was positively related with the correlation between EQA results. CONCLUSIONS: The proposed framework provides a quantitative evaluation of the impact of adding more EQA rounds or samples. A correlation exists and is higher for analyses performed closer in time, but the examples shown here did not show a detrimental effect on correctly evaluating laboratories.

Pre-surgical radiographic and clinical features as predictors for temporomandibular joint discectomy prognosis.

OBJECTIVES: This study aimed to identify potential clinical and radiological predictors associated with the outcome of discectomies. METHODS: In this retrospective observational study, the material comprised preoperative CBCT images and medical records of 62 patients with disc derangement disorders, who had undergone discectomy because of disc displacement with reduction (DDwR), disc displacement without reduction (DDwoR), systemic arthritis (SA), or joint hypermobility. Clinical and radiographic variables were analysed in relation to success rate determined by subjective, objective and combined outcomes. RESULTS: The success odds ratio was 11 times higher in patients with painful DDwR versus that of SA (p = 0.03), and even 25.9 times higher when considering solely objective outcome (p = 0.03). In the absence of subchondral pseudocyst, there were 5.2 times higher odds to have a successful subjective outcome (p = 0.04). Extensive bone apposition on the temporal joint component indicated a 9.3 times higher likelihood of a failed objective outcome (p = 0.04). CONCLUSIONS: There is a significant higher risk for combined outcome failure for the diagnosis SA involving the TMJ compared with DDwR. Predictors of importance based on CBCT findings related to the objective outcome failure were extensive bone apposition on the temporal joint component and condylar subchondral pseudocysts for the subjective outcome failure.

Strategy and Performance Evaluation of Low-Frequency Variant Calling for SARS-CoV-2 Using Targeted Deep Illumina Sequencing.

The ongoing COVID-19 pandemic, caused by SARS-CoV-2, constitutes a tremendous global health issue. Continuous monitoring of the virus has become a cornerstone to make rational decisions on implementing societal and sanitary measures to curtail the virus spread. Additionally, emerging SARS-CoV-2 variants have increased the need for genomic surveillance to detect particular strains because of their potentially increased transmissibility, pathogenicity and immune escape. Targeted SARS-CoV-2 sequencing of diagnostic and wastewater samples has been explored as an epidemiological surveillance method for the competent authorities. Currently, only the consensus genome sequence of the most abundant strain is taken into consideration for analysis, but multiple variant strains are now circulating in the population. Consequently, in diagnostic samples, potential co-infection(s) by several different variants can occur or quasispecies can develop during an infection in an individual. In wastewater samples, multiple variant strains will often be simultaneously present. Currently, quality criteria are mainly available for constructing the consensus genome sequence, and some guidelines exist for the detection of co-infections and quasispecies in diagnostic samples. The performance of detection and quantification of low-frequency variants using whole genome sequencing (WGS) of SARS-CoV-2 remains largely unknown. Here, we evaluated the detection and quantification of mutations present at low abundances using the mutations defining the SARS-CoV-2 lineage B.1.1.7 (alpha variant) as a case study. Real sequencing data were in silico modified by introducing mutations of interest into raw wild-type sequencing data, or by mixing wild-type and mutant raw sequencing data, to construct mixed samples subjected to WGS using a tiling amplicon-based targeted metagenomics approach and Illumina sequencing. As anticipated, higher variation and lower sensitivity were observed at lower coverages and allelic frequencies. We found that detection of all low-frequency variants at an abundance of 10, 5, 3, and 1%, requires at least a sequencing coverage of 250, 500, 1500, and 10,000×, respectively. Although increasing variability of estimated allelic frequencies at decreasing coverages and lower allelic frequencies was observed, its impact on reliable quantification was limited. This study provides a highly sensitive low-frequency variant detection approach, which is publicly available at https://galaxy.sciensano.be, and specific recommendations for minimum sequencing coverages to detect clade-defining mutations at certain allelic frequencies. This approach will be useful to detect and quantify low-frequency variants in both diagnostic (e.g., co-infections and quasispecies) and wastewater [e.g., multiple variants of concern (VOCs)] samples.

