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Context Molecular testing can refine the risk of malignancy in thyroid nodules with indeterminate cytology to decrease unnecessary diagnostic surgery. Objective This study was performed to evaluate the outcomes of cytologically indeterminate thyroid nodules managed with Afirma genomic sequencing classifier (GSC) testing. Methods Adult patients who underwent a biopsy at 3 major academic centers between July 2017 and June 2021 with Bethesda III or IV cytology were included. All patients had surgery or minimum follow-up of 1 year ultrasound surveillance. The primary outcomes were the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of GSC in Bethesda III and IV nodules. Results The median nodule size of the 834 indeterminate nodules was 2.1 cm and the median follow-up was 23 months. GSC sensitivity, specificity, PPV, and NPV across all institutions were 95%, 81%, 50%, and 99% for Bethesda III nodules and 94%, 82%, 65%, and 98% for Bethesda IV nodules, respectively. The overall false-negative rate was 2%. The NPV of GSC in thyroid nodules with oncocytic predominance was 100% in Bethesda III nodules and 98% in Bethesda IV nodules. However, the PPV of oncocytic nodules was low (17% in Bethesda III nodules and 45% in Bethesda IV nodules). Only 22% of thyroid nodules with benign GSC results grew during surveillance. Conclusion GSC is a key tool for managing patients with indeterminate cytology, including the higher-risk Bethesda IV category. GSC-benign thyroid nodules can be observed similarly to thyroid nodules with benign cytology. [ABSTRACT FROM AUTHOR]

There is a lack of documentation regarding cytopathology of renal neuroendocrine neoplasms (NENs) due to their rarity. Five cytology cases were gathered from 3 institutes. Cohort consisted of 4 females and 1 male. Fine needle aspiration biopsy and touch preparation slides of core needle biopsy revealed cellular samples, composed of round, plasmacytoid, or columnar cells. Tumor cells were present in nested, acinar, 3D cluster, and individual cell patterns. Tumor cells in 3 cases exhibited uniformly round to oval small nuclei with inconspicuous nucleoli, finely granular chromatin, and smooth nuclear membranes, whereas 2 other cases showed pleomorphic nuclei with conspicuous nucleoli, nuclear molding, and irregular nuclear membranes. Tumor cells displayed pale or granular cytoplasm, with 1 case showing small vacuoles. Examination of cores and cell blocks demonstrated tumor cells in sheets, nests, or acini. All tumor cells were positive for neuroendocrine immunomarkers. Based on mitotic count, Ki-67 index and morphology, 3 tumors were graded as well-differentiated neuroendocrine tumor (WDNET) (1 grade [G] 3, 1 G2, 1 G1) and 2 as large cell neuroendocrine carcinoma. Deletion of 7q, 10q, and 19q was detected in WDNETs. Two patients with large cell neuroendocrine carcinoma and 1 with WDNET G3 underwent chemotherapy due to aggressiveness, whereas nephrectomy was performed for patients with WDNET G1 and 2 without metastasis. Cytopathological characteristics of renal NENs closely resemble those affecting other organs. Despite its rarity, renal NENs should be kept in mind when confronted with morphological resemblances to NENs, to prevent misdiagnosis and inappropriate therapeutic interventions. • Morphology and immunocytochemical profile of renal neuroendocrine neoplasms (NENs) resemble those seen in other organs. • PAX8 is not useful in distinguishing primary renal NENs from metastatic NENs. [ABSTRACT FROM AUTHOR]

BACKGROUND: The DYNAMIC trial investigated the use of circulating tumor DNA (ctDNA) to guide adjuvant treatment decisions in stage II colon cancer. Despite the DYNAMIC trials assertion that a ctDNA-guided approach could minimize the use of adjuvant treatment without compromising recurrence-free survival (RFS), we raised concerns regarding the trials methodology and the practical implications of its findings in a Debate article. Here, we expand upon these concerns in a response to a correspondence by the authors of the DYNAMIC trial. MAIN BODY: We dispute the choice of a large non-inferiority margin in the DYNAMIC trial, simply because an 8.5 percentage points decrease in recurrence-free survival could result in significant harm to patients. We challenge the authors comparisons of the DYNAMIC trial outcomes with observational studies. Such comparison is subject to selection bias and changes over time that limit their relevance. The prognostic role of ctDNA do not automatically imply that more treatment in patients with ctDNA positivity would improve outcomes, which we highlight. In real-world settings, we anticipate a potential rise in chemotherapy use due to clinicians utilizing ctDNA alongside existing clinicopathologic factors, rather than using ctDNA as an entire replacement. Lastly, a key concern in DYNAMIC was an 350% higher use of oxaliplatin in the ctDNA arm compared with standard management (9.5% versus 2.7%, respectively), which poses a risk for long-term neuropathy. CONCLUSION: We look forward improvements in patient selection in the adjuvant setting, but we maintain our reservations about the DYNAMIC trial and the real-life implementation of its results. As an alternative to exploring de-escalation strategies with large margins non-inferiority trials, we propose that superiority trials in stage II patients could be a more effective strategy.

