MET), encoding a receptor tyrosine kinase protein, is amplified in a subpopulation of lung cancer patients. The aim of our consecutive study was to assess whether next-generation sequencing (NGS) is a reliable method for the detection of MET gene copy number. Our study confirmed that NGS is able to detect cases harboring a high-level MET gene amplification but is unreliable and fails to detect the various levels of MET gene amplification. Therefore, NGS cannot replace the gold standard method of fluorescence in situ hybridization for the detection of MET gene copy number.Lung cancer has a high incidence and affects both men and women. Targeted therapy options directed at certain mutant proteins, and which avoid systemic chemotherapy are already available and emerging. The gene mesenchymal epithelial transition . However, next-generation sequencing (NGS) is becoming more relevant to optimize therapy by revealing the mutational profile of each NSCLC. Using evaluable n = 205 NSCLC cases of a consecutive cohort, this study addressed the question of whether an amplicon based NGS assay can completely replace the FISH method regarding the classification of MET GCN status. Out of the 205 evaluable cases, only n = 9 cases (43.7%) of n = 16 high-level MET amplified cases assessed by FISH were classified as amplified by NGS. Cases harboring a MET GCN > 10 showed the best concordance when comparing FISH versus NGS (80%). This study confirms that an amplicon-based NGS assessment of the MET GCN detects high-level MET amplified cases harboring a MET GCN > 10 but fails to detect the various facets of MET gene amplification in the context of a therapy-induced resistance mechanism.In non-small cell lung cancer (NSCLC), approximately 1–3% of cases harbor an increased gene copy number (GCN) of the  MET  encodes an RTK protein, which is mainly expressed on the surface of epithelial cells and is physiologically activated by the binding of the HGF ligand  . The prorized in ,3). Bindrized in .MET gene represents one of the drivers of tumorigenesis due to genetic aberrations, including germline or somatic point mutations, splice-site mutations leading to the skipping of exon 14 (MET exon 14 skipping) or gene amplification [In a variety of malignant tumors, the fication ,4.MET gene locus is organized in a ladder-like structure characterized by many inverted repeats and therefore resembles an area of common chromosomal fragile sites [MET gene mainly occurs in lung cancer patients but can also develop in adenocarcinomas of the gastroesophageal junction (3.3%) or in glioblastoma (1.7%) [The genetic architecture of the le sites . These sa (1.7%) .MET GCN evaluation in advanced NSCLC is apparent since MET-amplified (non-squamous) NSCLCs has shown significantly poorer prognosis in previous studies [MET gene, a high-level amplification of MET can be found only in a low number of TKI-naïve NSCLC patients with frequencies ranging from 2.0–4.0% [MET varies between studies and the test method employed.The necessity of  studies ,8. Regar2.0–4.0% ,9,10,11.MET gene can be classified as a gain in the gene copy number (GCN), i.e., the MET gene is multiplied in relation to centromere 7 (CEN7) or in comparison to normal, healthy tissue. In addition, it can be classified as amplified due to polysomy, i.e., the MET gene and the centromere CEN7 are both increased in number [MET gene aberrations can occur either de novo as an early event in tumorigenesis or as an acquired resistance mechanism due to the use of EGFR tyrosine kinase inhibitors [MET gene amplification [MET-amplified lung cancer using a tyrosine kinase inhibitor (crizotinib) was reported in women harboring a high-level MET amplification, determined by FISH (MET/CEN7 ratio > 5) [MET gene and the associated efficiency of a drug response. In 2014, it was shown that a patient suffering from NSCLC harboring low-level amplification of the MET gene  had no response to the tyrosine kinase inhibitor crizotinib. In contrast, patients with tumors harboring an intermediate amplification  of the MET gene showed a durable response [Amplification of the n number ,13. Of chibitors ,10,14,15fication . The ampfication . In 2011tio > 5) . There aresponse .MET exon 14-mutated tumors of naïve, untreated NSCLC patients. In addition, it was especially effective against high-level MET amplified tumors (MET GCN ≥ 10) compared to low-level amplified tumors [Capmatinib, another MET tyrosine kinase inhibitor, showed high antitumor activity against d tumors .MET gene amplification is not always a result of treatment but can pre-exist and be selected for during TKI treatment [In a recent study, the authors claimed that reatment . The useMET copy number changes should be an integral part of the diagnostic workup in advanced NSCLC. For the evaluation of the MET amplification status, fluorescence in situ hybridization (FISH) is an adequate and well-established method that captures the various facets of MET gene amplification, including “true” high-level MET gene amplified cases characterized by a high MET GCN (≥6 per cell) without concomitant polysomy .Hence, the assessment of MET exon 14 skipping aberrations, which cannot be detected by FISH but is of high clinical relevance [According to various studies, multigene testing for nonsynonymous mutations, translocations and copy number variations of a variety of genes is recommended to identify optimal treatment options for patients with advanced tumors, including NSCLC ,22,23. Nelevance .MET GCN is able to replace the FISH-based approach. In addition, the present study aimed to investigate whether an NGS-based determination of the MET GCN status detects all the various facets of MET amplification.The present study utilized consecutive, real-world data to investigate whether a multiplex, PCR amplicon-based NGS-based determination of the n = 327 consecutive NSCLC cases diagnosed at the Institute of Pathology of the University Hospital Erlangen, Germany, between July 2016 and May 2018 were included in this study. All cases were therapy-naïve except for one patient who had undergone EGFR TKI therapy (Case #3) for one year. Of a total of n= 327 samples, n = 205 samples could be analyzed by next-generation sequencing (NGS) and fluorescence in situ hybridization (FISH) for genetic aberrations, including the status of the MET gene copy number (GCN) .FFPE specimens of MET gene, were enriched and amplified according to the manufacturers’ instructions, using a multiplex, PCR amplicon-based 15-gene panel . In detail, the regions of interest such as the MET gene or the ERBB2 gene were amplified using tagged, gene specific primers followed by target indexing. After this, libraries were cleaned up and the quality was checked via agarose gels or with the aid of a tape station. Finally, the various libraries were pooled and were run on a MiSeq, respectively (Illumina). The obtained sequences (.fastq files) were bioinformatically analyzed using the TruSight Tumor 15 application in the BaseSpace Sequence Hub (Illumina) and aligned to the reference sequence hg19. Molecular aberrations, including point mutations, deletions and insertions, were annotated and described using standard HGVS nomenclature. In addition, changes in gene copy numbers were determined by the CRAFT copy number variant caller (v1.0.0.12) algorithm incorporated in the TST15 application. This algorithm is designed for tumor samples without matched normal controls and can detect amplifications/deletions in three genes  above a 1.6-fold change.H&E staining was performed to confirm the diagnosis of NSCLC and to estimate the tumor cell content. After microdissection of the tumor tissue, DNA was isolated using standard techniques. Regions of interest, including the MET FISH protocol, established in-house for routine diagnostics, was performed on 1–2 µm-thick freshly cut sections from FFPE NSCLC tumor blocks using a MET dual-color probe  according to the manufacturers’ standard protocol  and the routine in-house standards. Pepsin digestion was performed at 37 °C for 9 min, first hybridization at 75 °C for 10 min and the second step at 37 °C overnight. The MET Dual Color Probe used comprises one orange fluorochrome (ZyOrange) directly labeled CEN7 probe binding specific to the alpha satellite centromeric region of chromosome 7 (D7Z1) and one green labeled (ZyGreen)  probe binding the MET gene located at 7q31.2.The MET FISH status was analyzed, blinded to NGS data, using a Leica fluorescence microscope at 1000× magnification . A DAPI filter was used to visualize nuclei, and a double-bandpass filter (green/orange) was used to quantify MET and CEN7 signals. After reviewing the H&E slide of each NSCLC case and reidentifying the tumor cells in DAPI, 50 nonoverlapping tumor nuclei were evaluated, and both green and orange signals per nucleus were counted to determine the mean GCN of MET and CEN7, respectively. Cases were considered invalid if signals were hardly detectable or missing or if background staining or autofluorescence was too strong. The MET gene copy number status was classified into four groups according to Schildhaus et al.  [CEN7 signals on average ≥3.6 were classified as polysomic [The plified) . Tumor colysomic .Venn diagrams were generated using the BioVenn-Web tool . The Oncn = 327 consecutive NSCLC cases in total, n = 107 cases could not be analyzed via FISH due to an insufficient quality of the fluorescence signal, limited amount of material or lack of the corresponding material  material from external pathologies). Out of the overall cohort (n = 327 NSCLC cases), n = 23 cases could not be analyzed via NGS due to a limited amount of material or due to insufficient coverage of the MET gene locus (minimum amplicon count of >200). Overall, n = 205 NSCLC cases could be directly compared using both NGS and FISH regarding the determination of the MET GCN status.Out of MET gene amplification assessed by FISH in the present study. High-level MET amplified cases, characterized by a high MET/CEN7 ratio (≥2.0) or cluster formation , accompanied by a polysomic state of the cells, were detected in the present study cohort . Focusing on the clinically relevant high-level MET amplified cases assessed by FISH, n = 16 cases of n = 42 cases showed a MET/CEN7 ratio ≥2.0 or an average MET GCN per cell ≥6.0 or ≥10% of tumor cells containing ≥15 MET signals .In the cohort of  by FISH . These c signals a. In then = 9 cases were classified as MET gene amplified assessed by the NGS approach . Focusing on both, the high-level MET amplified cases assessed by FISH and the MET amplified cases assessed by NGS showed a better concordance. Six of n = 16 cases classified as high-level MET amplified by FISH were classified as MET amplified by NGS . The high-level amplification of the MET gene in this case likely represents a resistance mechanism as a consequence of EGFR TKI therapy.Furthermore, looking at the overlap of n = 10 cases that were not classified as MET amplified by NGS but showed a clear high-level amplification by FISH demonstrates that not only polysomic MET amplified samples, but also cases harboring a high MET GCN >10 are not reliably detected by the NGS approach .With regard to the normal, non-approach b. Many oMET GCN revealed a discrepancy of n = 48 of n = 205 cases  in the MET gene itself.Investigation of a potential correlation of an NGS-based identification of in males a. No corient age b. IntereERBB2 and EGFR, as well as somatic point mutations in the proto-oncogene KRAS, were observed . Case #3 harbored an in-frame exon 19 deletion mutation in the EGFR gene. Case #1 was characterized by a high-level MET amplification assessed by FISH, whereas case #17 showed an intermediate-level amplification and case #19 showed a normal level of the MET gene. Another notable point relates to the fact that somatic SNVs in the EGFR gene are often accompanied by amplification of the gene itself. In contrast, amplification of the ERBB2 or MET gene is usually not associated with a concomitant SNV at the DNA level.Amplifications of n = 205 consecutive NSCLC cases from routine diagnostics regarding the evaluation of MET GCN and the type of MET gene copy aberration using either an amplicon-based, 15-gene NGS panel or the standard FISH method. Out of the evaluable n = 205 cases, n = 9 cases (4.4%) were classified as MET amplified by NGS. In contrast, n = 16 cases were classified as high-level MET amplified by FISH (including polysomic cases). Only n = 6 of the amplified cases, determined by NGS, were concurrently classified as MET amplified by FISH, yielding a discrepancy of n = 7 (43.7%) cases. Focusing on the cases harboring a MET GCN >10 as assessed by FISH, the NGS approach reliably classified 80.0% of these cases as MET amplified (n = 4/5).In this study, we directly compared MET amplified cases, but not reliably. In line with previously published data, our study showed that using an NGS approach, high-level MET amplified cases . In contrast, the NanoString copy number assay, ddPCR copy number assay and custom amplicon-based parallel sequencing showed a lower concordance, especially when looking at the low-level MET-amplified samples. Furthermore, they showed that high-level MET-amplified cases generally showed better concordance between NGS and FISH than intermediate- or low-level MET-amplified cases. They claim that only MET high-level amplified samples harboring a GCN ≥6 determined by FISH can be detected via alternative methods, including NGS. Additionally, other groups such as Guo et al. and Clavé et al. already compared the specificity and sensitivity of various methods for the detection of MET gene amplification [MET gene amplification or MET exon 14 skipping. They conclude that IHC is not a useful method for the detection of genomic changes of the MET locus as with gene amplification or MET exon 14 skipping. We strictly focused on the amplification of the MET gene, comparing the two methods, FISH versus NGS. Moreover, two institutes performing NGS used a hybrid capture based assay, not a PCR amplicon-based one as we did in our analysis. Using a hybrid capture based NGS assay, one investigates a larger gene panel and could lose some information of certain gene loci. In contrast, amplicon sequencing has a higher on target rate yielding a high specificity and deep coverage.Our study shows for the first time that using the small focus gene panel TST15 from Illumina one is able to detect N >10.0, ) can be problems . They cofication ,31. Guo n = 26 MET gene amplified  samples of in total n = 222 NSCLC cases, we used an unbiased consecutive, double blinded approach. In addition, they used a 500+ NGS gene panel (PGDx elioTM) and set a cut off for MET gene amplification of >3.0-fold change. In our study, cases harboring a fold change >1.6 were classified as MET gene amplified by NGS. For assessment of MET amplification by FISH, they referred to two individual FISH score references (Cappuzzo score and UCCC FISH-criteria). In our study, we referred to only one FISH scoring system [In contrast to  their big system .MET GCN >10, even in the presence of low polysomy (n = 2 cases). However, the NGS approach was not able to detect the various facets of MET amplification. No certain log fold changes could be defined that would segregate intermediate- to low-level MET amplification. Only n = 2 cases  harboring an intermediate- or low-level MET amplification assessed by FISH were also classified as amplified by NGS. In most cases, the NGS approach classified MET GCN as normal, although the corresponding FISH samples yielded intermediate- to low-level MET gene amplification.In our hands, the PCR amplicon based NGS approach was able to detect cases harboring a high MET gene amplified cases and intermediate- to low-level MET amplified cases is of clinical relevance is still debated. “True” high-level MET-amplified cells are characterized by a high MET/CEN7 ratio without concomitant polysomy. In a retrospective study, it was investigated whether “true” high-level MET amplified cases responded more efficiently to targeted therapy than intermediate- to low-level MET amplified cases [MET amplified cases harboring MET/CEN7 ratios ≥5.0 efficiently responded to targeted therapy.Whether a discrimination between “true” high-level ed cases . The stuMET gene locus. For example, the mean coverage of the ERBB2 gene in the present study comprised 15.000 reads, whereas the average coverage of the MET gene comprised a far lower amount of less than 5.000 reads (data not shown). Although amplification of the MET gene takes place, the number of amplified reads could still be low and could impede PCR amplification and subsequent detection.The discrepancy between the FISH and NGS approaches observed in the present study could be due to biological or technical reasons regarding the Another issue concerns the tumor content and tumor purity of a sample. If the tumor content is too low, NGS might show false negative results, as the normal cell content could distort the results. Therefore, carefully concerted microdissection of the tumor area is crucial to avoid contamination with normal tissue .MET gene itself is organized on the chromosome could be problematic. The ladder-like structure characterized by many inverted repeats makes the gene locus prone to gene amplification but may also complicate sophisticated primer design [MET locus could impair appropriate dissociation of the DNA strands during the PCR denaturation step.From a technical point of view, the way the r design . PerhapsMET gene assessed by FISH, whereas the NGS method classified these cases as normal. These n = 5 cases were all polysomic, thus not resembling the “true” high-level MET amplified samples. Looking at the coverage of the corresponding NGS amplicon files revealed high coverage values of the CEN7 control region and lower coverage values for the corresponding MET locus. This observation could suggest that the TST15 CRAFT algorithm missed these high-level MET amplified cases, probably due to a defined cut-off, which depends on the calculation of the median normalized bin count of the target gene versus the median bin count of the entire panel. In the case of a polysomic state of the cell, MET amplification could be masked.Our study also highlights the limitations of the NGS approach with regard to the polysomic state of the cells. For example, cases #7, 8 and 10–12 showed a high-level amplification of the ERBB2, EGFR, MET) for n = 14 breast cancer samples using the matched normal samples or a normal reference pool. Similarly, Niu et al. defined an ERBB2 breast cancer-specific cutoff for the NGS algorithm by sequencing n = 151 ERBB2 nonamplified FFPE samples [Using the corresponding normal healthy tissue of a tumor sample as a reference for the calculation of the ratios and a normal reference pool could circumvent such a problem. Grasso et al. used a PCR amplicon-based approach and developed an algorithm for the assessment of GCN alterations that used a sequence data pool of normal samples as a reference . They co samples . By thisMET amplification by NGS is that cells harboring cluster formation of the MET gene locus can be sparse and therefore can be masked by surrounding tumor cells without cluster formation  make use of the same CRAFT gene copy number algorithm, these new methods offer the customer values of fold changes and a possibility to determine the total gene copy number of a gene of interest. Furthermore, other technologies, such as the FoundationOne Assay, used in the study of Frampton et al., provide the customer with total gene copy number values. In this particular case, they defined samples harboring ≥6.0 gene copies of the mplified .MET gene amplification determined by NGS. The trend of more males having an amplification of the MET gene is in line with data from Okuda et al., showing that in a cohort of n = 213 NSCLC cases , all MET amplified cases were males [MET gene amplification, smoking behavior could also lead to the observed phenotype. Information concerning female smokers is lacking in this particular case.Our study also focused on a possible correlation between the sex and age of the patients and concomitant re males . AlthougMET amplification. In our study, n = 4 out of the n = 9 MET gene amplified cases determined by NGS showed a concomitant molecular alteration in the genes KRAS, EGFR and ERBB2. Focusing on the “true” high-level MET amplified cases classified by FISH, n = 6 of n = 9 cases showed a concurrent mutation of other oncogenic driver genes. These results are against the common consensus in the literature that a “true” high-level MET amplification is not simultaneously accompanied by a mutation in one of the known driver genes. The study of Noonan et al. revealed that the “true” high-level MET amplified cases explicitly did not show a concomitant mutation in another oncogenic driver gene, such as KRAS or EGFR [MET amplified by the FISH approach but revealed only a MET GCN of 4.4 accompanied by a GCN of CEN7 of 1.6 leading to a ratio of 2.7. Based on the classification system according to [MET amplified but probably does not reflect a “true” high-level MET amplified case.Another interesting issue, which was addressed by the present study, was