Regenerative Endodontic Procedure of Immature Permanent Teeth with Leukocyte and Platelet-rich Fibrin: A Multicenter Controlled Clinical Trial.

INTRODUCTION: The aim of this nonrandomized, multicenter controlled clinical trial was to evaluate the impact of leukocyte-platelet-rich fibrin (LPRF) on regenerative endodontic procedures (REPs) of immature permanent teeth in terms of periapical bone healing (PBH) and further root development (RD). METHODS: Healthy patients between 6-25 years with an inflamed or necrotic immature permanent tooth were included and divided between the test (= REP + LPRF) and control (= REP-LPRF) group depending on their compliance and the clinical setting (university hospital or private practice). After receiving REP ± LPRF, the patients were recalled after 3, 6, 12, 24, and 36 months. At each recall session, the teeth were clinically and radiographically (by means of a periapical radiograph [PR]) evaluated. A cone-beam computed tomographic (CBCT) imaging was taken preoperatively and 2 and 3 years postoperatively. PBH and RD were quantitatively and qualitatively assessed. RESULTS: Twenty-nine teeth with a necrotic pulp were included, from which 23 (9 test and 14 control) were analyzed. Three teeth in the test group had a flare-up reaction in the first year after REP. Except for 2 no shows, all the analyzed teeth survived up to 3 years after REP, and, in case of failure, apexification preserved them. Complete PBH was obtained in 91.3% and 87% of the cases based on PR qualitative and quantitative evaluation, respectively, with no significant difference between the groups with respect to the baseline. The PR quantitative change in RD at the last recall session with respect to the baseline was not significant (all P values > .05) in both groups. The qualitative assessment of the type of REP root healing was nonuniform. In the test group, 55.6% of the teeth presented no RD and no apical closure. Only 50% of the 14 teeth assessed with CBCT imaging presented complete PBH. Regarding volumetric measurements on RD 3 years after REP for the change with respect to the baseline in root hard tissue volume, mean root hard tissue thickness, and apical area, the control group performed significantly in favor of RD than the test group (P = .03, .003, and 0.05 respectively). For the volumetric change 3 years after REP with respect to the baseline in root length and maximum root hard tissue thickness, no significant difference (P = .72 and .4, respectively) was found between the groups. The correlation between the PR and CBCT variables assessing RD was weak (root lengthening) to very weak (root thickening). CONCLUSIONS: REP-LPRF seems to be a viable treatment option to obtain PBH and aid further RD of necrotic immature permanent teeth. Caution is needed when evaluating REP with PR.

Coverage of the national surveillance system for human Salmonella infections, Belgium, 2016-2020.

INTRODUCTION: The surveillance of human salmonellosis in Belgium is dependent on the referral of human Salmonella isolates to the National Reference Center (NRC). Knowledge of current diagnostic practices and the coverage of the national Salmonella surveillance system are important to correctly interpret surveillance data and trends over time, to estimate the true burden of salmonellosis in Belgium, and to evaluate the appropriateness of implementing whole-genome sequencing (WGS) at this central level. METHODS: The coverage of the NRC was defined as the proportion of all diagnosed human Salmonella cases in Belgium reported to the NRC and was assessed for 2019 via a survey among all licensed Belgian medical laboratories in 2019, and for 2016-2020 via a capture-recapture study using the Sentinel Network of Laboratories (SNL) as the external source. In addition, the survey was used to assess the impact of the implementation of culture-independent diagnostic tests (CIDTs) at the level of peripheral laboratory sites, as a potential threat to national public health surveillance programs. RESULTS: The coverage of the NRC surveillance system was estimated to be 83% and 85%, based on the results of the survey and on the two-source capture-recapture study, respectively. Further, the results of the survey indicated a limited use of CIDTs by peripheral laboratories in 2019. CONCLUSION: Given the high coverage and the limited impact of CIDTs on the referral of isolates, we may conclude that the NRC can confidently monitor the epidemiological situation and identify outbreaks throughout the country. These findings may guide the decision to implement WGS at the level of the NRC and may improve estimates of the true burden of salmonellosis in Belgium.