BackgroundAetiological diagnosis and targeted antibiotic therapy are essential to improve the prognosis of patients with infective endocarditis. Molecular tests on blood have been reported to be effective in identifying the causative organism and are recommended when blood cultures are negative. The role of molecular tests on the surgically excised valve is still unclear and needs further investigation.Materials and methodsIn this prospective, observational, single center study, we enrolled 100 consecutive patients with native or prosthetic valve endocarditis who underwent cardiac surgery between April 2020 and June 2023. Results of preoperative blood cultures, valve culture, 16s ribosomal RNA and histopathologic analysis of surgical samples were collected in a dedicated database.ResultsThe mean age of the study population was 60 ± 12.5 years, with a majority of men (73%). Previous cardiac surgery was reported in 31% of patients. Blood culture, valve culture, and 16srRNA were positive in 83%, 47%, and 76% of cases, respectively. The sensitivity of both valve culture and 16srRNA decreased significantly with prolonged preoperative antibiotic therapy. Of note, 16srRNA was the only positive result in 7% of cases, allowing aetiological diagnosis. In 33% of patients, the valve culture test was negative while the molecular test was positive. In these cases, histopathological analysis showed acute inflammation in most cases. In 10%, the molecular test helped in resolving discrepancies between the results of blood and valve cultures.ConclusionsThe molecular test showed significantly higher diagnostic sensitivity than valve culture and maintained this efficacy even after 28 days of preoperative antibiotic therapy. In addition to identifying the pathogen in 7% of cases with negative culture results, the molecular test demonstrated utility in other crucial situations. When valve cultures were negative, combining molecular testing and histopathological analysis they allowed the identification of patients who could benefit from prolonged antibiotic therapy. In addition, molecular testing guided the choice of antibiotic treatment when there was a discrepancy between blood culture and valve culture results. Based on these findings, molecular testing should be considered in all patients with infective endocarditis undergoing cardiac surgery. Clinical Trial Registration: ClinicalTrials.gov, identifier (NCT05791357).