the occurrence of concomitant driver gene mutations in the presence of  or EGFR . Lookingrding to , this caMET gene in the presence of MET gene amplification. An investigation of a cohort of n = 178 NSCLC cases by NGS showed that the MET gene itself is rarely mutated, and only n = 3 cases showed a somatic exon 14 deleting splice-site mutation [MET gene impedes a concurrent mutation of the gene itself.Regarding the MET gene itself, our cohort did not include cases that showed a concurrent mutation of the mutation . CurrentMET amplification (4.4%) using FISH is similar to the observed rate in the study by Okuda et al., where they obtained a rate of 5,6% MET gene amplified cases of n = 213 NSCLC patients [MET gene in 10,9% of NSCLC cases assessed by BISH  [MET gene amplification was higher compared to their results showing MET amplification in 2.4% of n = 380 NSCLC cases determined by FISH using the University of Colorado Cancer Center criteria (UCCCC) [MET gene amplified cases. Using MET IHC, they found that 13.7% of patients showed MET overexpression. In another study that investigated a large cohort of patients with surgically resected NSCLC for MET overexpression and gene amplification, Sterlacci et al. found MET amplification in 2.4% of cases assessed by FISH using tissue microarrays  [MET high-level amplified. However, low- and intermediate-level amplifications were assessed in 30% of investigated NSCLCs [MET amplification regarding the prognostic value and the various levels of MET amplification, respectively, is challenging.Our rate of clinically relevant “true” high-level patients . Tsuta er cells) . On the  (UCCCC) . Using tMET FISH). Moreover, we were not able to directly link the MET GCN alteration status with smoking behavior and prognosis of the respective NSCLC patients. In addition, we were not able to monitor whether these patients obtained targeted therapy. One reason for this lack of information is that some cases had been sent from external pathologies. Another point of criticism is the fact that 32.7% of our cohort could not be evaluated using MET FISH due to insufficient quality of the fluorescence signal, limited amount of material, or lack of the corresponding material. These limitations were also encountered in other studies where for example silver in-situ hybridization (SISH)--- was not assessable in 15% of patients [MET gene locus. This highlights a positive aspect of the NGS method as—although material can be sparse—the PCR amplification step and the highly sensitive sequencing platform make it possible to analyze a specimen, which could be potentially problematic for FISH.The disadvantages of our study comprise the partly retrospective character of the study (assessment of patients . RegardiMET amplification is still a rare event in NSCLC tumorigenesis. In the near future, however, it could become more prominent as a potential secondary resistance mutation in response to an increasing number of prescribed tyrosine kinase inhibitor therapies. Therefore, the need for a harmonized scoring system using FISH, which enables a comparison between laboratories and the dissection of high-, intermediate- and low-level amplified cases, is still crucial. In addition, one should be aware of the limitations that arise when MET GCNs or GNCs in general are assessed by NGS. The use of normal reference pools in the case of MET GCN determination should be carefully considered, as nonclinical relevant high-level polysomic cases could also be included in this way of therapy stratification. Of interest, TKI-induced, resistance-associated amplification of the MET gene is not exclusively characterized by high-level MET amplification, as intermediate- to low-level amplifications are also observed [MET GCN is currently inevitably necessary to detect the various facets of MET gene amplifications.In summary, our data agrees with previous studies in that a “true” high-level observed . TherefoMET gene copy number. Using FISH during routine diagnostics for the assessment of the MET gene copy number will remain necessary to reliably detect the various levels of MET gene amplification.The findings of the present study are of great importance, as the future of routine molecular diagnostics will be mainly based on NGS data. As gene amplifications can be obtained easily by NGS, one would be tempted to avoid a time- and tissue-consuming additional method such as FISH. However, NGS does not resemble a reliable substitute for the assessment of


The use of antibiotics in dentistry as prophylaxis and treatment is frequent. Their misuse has led to a major public health problem globally known as antibiotic resistance. This study aimed to assess the pattern of antibiotic prescription and its prophylactic use for systemic conditions. Besides, this study evaluated the awareness and adherence to antibiotic prescription guidelines and antibiotic prophylaxis guidelines along with awareness of antibiotic resistance across pediatric and general dentists.An overall of 378 pediatric and general dentists meeting the required eligibility criteria, fulfilled a pre-designed validated questionnaire. Data were collected, tabulated, and statistically analyzed.A significant statistical difference was found among the pediatric and general dentists regarding antibiotics prescription for most of the oral conditions where Amoxicillin with clavulanic acid was the most frequently prescribed antibiotic among the two groups . The majority of pediatric and general dentists, on the other hand, were aware of antibiotic resistance and prescribing recommendations.The present study showed a tendency to overprescribe and overuse antibiotics in certain dental conditions among the participants. The vast majority of dentists, especially general dentists do not have adherence to professional guidelines for antibiotics prescription in children despite their awareness of antibiotic resistance and prescription guidelines.The online version contains supplementary material available at 10.1186/s12903-021-01685-y. Antibiotics have been widely used in children and adults since Alexander Fleming discovered penicillin in 1928 and Howard Walter Florey began using it clinically in 1941. Dentists routinely prescribe antibiotics for therapeutic or prophylactic reasons for the management of oral and dental infections [From 2000 to 2015, the global antibiotic consumption in terms of defined daily doses has seen an increase of 65%, mainly in low middle-income countries where the use of antibiotics in developing countries has been observed to be much higher compared to the developed world .The success of antibiotics has been absolute, with a worldwide enhancement in morbidity and mortality. Though, this early success is being battered by the microbial capability to grow and renovate to protect themselves, thus neutralizing the therapeutic effects of antibiotics .In the twenty-first century, the increasing emergence of antibiotic-resistant bacteria is evolving into a major public health threat. This threat is no longer a prediction for the future but a current finding in every region of the world, claiming 700,000 lives each year, a figure that could rise to 10 million by 2050 , 5.Although there are lots of causes for the increased rates of resistance, the overuse of antibiotics is the most critical, even though the antibiotic prescription is still deemed an insignificant act , 7. UnsuIt was stated in 2015, that 40–50% of antibiotic prescriptions worldwide were unnecessary . The proA contribution from pediatric dentists (PD) has also been noted as an unwarranted use of antibiotics and was reported in children with orofacial infections , 10. SurThe observed prescribing behavior in reference to pediatric dentistry could be a result of the pressure of the parent where this parental pressure has been reported as one of the primary causes of unwarranted prescriptions. Other non-clinical situations acting as determinants for antibiotic use include uncertain diagnosis of the case, need for delay of treatment owing to unavailable appointments, problems like ineffective sterilization, and social relations. Also, it was suggested that practitioners assume that using antibiotics is the quickest way to resolve any form of consultation .Many studies in the literature described general and pediatric dentists’ antibiotic prescription patterns worldwide –14. HoweThis was a cross-sectional questionnaire study conducted among a random sample of Egyptian dentists, with an electronic link to the questionnaire produced by Google forms and distributed via email. The STROBE guidelines were used to ensure the reporting of this observational study.The current research was carried out in compliance with the Helsinki Declaration. Ethical approval was obtained from the Ethics Committee of Scientific Research, Faculty of Dentistry, Cairo University. Dentists were notified about the objective of the study and informed consent was acquired at the beginning of participation to answer the online survey questionnaire with the maintenance of confidentiality and anonymity by the settings of the online survey.According to the results of Konde et al.  where thGeneral dentists holding a Bachelor of oral and Dental medicine or a master's or doctorate in a specialty other than pediatric dentistry.Pediatric dentists holding a master's or doctorate.Any nationality except Egyptian.Dentists who aren't engaged in clinical practice.Dentists who didn't treat children.The antibiotic prescription patterns in case of pulpitis, draining sinus tract, localized intraoral swelling, acute facial swelling, dental trauma, pediatric periodontal diseases, pericoronitis, simple extraction, extraction by open method, periapical abscess, apical periodontitis, dry socket, evidence of anaerobic infection in children.Use of antibiotics as prophylaxis for systemic conditions including cardiovascular diseases, viral infections, juvenile diabetes, blood dyscrasias, respiratory disorders in children.The awareness and adherence to the antibiotic prescription guidelines.The awareness and adherence to guidelines for antibiotic prophylaxis.The awareness of antibiotic resistance across pediatric and general dentists.The dentist's perspective of possible causes of antibiotic misuse.This survey aimed to assess:An English pre-prepared validated self-administered questionnaire based on the previous work of Konde et al.  after taAn electronic link to the questionnaire was generated using Google forms and was distributed to a representative sample of 1512 dentists through emails. The study sample was chosen using a random number