Background: Serological screening tests play a crucial role to diagnose gambiense human African trypanosomiasis (gHAT). Presently, they preselect individuals for microscopic confirmation, but in future "screen and treat" strategies they will identify individuals for treatment. Variability in reported specificities, the development of new rapid diagnostic tests (RDT) and the hypothesis that malaria infection may decrease RDT specificity led us to evaluate the specificity of 5 gHAT screening tests. Methods: During active screening, venous blood samples from 1095 individuals from Côte d'Ivoire and Guinea were tested consecutively with commercial (CATT, HAT Sero-K-SeT, Abbott Bioline HAT 2.0) and prototype (DCN HAT RDT, HAT Sero-K-SeT 2.0) gHAT screening tests and with a malaria RDT. Individuals with ≥ 1 positive gHAT screening test underwent microscopy and further immunological (trypanolysis with T.b. gambiense LiTat 1.3, 1.5 and 1.6; indirect ELISA/T.b. gambiense; T.b. gambiense inhibition ELISA with T.b. gambiense LiTat 1.3 and 1.5 VSG) and molecular reference laboratory tests (PCR TBRN3, 18S and TgsGP; SHERLOCK 18S Tids, 7SL Zoon, and TgsGP; Trypanozoon S2-RT-qPCR 18S2, 177T, GPI-PLC and TgsGP in multiplex; RT-qPCR DT8, DT9 and TgsGP in multiplex). Microscopic trypanosome detection confirmed gHAT, while other individuals were considered gHAT free. Differences in fractions between groups were assessed by Chi square and differences in specificity between 2 tests on the same individuals by McNemar. Results: One gHAT case was diagnosed. Overall test specificities (n = 1094) were: CATT 98.9% (95% CI: 98.1–99.4%); HAT Sero-K-SeT 86.7% (95% CI: 84.5–88.5%); Bioline HAT 2.0 82.1% (95% CI: 79.7–84.2%); DCN HAT RDT 78.2% (95% CI: 75.7–80.6%); and HAT Sero-K-SeT 2.0 78.4% (95% CI: 75.9–80.8%). In malaria positives, gHAT screening tests appeared less specific, but the difference was significant only in Guinea for Abbott Bioline HAT 2.0 (P = 0.03) and HAT Sero-K-Set 2.0 (P = 0.0006). The specificities of immunological and molecular laboratory tests in gHAT seropositives were 98.7–100% (n = 399) and 93.0–100% (n = 302), respectively. Among 44 reference laboratory test positives, only the confirmed gHAT patient and one screening test seropositive combined immunological and molecular reference laboratory test positivity. Conclusions: Although a minor effect of malaria cannot be excluded, gHAT RDT specificities are far below the 95% minimal specificity stipulated by the WHO target product profile for a simple diagnostic tool to identify individuals eligible for treatment. Unless specificity is improved, an RDT-based "screen and treat" strategy would result in massive overtreatment. In view of their inconsistent results, additional comparative evaluations of the diagnostic performance of reference laboratory tests are indicated for better identifying, among screening test positives, those at increased suspicion for gHAT. Trial registration: The trial was retrospectively registered under NCT05466630 in clinicaltrials.gov on July 15 2022. [ABSTRACT FROM AUTHOR]

Simple Summary: Sarcomas represent a group of rare tumors consisting of more than 100 different entities. A precise diagnosis is crucial to optimal treatment. In addition, a significant proportion of sarcomas are characterized by a specific genetic abnormality. This study was designed by the Hellenic Group of Sarcoma and Rare Cancers to assess the effect of expert pathology review, coupled with the application of molecular methods to detect genetic abnormalities, on the diagnosis and management of sarcoma patients in Greece. We found that histological diagnosis review by a sarcoma pathologist led to modifications in diagnosis in almost one-third of patients, resulting in modifications in management in 14% of patients. The use of molecular tests led to modifications in diagnosis in 26% of patients, resulting in modifications in management in almost 11% of patients. Our study highlights the importance of expert pathologists, assisted in some cases by molecular methods, in sarcoma diagnosis and treatment. Precise classification of sarcomas is crucial to optimal clinical management. In this prospective, multicenter, observational study within the Hellenic Group of Sarcoma and Rare Cancers (HGSRC), we assessed the effect of expert pathology review, coupled with the application of molecular diagnostics, on the diagnosis and management of sarcoma patients. Newly diagnosed sarcoma patients were addressed by their physicians to one of the two sarcoma pathologists of HGSRC for histopathological diagnostic assessment. RNA next-generation sequencing was performed on all samples using a platform targeting 86 sarcoma gene fusions. Additional molecular methods were performed in the opinion of the expert pathologist. Therefore, the expert pathologist provided a final diagnosis based on the histopathological findings and, when necessary, molecular tests. In total, 128 specimens from 122 patients were assessed. Among the 119 cases in which there was a preliminary diagnosis by a non-sarcoma pathologist, there were 37 modifications in diagnosis (31.1%) by the sarcoma pathologist, resulting in 17 (14.2%) modifications in management. Among the 110 cases in which molecular tests were performed, there were 29 modifications in diagnosis (26.4%) through the genomic results, resulting in 12 (10.9%) modifications in management. Our study confirms that expert pathology review is of utmost importance for optimal sarcoma diagnosis and management and should be assisted by molecular methods in selected cases. [ABSTRACT FROM AUTHOR]