generator from a list containing all dentists fulfilling the inclusion and exclusion criteria obtained from the official registry of the Faculty of Dentistry, Cairo University.The questionnaire was prefaced with the consent section which explained the study's purpose, nature of the survey, study objectives, and voluntary participation. Informed consent was obtained from each participant in the form of answering a question (Yes/No) before proceeding with answering the questionnaire.The questionnaire consisted of close-ended questions (Yes/No) or multiple choices. Four questions related to demographic details and practice information including specialty, graduation university, clinical experience, and work practice.Twenty questions describing clinical situations for which antibiotics were prescribed routinely, the most commonly prescribed antibiotics, the duration of the antibiotic course, prescription in presence of anaerobic infection, and systemic conditions.Along with awareness of antibiotic resistance, the awareness and adherence to the AAPD guidelines for use of antibiotic therapy for pediatric dental patients  and antiPossible causes of antibiotic misuse from the dentist's perspective were evaluated in six questions with Yes/No answers.All dental practitioners who took part in the study were chosen randomly and asked to complete a self-administered questionnaire anonymously to limit selection bias. All participants were given the same explanation about the study's nature and purpose to limit information bias .The statistical analysis program Statistical Package for Social Sciences (SPSS) version 22.0 was used to undertake all the statistical analysis. For the categorical data, descriptive statistics were used to calculate numbers, frequencies, and percentages for each category. A Chi-square test was utilized for comparing prescription patterns between general and pediatric dentists.Out of 1512 mailed questionnaires, only a total of 378 dentists completed the questionnaire where 154 (40.7%) were pediatric dentists and 224 (59.3%) were general dentists with a 25% response rate. The demographic data and practice information of the participants were displayed in Table Clinical situations for routine antibiotic prescriptions were demonstrated in Table The antibiotic prescription patterns among the pediatric and general dentists were displayed in Table A large number of pediatric dentists (90.3%) and general dentists (89.3%) prescribe prophylactic antibiotics in cases of cardiovascular diseases with no statistical significance between both groups (p = 0.760) while the majority of pediatric and general dentists didn’t prescribe antibiotics in case of viral infections, juvenile diabetes, blood dyscrasias, and respiratory disorders with no statistical significance between both groups as demonstrated in Table Concerning the awareness of the guidelines, 67.5% of pediatric dentists, and 62.1% of general dentists were aware of the antibiotic prescription guidelines with no statistical significance between both groups (p = 0.275) while 74.7% of pediatric dentists and 70.1% of general dentists were aware of the antibiotic prophylaxis guidelines with no statistical significance between both groups (p = 0.329) as displayed in Table Regarding adherence to guidelines, 68.2% of pediatric dentists and 53.6% of general dentists were adherents to the antibiotic prescription guidelines with statistical significance between both groups (p = 0.004) while 68.2% of pediatric dentists and 73.3% of general dentists were adherents to the antibiotic prophylaxis guidelines with no statistical significance between both groups (p = 0.247) as displayed in Table Nearly all pediatric and general dentists were aware of antibiotic resistance and most of them grasped that self-medication and injudicious use of antibiotics lead to the development of antibiotic resistance with no statistical significance between both groups as revealed in Table Before prescribing antibiotics, the majority of participants ask whether the patient has taken antibiotics in the previous week and instruct them to adhere to the dose as demonstrated in Table Only 5% of general and pediatric dentists prescribed antibiotics because of insisting parents or the presence of too many patients in the waiting area and less than 15% prescribed antibiotics to sustain the patient until the specialist treats the patient with no statistical significance between both groups as revealed in Table Antibiotic resistance is growing to alarmingly high levels worldwide, endangering our capability to treat frequent infectious diseases. This antibiotic resistance is exacerbated by antibiotic misuse and overuse, as well as ineffective infection prevention and control .Reinforcement of the global antimicrobial resistance surveillance is crucial for establishing global strategies, monitoring the effectiveness of public health interventions, and detecting new trends and threats. Hence, This study is an attempt to contribute to the available literature on antibiotic use and abuse, particularly when treating children in the Middle East .The present study showed a tendency to overprescribe and overuse antibiotics in certain conditions like pulpitis, draining sinus tract, localized intraoral swelling, periapical abscess, apical periodontitis, dry socket which occurred more by general dentists in comparison to pediatric dentists. These findings were in accordance with several studies which indicate that inadequate understanding of the disease, uncertain diagnosis, time pressure, patient expectation, parental pressure, and refusal of operative treatment may be the primary reasons , 13, 20.Amoxicillin with clavulanic acid was the most frequently prescribed antibiotic followed by Amoxicillin. It may be contributed to the effectiveness of Amoxicillin against Streptococci and oral anaerobes which make them appropriate for the treatment of odontogenic infections and the advantage of Amoxicillin with clavulanic acid to preserve the activity against the Beta-lactamases commonly produced by microorganisms associated with odontogenic infections , 21.According to the American Academy of Pediatric Dentistry, Metronidazole may be prescribed as a supplementary antimicrobial treatment in the presence of anaerobic bacterial involvement . This maRegarding the duration of antibiotic prescription, the majority of the dentists in both groups prescribed antibiotics for 5–7 days allowing resolution of signs and symptoms with no risk of clinical and microbiological relapse. Moreover, antibiotic prescription for the correct duration lowers the unfavorable results and relieves the dilemma of antibiotic resistance , 15, 21.Concerning antibiotic prescription for systemic conditions, a majority of dentists would prescribe antibiotics in cases of cardiovascular diseases while in case of viral infections, juvenile diabetes, blood dyscrasias, respiratory disorders majority of dentists declared they would not prescribe any antibiotics.While there is a possibility that oral microorganisms can germ and infects distant tissues after oral practices, there is no proven evidence that this happens. Therefore, the fact of when and for which situations systemic prophylactic antibiotics are needed is debatable , 22.The American Heart Association (AHA) proposes antibiotic prophylaxis for patients with cardiac disorders as they have the greatest risk of an unfavorable outcome , 17.The awareness of guidelines for the prescription and prophylaxis of antibiotics was found to be around 70% of the study population where only 30% felt inadequately informed and trained regarding antibiotic use with no statistical significance between both groups in line with previous studies , 20.Pediatric dentists showed comparatively better adherence to the guidelines as compared to general dentists in our study which may be contributed to the fact that pediatric dentists treated children more often and usually have more years of education in treating children .But still, adherence of pediatric dentists to guidelines was considered low (less than 60%). This finding was in accordance with previous studies that have demonstrated varying low levels of adherence among pediatric dentists in various parts of the world, ranging from 10 to 56% , 11–13.Despite the awareness of antibiotic resistance and prescription guidelines among most of the participants, still there is a misuse in antibiotics prescription. This might be explained by the fact that although knowledge of clinical guidelines and research evidence directly influence antibiotic prescribing, other barriers or competing factors exist that hinder their use in the oral health setting .The results of the present study showed that the majority of the participants inquire the patient about taking antibiotics in the past week before prescribing antibiotics and advise them to adhere to the dosage regimen. Also, the presence of too many patients in the waiting areas or the need to sustain the patient till the next appointment were not reasons for prescribing antibiotics. Dentists demonstrate a lack of worry in limiting this major problem which may be defended by the statement of self-medication as a trigger for its advancement.This may be explained by the socially desirable responding phenomena, that is, when inquired about unprofessional acts, participants may reply in a way that they feel socially acceptable rather than disclosing information about their true behavior .The data collected was self-reported by the participants and did not examine the actual prescription from the patient files. Furthermore, reporting bias is a concern, as dentists’ responses may not truly reflect their actual practice. In addition, non-response bias is another possible drawback of self-administered questionnaire research. Respondents' answers may have varied more positively or negatively from those of non-respondents, making it difficult to predict which direction the non-responders will lean.The present study showed a tendency to overprescribe and overuse antibiotics in certain dental conditions among the participants. The vast majority of dentists, especially general dentists do not have adherence to professional guidelines for antibiotics prescription in children despite their awareness of antibiotic resistance and prescription guidelines. Nearly all pediatric and general dentists were aware of antibiotic resistance, and the majority of them were also aware of the guidelines for the prescription and prophylactic use of antibiotics for systemic conditions.Additional file 1. Questionnaire used in the present study.