With the continuous spread of new SARS-CoV-2 variants of concern (VOCs), the monitoring of diagnostic test performances is mandatory. We evaluated the changes in antigen diagnostic tests' (ADTs) accuracy along the Delta to Omicron VOCs transition, exploring the N protein mutations possibly affecting ADT sensitivity and assessing the best sampling site for the diagnosis of Omicron infections. In total, 5175 subjects were enrolled from 1 October 2021 to 15 July 2022. The inclusion criteria were SARS-CoV-2 ADT combined with a same-day RT-PCR swab test. For the sampling site analysis, 61 patients were prospectively recruited during the Omicron period for nasal and oral swab analyses by RT-PCR. Next-Generation Sequencing data were obtained to evaluate the different sublineages. Using RT-PCR as a reference, 387 subjects resulted in becoming infected and the overall sensitivity of the ADT decreased from 63% in the Delta period to 33% in the Omicron period. This decrease was highly statistically significant (p < 0.001), and no decrease in viral load was detected at the RNA level. The nasal site presented a significantly higher viral load than the oral site during the Omicron wave. The reduced detection rate of Omicron infections by ADT should be considered in the global testing strategy to preserve accurate diagnoses across the changing SARS-CoV-2 variants. [ABSTRACT FROM AUTHOR]

In the adjuvant setting of cancer treatment, de-escalation strategies have the goal of omitting or minimizing treatment in patients, without compromising outcomes. Historically, eligibility for adjuvant treatment solely relied on the patient's clinical and tumor's pathological characteristics. At the turn of the century, based on new biological understanding, molecular-based strategies were tested and sometimes implemented. However, we illustrate how molecularly based de-escalation strategies may paradoxically lead to overtreatment. This may happen when the novel approach is tested in lieu of standard management and may not yield the same results when being implemented in addition to usual practice. In the DYNAMIC trial, adjuvant chemotherapy decision in stage II colon cancer was compared between a circulating tumor DNA (ctDNA)-based approach and the standard care. We show this may result in more patients receiving oxaliplatin-based chemotherapy and may expose a similar proportion of patients to chemotherapy if the novel strategy is implemented in addition to usual practice. The other potential risk is undertreatment. We provide an illustration of early breast cancer, where the decision of adjuvant chemotherapy based on the gene expression signature MammaPrint may lead to inferior outcomes as compared with the clinico-pathologic strategy. This may also happen when non-inferiority designs have large margins. Among solutions, it should be acknowledged that clinico-pathological features, like T4 in colon cancer, may not be abandoned and replaced by novel strategies in real-life practice. Therefore, novel strategies should be tested in addition to standard of care, and not in lieu of. Second, de-escalation trials should focus on the settings where the standard of care has a widespread agreement. This would avoid the risk of testing non-inferiority against an ineffective therapy, which guarantees successes without providing informative data. Simply because a molecular test is rational does not mean it can improve patient outcomes. Here, we highlight how molecular test-based strategies may result in either overtreatment or undertreatment. In the rapidly evolving field of medicine, where technological advances may be transformative, our piece highlights scientific pitfalls to be aware of when considering running such trials or before implementing novel strategies in daily practice.

Background Cytologically indeterminate thyroid nodules (ITN) pose a management challenge. Here we analyze if adding ultrasound characteristics to Afirma Genome Sequence Classifier (GSC) results increases GSC diagnostic performance. Methods We retrospectively analyzed 237 GSC-tested Bethesda III/IV ITNs between July 2017 and December 2019 and classified them by American Thyroid Association (ATA) and the Thyroid Imaging Reporting and Data System (TIRADS) of the American College of Radiology. Results The benign call rate was higher in Bethesda III ITNs with TIRADS <5 vs TIRADS 5 (89% vs 68%. P =.015). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of GSC in ATA high-risk Bethesda III ITNs vs lower were 100% vs 80% (P = 1), 89.5% vs 91.5% (P =.67), 66.7% vs 25% (P =.13), and 100% vs 99.2% (P = 1), respectively, and for TIRADS 5 vs <5 were 100% vs 80% (P = 1), 88.2% vs 91.4% (P =.65), 71.4% vs 23.5% (P =.06), and 100% vs 99.3% (P = 1). The sensitivity, specificity, PPV, and NPV of GSC in high-risk ATA Bethesda IV ITNs vs lower were 66.7% vs 100% (P =.42), 83.3% vs 85.7% (P = 1), 66.7% vs 64.3% (P = 1), and 83.3% vs 100% (P =.3), respectively, and for TIRADS 5 vs <5 were 66.7% vs 90% (P =.42), 88.9% vs 83.8% (P = 1), 66.7% vs 60% (P = 1), and 88.9% vs 96.9% (P =.39). Conclusion Sensitivity, specificity, NPV, and PPV of GSC were not significantly different in ATA high-risk and TIRADS 5 ITNs compared to ATA < high-risk and TIRADS 1-4 ITNs. [ABSTRACT FROM AUTHOR]

Brucellosis is a significant infection that causes abortion, decreased milk production, and sterility in livestock, which greatly affects the industry. This study aimed to determine the prevalence of Brucella in buffalo milk samples across various regions of Iran, utilizing serological, molecular, and cultural analyses. A total of 1860 buffalo milk samples were collected from industrial, semi-industrial, and traditional buffalo farms in four major buffalo breeding provinces. The milk ring test agglutination test (MRT) was initially conducted on all milk samples, followed by culture and molecular testing for positive and negative samples in MRT. The study revealed positive results for the presence of Brucella DNA in various provinces of Iran. The MRT had a relatively low sensitivity, with results ranging from 0 to 0.7% in different provinces. However, the AMOS PCR method showed a significantly higher presence of Brucella DNA, ranging from 13 to 46% in these provinces. The highest abundance of Brucella bacterial DNA was found in Ardabil province, while the lowest was in West Azerbaijan province. Brucella abortus was the most commonly detected bacteria, followed by Brucella melitensis. Interestingly, the B. abortus vaccine strain RB51 was detected in 26.3% of positive samples of B. abortus. The culture assay of milk samples further confirmed the presence of B. melitensis biovar 1 in one sample from Khuzestan province. Overall, the study emphasizes that the AMOS PCR method is the most sensitive in detecting Brucella-exposed milk, while the sensitivity of milk sample culture and MRT is relatively lower. [ABSTRACT FROM AUTHOR]

Background: We assessed the laboratory diagnosis and treatment of invasive fungal disease (IFD) in Italy to detect limitations and potential for improvement. Methods: The survey was available online at www.clinicalsurveys.net/uc/IFI management capacity/, and collected variables such as (a) institution profile, (b) perceptions of IFD in the respective institution, (c) microscopy, (d) culture and fungal identification, (e) serology, (f) antigen detection, (g) molecular tests, (h) susceptibility testing and (i) therapeutic drug monitoring (TDM). Results: The laboratory capacity study received responses from 49 Italian centres, with an equitable geographical distribution of locations. The majority of respondents (n = 36, 73%) assessed the occurrence of IFD as moderate-high, with Aspergillus spp. being the pathogen of highest concern, followed by Candida spp. and Mucorales. Although 46 (94%) of the institutions had access to microscopy, less than half of them performed direct microscopy on clinical specimens always when IFD was suspected. Cultures were available in all assessed laboratories, while molecular testing and serology were available in 41 (83%), each. Antigen detection tests and antifungal drugs were also generally accessible (> 90%) among the participating institutions. Nevertheless, access to TDM was limited (n = 31, 63%), with a significant association established between therapeutic drug monitoring availability and higher gross domestic product per capita. Conclusions: Apart from TDM, Italy is adequately prepared for the diagnosis and treatment of IFD, with no significant disparities depending on gross domestic product. Future efforts may need to focus on enhancing the availability and application of direct microscopic methods, as well as TDM, to promote optimal treatment and better patient outcomes. [ABSTRACT FROM AUTHOR]

Summary: The emergence of next-generation sequencing technologies and computational advances have expanded our understanding of gene expression regulation (i.e., the transcriptome). This has also led to an increased interest in using transcriptomic biomarkers to improve disease diagnosis and stratification, to assess prognosis and predict the response to treatment. Significant progress in identifying transcriptomic signatures for various clinical needs has been made, with large discovery studies accounting for challenges such as patient variability, unwanted batch effects, and data complexities; however, obstacles related to the technical aspects of cross-platform implementation still hinder the successful integration of transcriptomic technologies into standard diagnostic workflows. In this article, we discuss the challenges associated with integrating transcriptomic signatures derived using high-throughput technologies (such as RNA-sequencing) into clinical diagnostic tools using nucleic acid amplification (NAA) techniques. The novelty of the proposed approach lies in our aim to embed constraints related to cross-platform implementation in the process of signature discovery. These constraints could include technical limitations of amplification platform and chemistry, the maximal number of targets imposed by the chosen multiplexing strategy, and the genomic context of identified RNA biomarkers. Finally, we propose to build a computational framework that would integrate these constraints in combination with existing statistical and machine learning models used for signature identification. We envision that this could accelerate the integration of RNA signatures discovered by high-throughput technologies into NAA-based approaches suitable for clinical applications.