High temperature conditions, such as fire, are detrimental factors to the mechanical and chemical properties of concrete. In this paper, the authors developed a new type of coarse aggregate, named PCM/SiC composite aggregate (enhanced aggregate: EA), to improve fire-resistance performance. To investigate the validity of EA for construction materials, a compressive strength test, static modulus of elasticity, X-ray diffraction (XRD), and scanning electron microscopy (SEM) were conducted. In addition, this EA has been developed to improve residual performance after exposure to high temperature, with residual compressive strength and internal temperature measurement tested at 1000 °C. Furthermore, chemical properties after heating were investigated by XRD and SEM-EDAX. The results show that the percentage of residual compressive strength of heated concrete with EA is higher than plain concrete. The concrete with EA exhibited primary cement composites such as C-H and C-S-H after exposure to high temperature through XRD and SEM-EDAX. On the other hand, major hydration products could not be observed in plain concrete. PCM and SiC offer an opportunity to delay the increase in concrete temperature. From evaluation of the results, we can see that EA enhanced the residual performance of concrete after exposure to high temperature conditions. Concrete structures are incombustible construction materials compared with wood and steel when exposed to fire or high-level temperatures. Though concrete is a nonflammable material, the mechanical, chemical and durability properties of concrete can be detrimental at elevated temperature conditions . These pThere is previous research that has studied the thermal performance of concrete blended with SCMs, such as fly ash (FA), silica fume (SF), and grounded granulated blast furnace slag (GGBS) to improve the properties of concrete after exposure to high temperatures. Pozzolanic concrete with fly ash (30%) and blast furnace slag (40%) have shown a higher retained residual strength in normal concrete at high temperatures of around 600 °C  have evaIn addition, many researchers have investigated the thermal performance of concrete when adding diverse fiber types at elevated temperatures. Lau et al.  comparedFinally, the impact of aggregate type of residual performance on concrete has been studied by various researchers. Generally, natural aggregate has poor performance in residual compressive strength compared with recycled concrete aggregate (RCA) at high temperature because natural aggregate’s composition of siliceous mineral expands at elevated temperature. Salahuddin et al.  investigResearch on improving residual mechanical properties in the above methods is being actively conducted. However, little is known about the new type of developed aggregate. For this reason, this study developed a new type of aggregate to improve residual performance after exposure to high temperatures. The new type of developed composite aggregate in this research was called enforced aggregate (EA), which refers to PCM/SiC composite aggregates. This EA was used for coarse aggregate in concrete, while concrete that contains EA shows a delay in the time it takes to increase the internal temperature when exposed to elevated temperatures. Therefore, first of all, the mechanical and chemical properties of concrete with EA at normal conditions were examined to evaluate EA’s usefulness as a construction material. This was undertaken through compressive strength test, X-ray diffraction (XRD), scanning electron microscopy and energy dispersive spectroscopy (SEM-EDAX). In addition, to investigate the effect of EA on the behavior of concrete at high temperatures, residual compressive strength was examined after various high temperature and internal temperature measurements of concrete with EA were conducted in an electric furnace at 1000 °C. XRD and SEM-EDAX were performed to explore chemical properties after exposure high temperature.3, water absorption: 1.11%) was used as fine aggregate. Paraffin wax and silicon carbide (SiC) were used for developing the high thermal resistance aggregate as impregnating material and coating, respectively. SiC  was obtained from a local company . High porosity of air-cooled blast furnace slag aggregate (ACBFS) are selected as a phase-change material (PCM) carrier which was offered by manufacturer  . Paraffin wax produced by the manufacturer  is well known for having a mixture composed of hydrogen and carbon molecules with the configuration (CnH2n+2). It has a high specific fusion heat, is reliable, less expansive and is chemically stable when it is flashed. Paraffin wax has a melting point of 47 °C and a flash point of 202 °C.This study used ordinary Portland cement (OPC), which follows ASTM C150  as the cThe processes of coating and impregnation were conducted as follows;(1)Prepared ACBFS in the bowl with melted paraffin wax at 80 °C in dry oven for 4 h at 100 °C.(2)After 4 h, the dredged impregnated slag aggregates are put in an ice chamber quickly and left to congeal for 10 min.(3)After the prior steps, SiC is coated on the surface of the impregnated aggregates with mixed polymer and PVA water soluble film.3). In the other case, 5% of the SiC was replaced by the natural fine aggregate which, with its finer particle size compared with normal fine aggregate, could improve the mechanical properties. Then, these concretes were evaluated for their mechanical and chemical properties and their post-fire residual properties. The concrete mixes were designed with a target strength of 30 Mpa . The sluSpecimens were prepared with 100 mm (diameter) × 200 mm (height) cylinder concretes to perform compressive strength tests after 3, 7, and 28 days as per ASTM C39 . In addiIn the case of concrete, physical and mechanical properties are very important when it is exposed to high temperature. In particular, concrete which is exposed to high temperature undergoes severe damage and a change of its chemical composition. These changes usually result in the generation of a hardened cement paste by the dissociation of calcium hydroxide (C-H) at 400 °C. Afterward, it continues toward a complete decomposition and destruction of the hydration products, such as calcium silicate hydrate (C-S-H) gel, at around 900 °C. Furthermore, the effects of exposure to the high temperature could be revealed in the forms of cracking, spalling and disintegration on the surface which is destructive to the concrete structure. Therefore, residual strength is a considerably important factor for evaluating the deterioration degree of the concrete. The residual compressive strength test was conducted after exposure to temperatures of 200, 400, 600 and 800 °C. Specimens were taken out of the furnace and then a compressive strength test was conducted after cooling, upon reaching each temperature interval. In the cases of those exposed to high temperature, the internal temperature condition of the concrete is significantly important point in terms of its structural safety. The use of PCM in concrete can improve its high thermal resistance. The PCM hinders the increasing temperature of concrete because it absorbs heat during two steps of their phase changing, solid to liquid (around melting point) and liquid to gas (around flash point) . TherefoTo evaluate the performance of the physical properties of specimens with EAs, a compressive strength test was conducted after 3, 7 and 28 days. The results of the compressive strength tests are shown in From the results, the compressive strength values of the specimens EAs were decreased in comparison with the plain specimen. Plain specimen was exposed to 26.78 MPa after 3 days. Finally, it was exposed to 41.04 and 46.24 after 7 and 28 days, respectively. However, the specimen with 50% of EA was exposed to 21.2, 30.4 and 36.7 MPa after 3, 7 and 28 days respectively, reflecting a decrease of 21, 27 and 21% in comparison with the plain specimen after 3, 7 and 28 days respectively. In addition, the specimen composed of 100% EA showed a decrease of 19, 18 and 19% in comparison with plain specimen after 3, 7 and 28 days respectively. The compressive strength decreased slightly in comparison with the plain specimen with increased usage of EAs. On the other hand, the P+SiC specimen demonstrated a compressive strength of 28.54 after 3 days, while after 7 and 28 days, was at 43.12 and 49.27 respectively. The compressive strength of the EA100+SiC specimen reached 23.5, 36.4 and 40.71 MPa after 3,7 and 28 days, respectively, which was lower than P+SiC by about 21%. Thus, compressive strength decreased with the increased usage of EAs. The reduction of this mechanical property is caused by comprehensive effects. In this study, the coarse aggregate was replaced with EAs . TherefoFrom the results of the stress-and-strain curves in The highest vulnerability of the deformation was observed under a short-term loading with each replacement ratio of EAs. Furthermore, the highest effect of the deformation under load is greatly related to specimens with a low modulus of elasticity. It is assumed that the lower adhesion of EA compared with natural aggregate with cement paste can be attributed to the oil contents of paraffin wax.As mentioned in the compressive strength section, the poor adhesion between paste and EAs could be attributed to the reduction of the mechanical properties. On the other hand, the use of SiC is slightly affected by the improvement of the mechanical properties of the deformation. The static modulus of elasticity of the specimens with SiC was increased in comparison with those without SiC. However, the reduction of static modulus of elasticity in the EA100 and EA100+SiC specimens was slightly minor. Therefore, the use of EA is reasonable to be applied in concrete structures. To evaluate hydration characteristic and chemical composition of specimens with EAs, XRD patterns were analyzed after 3, 7 and 28 days. The XRD patterns of hydrated samples are shown in The clinker materials can be seen in To analyze the hydration products of specimens with or without EAs, SEM-EDAX analysis was conducted after 3, 7 and 28 days. The results are shown in Various hydrated phases were observed in all figures, such as C-H, Ettringite, and C-S-H formation. These products are commonly observed in plain and EA samples after 3 days. In case of samples with SiC, C-H and ettringite formation was also observed after 3 days. The phases are much denser after 7 days, with C-H formation in particular much bigger than at 3 days.However, C-S-H was observed around SiC particles in P+SiC and EA100+SiC samples after 7 days. SiC particles were verified by EDAX analysis. As per the results of the EDAX analysis, it can be confirmed that the peak of O (oxygen) is very low. In addition, it can be confirmed that paste and SiC are well combined in the matrix.In addition, by day 28, the structure was considerably denser than at 7 days. Furthermore, the Si peak of the EA100 sample was lower than in the plain sample per the results obtained by the EDAX analysis. The effect of paraffin wax on hydration can be observed by the mass of paraffin wax lost to hydration. Therefore, this can be affected to reduce the hydration products at the initial stage. This result can be verified with the results of EDAX for the EA100+SiC sample in comparison with the P+SiC sample. All chemical elements are lower than P+SiC. However, SiC particles are well combined in the cement paste. Thus, it can be proved that the mechanical properties were improved with the use of SiC as a filler.To confirm the condition on the surface of samples after high-temperature exposure (1000 °C), the damaging effect on the surface of samples was observed by microscope, 160 times magnification. The results of the observation on the samples are shown in From the results, a lot of cracks on the surface of the plain sample can be observed after high-temperature exposure. Verification of the concrete damaged by high temperature can be seen with visual observation of the color changes , as wellIn the case of the heated concrete surface, the damage was significantly affected by parallel degradation of the concrete strength and pressure of concrete pores . In addiSiC particles (green) around aggregate can be observed in Mechanical and physical properties are dramatically decreased with high temperature exposure. Therefore, residual compressive strength tests were conducted after various temperature  exposures. Specimens were cooled at room temperature for this test. These were calculated by using the percentage of retained