Thyroid fine needle aspiration biopsy (FNAB) remains indeterminate in 16–24% of the cases. Molecular testing could improve the diagnostic accuracy of FNAB. This study examined the gene mutation profile of patients with thyroid nodules and analyzed the diagnostic ability of molecular testing for thyroid nodules using a self-developed 18-gene test. Between January 2019 and August 2021, 513 samples (414 FN ABs and 99 formalin-fixed paraffin-embedded (FFPE) specimens) underwent molecular testing at Ruijin Hospital. Sensitivity (Sen), specificity (Spe), positive predictive value (PPV), negative predictive value (NPV), and accuracy were calculated. There were 457 mutations in 428 samples. The rates of BRAF, RAS, TERT promoter, RET/PTC, and NTRK3 fusion mutations were 73.3% (n = 335), 9.6% (n = 44), 2.8% (n = 13), 4.8% (n = 22), and 0.4% (n = 2), respectively. The diagnostic ability of cytology and molecular testing were evaluated in Bethesda II and V–VI samples. For cytology alone, Sen, Spe, PPV, NPV, and accuracy were 100%, 25.0%, 97.4%, 100%, and 97.4%; these numbers were 87.5%, 50.0%, 98.0%, 12.5%, and 86.2% when considering positive mutation, and 87.5%, 75.0%, 99.0%, 17.6%, and 87.1% when considering positive cytology or and positive mutation. In Bethesda III–IV nodules, when relying solely on the presence of pathogenic mutations for diagnosis, Sen, Spe, P PV, NPV, and AC were 76.2%, 66.7%, 94.1%, 26.8%, and 75.0%, respectively. It might be necessary to analyze the molecular mechanisms of disease development at the genetic level to predict patients with malignant nodules more accurately in different risk strata and develop rational treatment strategies and definite management plans.

Background: Staphylococcus aureus is a typical pathogenic agent causes several morbidities and mortalities which variable largely following the severity of bacteria and activity of host immunity. Aim: Isolation of S. aureus from different cattle infections, molecular detection of nuc gene in positive S. aureus isolates and identification the effectiveness of phagocytic activity. Methods: Totally, 100 cattle with various infections (25 wounds, 25 abscesses, 25 nasal discharge, and 25 ear swaps) were selected and subjected for collection of swabs under controlled conditions. All collected samples were cultured on Mannitol salt agar (MSA) and assessed by the biochemical tests. Targeting the nuc gene, all study MSA positive isolates were examined molecularly by conventional polymerase chain reaction (PCR), and then subjected for antibiotic susceptibility test. Jugular venous blood was collected from all infected animals in addition to 20 healthy cattle that selected as a control group to estimate the phagocytic activity of S. aureus isolates. Results: The findings of MSA culture revealed a totally 80 positive samples to S. aureus as 23, 21, 20, and 16 positive isolates for nasal discharge, abscess, wound and ear swab, respectively; based on its morphology, cultural trait, and biochemical test. Subsequently, PCR assaying of MSA positive isolates was demonstrated an overall 59 positive samples as 14, 16, 12 and 17 positive isolates for nasal discharge, abscess, wound and ear swab, respectively. Antibiotic susceptibility testing of S. aureus positive PCR isolates reported a significant high sensitivity to chloramphenicol and vancomycin, and a high resistance to penicillin. Finally, there was a considerable decline in phagocytic activity in particular 2 weeks post infection as a result of bacterial invasion. Conclusion: This study shows a high prevalence of S. aureus in cattle infections, and protocol includes regular screening of cattle infection and suitable therapy based on antibacterial susceptibility test is of great importance in long-term control of pathogen. However, additional molecular studies targeting other genes of S. aureus and the role of immune markers in different infections should be aimed [Open Vet J 2023; 13(8.000): 1021-1026]