strength in comparison with unheated specimens. The results show that the residual rate of compressive strength on every specimen was significantly decreased. Residual rate of the compressive strength of the plain specimen was 33.4% at 800 °C. In the case of the P+SiC specimen, this was at 35.2% after exposure to 800 °C. However, the EA100 and EA100+SiC specimens were higher than the plain and P+SiC specimens. The EA100+SiC specimen was at 56.24% after being exposed to 800 °C. The compressive strength could be reduced by the dehydration caused by evaporating free water due to the high temperature ,27,29. FThe results of internal temperature measurements of the specimens are shown in In In order to evaluate the chemical analysis of the samples after heating, an XRD analysis was conducted. The results of the XRD analysis of the heated samples are shown in 3S and C2S is higher than EA samples in the results. The results show that C-S-H phase decomposed due to the high temperature at around 800 °C. At that time, the intensity of C3S and C2S increased whereas the intensity of C-S-H decreased. The C-S-H intensity of the plain sample was not observed, whereas C-S-H intensity of EA100 sample was observed around 28°. On the other hand, the intensity of the paraffin wax was indicated in the EA samples. Carbon intensity was observed at around 27° because of the paraffin wax.From the results, C-S-H intensity was not observed in the plain sample. In addition, intensity of CIn addition, SiC intensity was observed in the EA samples at around 36°. Every sample for XRD analysis was produced after internal temperature experiment was conducted to 1000 °C. Thus, SiC intensity can be seen in the result. In addition, paraffin wax remained in the samples with EAs after high-temperature exposure. As result, paraffin wax and SiC on the surface of the aggregate can affected a much lower decomposition and dehydration around the ITZ parts. Thus, EAs can prevent rapid reduction of the mechanical and physical properties in a concrete system at high-temperatures exposure.The microstructure and physical state of heated samples was analyzed by SEM-EDAX. The results of SEM-EDAX are shown in The results show that the C-S-H phases disappeared. Also, many cracks can be observed in On the other hand, the EA100 sample showed the C-S-H and CH products that can be observed in However, EA100 sample can be seen with C-S-H gels and CH. As shown by the results of the plain and P+SiC EDAX analyses, the peak and weight of Ca is much higher than in the EA100 sample. However, in the case of the Si peak and weight, EA100 and EA100+SiC samples are much higher than the P and P+SiC samples. In the case of the EA100 and EA100+SiC samples, SiC particles can be verified around hydration products. As shown by the results of the EDAX analysis, there are Si and C peaks. Furthermore, the O peak is much lower than the others. Therefore, it can be proved that SiC particles remained after high-temperature exposure. Thus, SiC particles on the surface of the aggregate can be attributed to a delay of, and protection against, the decomposition and dehydration processes. Finally, the concrete with EA has demonstrated that it has a good ability to improve fire resistance. These results demonstrate that a delay in the decomposition time and decrease of strength can be attributed to paraffin wax and SiC coating.The main purpose of this study was to develop a PCM/SiC composite aggregate called EA. The properties of concretes containing EA were investigated before and after exposure to high temperatures.The compressive strength of concrete with EA reduces gradually according to the increasing replacement of natural aggregate with EA. However, compressive strength tended to increase by adding SiC whose particle size is smaller than the fine aggregate. SiC can lead to a bridging effect and the formation of voids after hydration.The reason for the deformation under load is highly related to the low modulus of elasticity of specimens. This shows that poor adhesion of EA between the surface of EA and cement paste can be attributed to the oil contents of paraffin wax. However, SiC is slightly affected by the improvement of the mechanical properties of the deformation.From the XRD results, the tendency of plain concrete and concrete containing EA are similar because PCM and SiC are non-reactive materials.In the SEM-EDAX section, main gradients in concrete, such as C-H and C-S-H were observed. Also, SiC particles were well bonded to the cement matrix, which can improve its mechanical properties.Properties of concrete before exposure to high temperatures.EA100+SiC has the lowest reduction in compressive strength after high-temperature exposure. This is because paraffin wax absorbs the heat that would otherwise power aggregate expansion and SiC maintains the bridge effect even after heating.According to the internal temperature measurement test, the temperature behavior of EA100 and EA100+SiC specimens show a lower temperature trend compared with plain concrete. Also, the EA100+SiC specimen was delayed by 16 min in reaching 200 °C, which means that EAs in concrete hamper the increasing temperature at the initial stage in elevated temperature conditions.In SEM-EDAX, after exposure to high temperature, C-S-H phases disappeared and there were easily observed cracks. However, the concrete specimen with EA displayed S-C-H gels and C-H. Delay and protection against the decomposition of chemical products can be attributed to EA.Properties of concrete after exposure to high temperatures.


Despite the great progress made over the last decades, stronger structural interventions are needed to end the HIV/AIDS pandemic in Low and Middle-Income Countries (LMIC). Brazil is one of the largest and data-richest LMIC, with rapidly changing socioeconomic characteristics and an important HIV/AIDS burden. Over the last two decades Brazil has also implemented the world’s largest Conditional Cash Transfer programs, the Bolsa Familia Program (BFP), and one of the most consolidated Primary Health Care (PHC) interventions, the Family Health Strategy (FHS).We will evaluate the effects of socioeconomic determinants, BFP exposure and FHS coverage on HIV/AIDS incidence, treatment adherence, hospitalizations, case fatality, and mortality using unprecedently large aggregate and individual-level longitudinal data. Moreover, we will integrate the retrospective datasets and estimated parameters with comprehensive forecasting models to project HIV/AIDS incidence, prevalence and mortality scenarios up to 2030 according to future socioeconomic conditions and alternative policy implementations.We will combine individual-level data from all national HIV/AIDS registries with large-scale databases, including the “100 Million Brazilian Cohort”, over a 19-year period (2000–2018). Several approaches will be used for the retrospective quasi-experimental impact evaluations, such as Regression Discontinuity Design (RDD), Random Administrative Delays (RAD) and Propensity Score Matching (PSM), combined with multivariable Poisson regressions for cohort analyses. Moreover, we will explore in depth lagged and long-term effects of changes in living conditions and in exposures to BFP and FHS. We will also investigate the effects of the interventions in a wide range of subpopulations. Finally, we will integrate such retrospective analyses with microsimulation, compartmental and agent-based models to forecast future HIV/AIDS scenarios.The unprecedented datasets, analyzed through state-of-the-art quasi-experimental methods and innovative mathematical models will provide essential evidences to the understanding and control of HIV/AIDS epidemic in LMICs such as Brazil. It is estimated that 37.7 million people are currently living with the HIV/AIDS and 680 thousand die annually worldwide . While, Brazil is one of the largest and most inequal Low and Middle-Income countries, it implemented during the last decades a number of well-evaluated programs intended to meet the needs of people living with HIV/AIDS, and its comprehensive approach has been regarded as relatively successful , 15. NevThe Brazilian conditional cash transfer (CCT) program, Bolsa Familia Program (BFP), is the world`s largest CCT and targets poor households earning between US$18–36 per person per month. Eligibility is assessed at the federal level to minimize errors and fraud. The monthly cash benefits range from US$17 to a maximum of US$41 (depending on household size and composition) and payments are credited directly to a beneficiary debit card –23. BrazAim 1To integrate aggregate and individual-level large datasets to evaluate the effect of a broad range of socioeconomic determinants on incidence, treatment adherence, hospitalization, case fatality and mortality rates from HIV/AIDS;Aim 2To assess the impact of BFP exposure and FHS coverage on incidence, treatment adherence, hospitalizations, case-fatality, and mortality from HIV/AIDS, evaluating their individual and synergistic effects on a broad range of subpopulations;Aim 3To forecast HIV/AIDS incidence, prevalence and mortality scenarios in Brazil using integrated microsimulation (MS), compartmental model (CM) and agent-based models (ABM) that leverages the large set of results from Aims 1 and 2, to explore the impact of different socioeconomic and policy scenarios on HIV/AIDS morbidity and mortality up to 2030.Brazil is also an extremely data-rich country, where demographic, socioeconomic and interventions coverage data are available in terms of both quantity and adequate quality. Several impact evaluations of CCT have been performed using publicly available secondary aggregate-level longitudinal data and, more recently, individual-level data using the 100 Million Brazilian Cohort . For theThis study breaks new ground by providing a unique opportunity to use unprecedented big longitudinal dataset of linked socioeconomic and health data to assess the effects of socioeconomic conditions, PHC and CCT on all sequential outcomes of HIV/AIDS morbidity and mortality. Moreover, this study provides the unique opportunity to assess potential effect modification and synergistic effects of the simultaneous receipt of CCT and PHC, and how baseline socioeconomic conditions influence the effectiveness of both types of programs on the burden of HIV/AIDS. The longitudinal aspect of the cohort, which has a long follow-up period of 18 years of socioeconomic and health data, will allow us to explore and assess in-depth dynamics, lagged and long-term effects of changes in living conditions and in exposures to BFP and PHC. One of the study’s most relevant potential contribution to translational science will be the systematic integration of the large dataset and results from the retrospective impact evaluations with the prospective mathematical models, developing more reliable forecast scenarios than models which use parameters from other studies or populations. Moreover, the creation of a pioneering hybrid microsimulation, compartment and agent-based model (MS-CM-ABM), which will be supported by the very large number of parameters available from the retrospective big data evaluation, will be an innovation in the epidemiology of infectious diseases, and will be essential to the effort of modelling the effects of social determinants, CCT and PHC on HIV/AIDS. To date, very few studies in HIV/AIDS have introduced poverty and CCT as variables in their predictive models, and they have not explored their influence in depth , 35. RegThe conceptual framework for the project is presented in In the case of Brazil, the FHS could complement HIV clinic-based providers of HIV prevention and care services by increasing the proportion of its patients who have been screened for HIV, opening dialogues around sexual health, including asking about sexual orientation and gender identity, and prescribing antivirals as pre- and post-exposure prophylaxis for their non-HIV-infected patients . PHC couCadastro Unico (CadUnico), a national administrative system containing information on all individuals, and their families, applying for any social programs in Brazil [This study builds upon the integration of a wide range of datasets. The aggregate-level longitudinal data on municipalities conditions, infrastructures, healthcare coverage and morbidity and mortality levels will be integrated with large individual-level dataset constituted by the linkage between AIDS-specific clinical and treatment longitudinal data with the 100 Million Brazilian Cohort, built by the Centre for Data and Knowledge Integration for Health (CIDACS), Salvador, Brazil , 41. Fign Brazil . To be en Brazil . The datCadUnico with the Brazilian Mortality Information System (SIM), that provides all death notifications by age, sex, cause (including AIDS), and municipality of residence of the deceased. More recently, the CadUnico was also linked with the Brazilian Hospitalization Information System (SIH), that provides registers for all, public hospitalizations, including for AIDS.The 100 Million Brazilian Cohort is based on the linkage between the The health datasets related to HIV/AIDS and that will be linked to 100 Million Brazilian Cohort and be used in this study are described below.The National System of Disease Notification for AIDS (SINAN), that includes information on all notified AIDS cases in the country.The Logistic Control System of Medicines (SICLOM), that manages the logistics of all antiretroviral drugs in Brazil and report when and how many doses are provided to the patients.The National Network System of Laboratories of CD4 + CD8 + Lymphocyte Count and Viral Load (SISCEL), that records all the CD4/CD8 counts and viral load among people living with HIV and AIDS.CadUnico and SIM  have been extensively evaluated and validated [Universidade Federal da Bahia (UFBA), Salvador, Brazil .The CIDACS has developed algorithms and codes to perform efficient, highly sensitive and specific probabilistic linkages using each individual’s name, date of birth, sex, and municipality of residence between these systems, and has successfully tested linkages , 45. Thealidated , 47. SINper capita family income. Proximal variables are related to household conditions at the family level and include building material, water supply, sanitation, electricity, waste disposal destination, and household density. Following a hierarchical analytical approach, three adjusted Poisson regression models will be used with cluster-robust standard errors (SEs) [To achieve the three main objectives of this study, several retrospective and forecasting methods will be integrated. The detailed steps of each objective are described below. As discussed before, the Aim 1 is to use very large datasets to evaluate the effect of a broad range of socioeconomic determinants on incidence, treatment adherence, hospitalization, case fatality and mortality from HIV/AIDS. A hierarchical framework to investigate the social determinants of HIV/AIDS , 48 willrs (SEs) –51, i.e.Step 1. To analyze the differential impact and dose-response of BFP according to the duration of the intervention, amount of money received and compliance with conditionalities. The retrospective impact evaluation will use arguably one of the most robust study designs for causal inference in quasi-experimental studies: the regression discontinuity design (RDD), which allows a quasi-randomization of the subjects under study around the eligibility threshold [per capita) with vulnerable members (children or pregnant women), the second for extremely poor families (below 77 BRL per capita), even without any vulnerable member. BFP is one of the most accurately targeted CCTs, and if inclusion/exclusion errors are identified, a fuzzy-instead of sharp-RDD will be used. The presence of two different cut-offs will allow to evaluate the different impact of the BFP on the two different thresholds: between eligible and non-eligible poor and eligible and non-eligible extremely poor families. In order to explore the impact in subjects outside of the RDD selected bandwidth, analyses using Random Administrative Delays (RAD) and Propensity Score Matching (PSM) [hreshold , 53. Theng (PSM) –56 will ng (PSM) . Where png (PSM) . The comStep 2. To measure the BFP impact on a broad and detailed range of different subpopulations defined by individual demographic and socioeconomic characteristics and by local geographic regions, and municipality development. The evaluation designs and methods described above will be used for stratified analyses with each subpopulation of interest: age groups, gender, illiteracy and educational level. Further stratifications will be based on the characteristics of the municipality: human development index, GDP per capita and Gini index of income inequality.Step 3. To analyze the dose-response of FHS according to the extent and duration of the municipal coverage. Using the same structured dataset from the previous analyses of Aim 2, and considering that coverage of FHS is only available at the municipal level, the FHS impact will be evaluated using a Propensity Score (PS) weighted multilevel negative binomial regression design with individual level-variables from Aim 1, including BFP as adjusting variable [variable . To avoiStep 4. To measure the FHS impact on a broad range of different subpopulations defined by individual demographic and socioeconomic characteristics and by local geographic regions. As in the Step 2 of the Aim 2, the evaluation designs and methods will be used for each subpopulations of interest: age groups, gender, illiteracy and educational level. Moreover, further stratifications also will be based on the characteristics of the municipality.Step 5. To evaluate the synergistic individual-level exposure to BFP with municipal-level coverage of FHS on incidence and mortality from HIV/AIDS. Using the same multilevel and multivariable regression models above, the interaction effects of BFP with FHS will be evaluated with product terms and stratification analyses.The second aim of this study is to assess the impact of the Bolsa Familia Program exposure and Primary Health Care coverage on incidence, treatment adherence, hospitalization, case fatality, and mortality from HIV/AIDS, evaluating their impact, alone and in a synergistic fashion, on a broad range of subpopulations. To achieve this goal, the following steps will be performed:® and R Studio 3.6®.The main limitation of Aim 1 and Aim 2 is the usage of the population registered in the CadUnico that will be used rather than the overall Brazilian population to evaluate the impact of the interventions under study. While for the evaluation of the BFP the use of a control group which is part of the poorest half of the Brazilian population—instead of the overall population—is supposed not to influence the impact estimates of RDD and PSM designs (which uses controls comparable to the exposed), for FHS this is not necessarily the case. While the use of PS weighted regression will be able to weight observations and allow an unbiased estimation of the FHS effects (in relation to implementation bias), we assume that the FHS effect in the upper income group of the CadUnico—which includes a great part of the Brazilian middle-class—will not be significantly different from the richest half of the Brazilian population not included in the CadUnico dataset. All analyses for Aims 1 and 2 will be performed with STATA 16Step 1. To evaluate the most adequate compartmental structure for the mathematical models. In the first exploratory stage we will use compartmental models (CM) to evaluate which structures and compartments are more adequate to represent the influence of the wide range of social determinants, the effects of BFP and FHS on incidence, prevalence, compliance to treatment in the HIV/AIDS epidemic in Brazil as a whole.Step 2. To develop an Agent-Based Model (ABM) to evaluate the effect of interactions in HIV/AIDS dynamics. ABM will be used to explore patterns of interactions between individuals [ividuals . ABM focividuals –61 and iividuals –65.Step 3. Develop an integrated Microsimulation, Compartmental and Agent-Based Model (MS-CM-ABM) and forecast the impact of alternative economic and policy scenarios on HIV/AIDS outcomes. The third stage of modelling of the Aim 3 will integrate the two analyses above with microsimulation (MS), creating an integrated and comprehensive model. MS-CM-ABM can provide more accurate estimates of policy effects than traditional compartmental models or time-series forecasting models, which use only the average values in the population. Both MS and ABM use the micro-level description to represent the real phenomena, but while ABM is focused on the understanding the interaction dynamics in relatively small population, MS is used to recreate large synthetic populations from retrospective datasets. Their integration can be particularly useful—as in our case—when both the heterogeneity and dynamic interactions of the population are relevant for the outcome under study. A representation of the proposed MS-CM-ABM model is presented in Finally, to achieve Aim 3, some steps are needed to be developed. This objective is to forecast HIV/AIDS incidence, prevalence and mortality scenarios, using the broad range of subpopulations estimates and parameters from the retrospective analyses, according to socioeconomic changes and coverage intervention scenarios up to 2030. Leveraging the richness and granularity of parameters obtained from Aim 1 and 2, mathematical models will fully explore and comprehensively evaluate the effect of several scenarios using compartmental models, microsimulation, and agent-based models. The steps are described below.2) explained will be assessed, and will be verified that all observed values will be included in the 95% CIs of the simulation estimates, among other validation criteria [For each outcome and each scenario, 10,000 Monte Carlo simulations will be performed allowing all parameters to vary by their underlying distribution—measured in the retrospective analysis—and consequently obtaining the Predictive Intervals of each estimate. The linear regression of predicted versus observed values and the proportion of variance Click here for additional data file.


Esophageal cancer is the seventh most commonly diagnosed cancer and the sixth leading cause of cancer mortality in the world.Esophageal cancer has a unique immune microenvironment,Clinical Cancer Research, entitled “Neoadjuvant chemoradiotherapy combined with atezolizumab for resectable esophageal adenocarcinoma: A single arm phase II feasibility trial (PERFECT)”, Ende et al. investigated the feasibility and efficacy of neoadjuvant chemoradiotherapy combined with PD‐L1 inhibitor (atezolizumab) for resectable EAC. A total of 40 patients were enrolled in this study. Among them, 34 (85%) patients received all cycles of atezolizumab and 33 (83%) underwent surgery. Immune‐related adverse events (IRAEs) were observed in six (15%) patients. Reasons for not undergoing surgery were disease progression , patient choice , and death . A total of 10 (25%) patients had achieved complete pathological remission. There was no statistically significant difference in response or survival between the PERFECT and neoadjuvant chemoradiation cohort. The baseline IFNγ‐signature . The common characteristic of nonresponders receiving treatment is the presence of a large number of cytotoxic lymphocytes with transcriptional signature consistent with the expression of immune checkpoints, or a small number of cytotoxic lymphocytes.In the PERFECT trial recently published in This is the first trial combining CROSS ‐based neoadjuvant chemoradiotherapy with PD‐L1 inhibitor (atezolizumab) for resectable EAC. Most of the participants (85%) in this study received all planned cycles of atezolizumab suggesting that the treatment regimen is feasible. The neoadjuvant chemotherapy‐related advanced events and postoperative complications reported in the PERFECT trial were basically the same as those reported in the CROSS trial, apart from the immune‐related adverse events associated with PD‐L1 inhibition.In previous studies, the expression of a six‐gene IFNγ‐signature was used to verify the efficacy of pembrolizumab treatment in several cancer types.In summary, the PERFECT trial proved that it is feasible to add atezolizumab to CROSS‐based neoadjuvant chemoradiotherapy without affecting the surgical outcomes. Additionally, the immune‐related toxicity of this combination therapeutic strategy was manageable. Compared to the responders of neoadjuvant chemoradiotherapy, the responders in the PERFECT trial had a numerically longer overall survival and progression‐free survival, but the patients treated with neoadjuvant chemoradiation combined with atezolizumab had similar overall survival and progression‐free survival as those who were treated with neoadjuvant chemoradiotherapy alone. Moreover, the six‐gene IFNγ‐signature was associated with the treatment response to neoadjuvant ICI therapy. All these findings lay the foundation for the application of neoadjuvant chemoradiation plus PD‐L1 inhibitor in resectable EAC.The author declares no competing interests.


The purpose of this study was to evaluate the impact of full-spectrum light-emitting diodes mimicking sunlight (Sunlike LEDs) on ocular growth and refractive error development in a chicken model of myopia.n = 39) were distributed into 3 groups and raised for 28 days in isoluminant (approximately 285 lux) fluorescent  or Sunlike LED  white lighting environments. Form-deprivation myopia was induced monocularly from day 1 post-hatching (D1) until D14. On D14, form deprivation was halted and the recovery of form-deprived (FD) eyes was monitored until D28. Axial length (AL), refraction, choroidal thickness, and anterior chamber depth were measured in vivo on D1, D7, D14, D22, and D28. Differences in outcome measures between eyes and groups were compared using 2-way repeated-measures ANOVA.One-day old chicks (P < 0.001 and D28: P < 0.001) and SL-6500 (D22: P = 0.006 and D28: P < 0.001) recovered faster from axial elongation compared with animals raised under FL-4000. The refractive status of FD eyes reared under SL-6500, not under FL-4000 or SL-4000, was similar to control eyes on D28 (P > 0.05). However, SL-4000 and SL-6500 exhibited similar refraction on D28 than FL-4000 (P > 0.05). Choroidal thickness was significantly greater in FD eyes of chickens raised under SL-6500 than in animals raised under FL-4000 (P = 0.03).AL and myopic refraction of FD eyes increased similarly among groups during form-deprivation. FD eyes of animals raised under SL-4000 (D22: Compared to fluorescent light, moderate intensities of full-spectrum Sunlike LEDs can accelerate recovery from form-deprivation myopia in chickens, potentially through a change in the choroid-mediated pathway. The humidity of the dark room where the chicken enclosure was placed was maintained between 50% and 55%. Monocular FDM was induced over one randomly selected eye starting on D1 by placing a dome shaped 3D-printed translucent diffuser made of veroclear material  having a diameter of 13 mm, a thickness of 1.1 mm, and a radius of curvature of 7.5 mm. Different diffusers used for different animals yielded a very similar light spectral transmittance that was between 70% and 80% across the visible spectrum, except at the shortest wavelengths (<425 nm) where it was reduced to 50% to 70% C. Diffusn = 18) was reared under fluorescent light  with peaks in the spectral distribution of light at 435 nm, 545 nm, and 611 nm ; group SL-4000 (n = 12) was reared under a full-spectrum Sunlike LEDs  with a continuous spectrum from 400 to 775 nm peaking at 618 nm ; and Group SL-6500 (n = 9) was reared under a full-spectrum SL LEDs  with a continuous spectrum from 400 to 775 nm peaking at 450 nm  (Chicks were reared under a 12:12 hour light/dark cycle (0700–1900 hours) from day one (D1) until D28. On D1, chickens were divided into 3 groups based on the spectrum and CCT of isoluminant (approximately 285 lux) ambient lighting conditions: group FL-4000 (o. Ltd.) A, 1B. Ado. Ltd.) B. AveragAll ocular measurements were carried out between 12 PM and 5 PM and the animals were evaluated in a random order to minimize any circadian impact on the outcome measures. Non-cycloplegic refraction was assessed using an automated version of chicken infrared photoretinoscopy, as previously described by Schaeffel and Howland.P < 0.05. Statistics were performed using Sigmaplot version 14.0 , and plots were drawn using GraphPad Prism 6 .Results are presented as average ± standard deviation (SD). Changes in the measured parameters  of the form-deprived (FD) eye over the duration of the experimental procedure (D1 to D28) were compared with the control eyes  then expressed as interocular differences (IODs) between the FD and the control eye (FD eye − control eye) and compared between experimental groups. After confirming normal distribution of the variables, ocular parameters and IODs were compared between eyes within the same group and among groups using a 2-way repeated-measures analysis of variance (2-way RM-ANOVA) with day and eye or day and group as within- and between-subject factors, respectively. Whole-body weights of animals in different groups were also compared using a 2-way RM-ANOVA. For those comparisons in which the omnibus test reached statistical significance, pairwise multiple comparison procedures were performed using the Holm-Sidak method. For all statistical tests, the level of significance was set at Average illuminances, measured in all directions of gaze within the enclosure, were maintained at 281.8 lux (FL-4000), 284.5 lux (SL-4000), and 287.9 lux (SL-6500) throughout the experimental period. The relative spectra of SL LEDs (4000 K and 6500 K) were similar to that of outdoor sunlight/daylight , being broader and more homogeneously distributed than that of FL-4000, which was discontinuous with sharp and spike-like peaks see A, 1B. Th(P > 0.05) throughout the experimental protocol  and SL-6500 (P = 0.34), compared with control eyes, were no longer statistically significant (P < 0.001).Compared with control eyes, FD eyes showed an increase in AL on D7 and D14 in animals reared under FL-4000, SL-4000, and SL-6500 ; FL-4000 versus SL-4000 (P = 0.04), and FL-4000 versus SL-6500 (P = 0.049), but was not significantly different between the SL groups (see P > 0.05) different throughout the experimental period  = 244.44, P < 0.001]; SL-4000:  and SL-6500; ). This myopic shift predominantly took place during the form-deprivation period , whereas the refractions of FD eyes exposed to FL-4000 (see P < 0.001) or SL-4000 (see P = 0.04) remained significantly different from those of the control eyes. IODs in refraction among the three groups were not significantly different  = 27.63, P < 0.001]; SL-4000:  and SL-6500: ; P = 0.04]; ) and D14  = 3.92, P = 0.03). Pairwise comparison revealed that the IOD in the choroidal thickness of the eyes exposed to SL-6500 was increased compared with eyes exposed to FL-4000  or FL-4000 and SL-4000 , whereas at D7, retinal thinning was found only in the FD eyes of animals raised under FL-4000  = 24.56, P < 0.001]; SL-4000: ; and SL-6500: ; P > 0.05) different throughout the experimental period  accelerated recovery from the excessive axial elongation induced by form-deprivation, more completely than did isoluminant fluorescent light. None of the lights tested, however, halted the development of FDM, and only the higher-CCT continuous-spectrum LED light (6500 K) promoted a recovery from myopic refractive error to measures that were not significantly different from control eyes by the end of the experimental protocol. Furthermore, FD eyes of chicks reared under the higher-CCT continuous-spectrum LED light (6500 K) had thicker choroids than FD eyes exposed to fluorescent light, and did not exhibit any choroidal thinning on D7 and D14 of form-deprivation.–,,Epidemiological investigations have highlighted the protective effect of spending time outdoors against human myopia.In our study, Sunlike LEDs did not stop the development of FDM. These findings are in agreement with Li et al.,,,,,,,In our study, moderate levels of Sunlike LEDs (SL-6500 and SL-4000) accelerated recovery from FDM. These findings may reflect an improved emmetropization process under sunlike artificial light compared to florescent light, even when matched for CCT. These findings are in agreement with statements by Rucker et al.,,,Choroidal thinning is a particular anatomical change related to myopia in humansAberrant axial elongation due to form-deprivation impacts the anterior segment of the eye. Although anterior segment changes have been observed in experimental myopia models, they are not directly related to the visual regulation of refractive state, but are rather epiphenomena of considerable changes in the posterior segment of the eye during form-deprivation.,,The spectral composition of light, more specifically narrow-band light, can affect ocular growth and refractive development of uncovered control eyes.,GNB3) and dopaminergic actions (DRD1). These are involved in the light-dependent retina-to-sclera signaling cascade and subsequent visual regulation of ocular growth.One of the potential pathways for light-driven myopia-control is through retinal signaling molecules, acting via the retinal pigment epithelium and choroid to regulate scleral structure and biomechanics.Our study has a few limitations. First, we compared the impact of broad-, full-spectrum Sunlike LEDs to that of discontinuous-spectrum fluorescent light. Future studies should compare the effects of Sunlike LEDs to those of standard LEDs used in households. Second, owing to differences in the ocular systems of chicks and humans, our findings are not directly translatable to understanding and treating human myopia. Third, changes to the corneal curvature in response to FDM and different light exposures were not recorded in this study. These changes may have affected the refractive measurements and should be investigated in future studies. Further studies, benefitting from insights provided by non-primate animal studies such as ours, should investigate the impact of full-spectrum high CCT lighting on ocular growth in primate models.This study, using a chicken model of FDM, reveals that moderate levels of continuous-, full-spectrum LED lights that mimic sunlight can promote recovery from myopia and promote emmetropization after restoring normal viewing; and that full-spectrum light with higher CCT can prevent choroidal thinning that accompanies FDM on days 7 and 14. Our study supports previous reports showing that the spectral composition of indoor light could affect ocular growth and emmetropization, and open new research avenues for light-centered, passive myopia-control.