Background: Staphylococcus aureus is a typical pathogenic agent that causes several morbidities and mortalities which variable largely following the severity of bacteria and activity of host immunity. Aim: Isolation of S. aureus from different cattle infections, molecular detection of nuc gene in positive S. aureus isolates, and identification of the effectiveness of the phagocytic activity. Methods: Totally, 100 cattle with various infections (25 wounds, 25 abscesses, 25 nasal discharges, and 25 ear swaps) were selected and subjected to collection of swabs under controlled conditions. All collected samples were cultured on mannitol salt agar (MSA) and assessed by biochemical tests. Targeting the nuc gene, all study MSA positive isolates were examined molecularly by conventional polymerase chain reaction (PCR), and then subjected to antibiotic susceptibility test. Jugular venous blood was collected from all infected animals in addition to 20 healthy cattle that were selected as a control group to estimate the phagocytic activity of S. aureus isolates. Results: The findings of MSA culture revealed a total of 80 positive samples of S. aureus as 23, 21, 20, and 16 positive isolates for nasal discharge, abscess, wound, and ear swab, respectively; based on its morphology, cultural trait, and biochemical test. Subsequently, PCR assaying of MSA-positive isolates demonstrated an overall 59 positive samples as 14, 16, 12, and 17 positive isolates for nasal discharge, abscess, wound, and ear swabs, respectively. Antibiotic susceptibility testing of S. aureus-positive PCR isolates reported a significantly high sensitivity to chloramphenicol and vancomycin, and a high resistance to penicillin. Finally, there was a considerable decline in phagocytic activity in particular 2 weeks post-infection as a result of bacterial invasion. Conclusion: This study shows a high prevalence of S. aureus in cattle infections, and the protocol includes a regular screening of cattle infection and suitable therapy based on antibacterial susceptibility test is of great importance in the long-term control of the pathogen. However, additional molecular studies targeting other genes of S. aureus and the role of immune markers in different infections should be aimed. [ABSTRACT FROM AUTHOR]

Abstract Background Frequent serial monitoring of plasma cytomegalovirus (CMV) viral load caused unnecessary budgets for laboratory testing without changes in treatment. We aimed to implement diagnostic stewardship to limit CMV viral load testing at appropriate intervals. Methods A quasi-experimental study was performed. To avoid unnecessary plasma CMV viral load testing, the inpatient electronic pop-up reminder was launched in 2021. In cases with plasma CMV viral load testing was ordered in intervals of less than five days, telephone interview and feedback were performed. Pre-post intervention data was compared in terms of clinical and monetary outcomes. The rate of plasma CMV viral load testing performed in intervals of less than five days was compared between 2021 and 2019 using the Poisson regression model. Results After the protocol implementation, there was a significant decrease in the rate of plasma CMV viral load test orders in intervals of less than five days from 17.5% to 8.0% [incidence rate ratio 0.40, p

Yanghui Jin,1 Shengping Hu,1 Junkai Feng,2 Jianjun Ni3 1Department of Orthopaedics, Affiliated Hangzhou Chest Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, People’s Republic of China; 2The Second Clinical Medical College, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, People’s Republic of China; 3Department of Pharmacy, Affiliated Hangzhou Chest Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, People’s Republic of ChinaCorrespondence: Jianjun Ni, Department of Pharmacy, Department of Orthopaedics, Affiliated Hangzhou Chest Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, People’s Republic of China, Email njj19700912@163.comPurpose: To evaluate the accuracy of metagenomic next-generation sequencing (mNGS) for rapid diagnosis of spinal tuberculosis using spinal tissue specimens.Methods: Medical data regarding suspected spinal tuberculosis were retrospectively analyzed. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and area under the curve (AUC) of mNGS were calculated to determine its diagnostic efficacy, and these values were compared with culture and composite reference standard (CRS).Results: Two hundred and three cases of spinal tuberculosis were included for analysis. The sensitivity, specificity, PPV, NPV, and AUC of mNGS compared with culture were all very good. When CRS was used for the comparison, the sensitivity of mNGS and culture were 71.2% and 73.0%; the specificity and PPV were 100% in all cases; the NPV were 74.2% and 75.4%; the AUCs were all 0.86. The sensitivity and NPV of culture were slightly higher than that of mNGS; however, the diagnostic efficacy of mNGS and culture was consistent (P > 0.05).Conclusion: Spinal tissue specimens for mNGS testing had very good accuracy for diagnosing spinal tuberculosis.Keywords: spinal tuberculosis, culture, mNGS, molecular test, diagnostic accuracy

Abstract Background The DYNAMIC trial investigated the use of circulating tumor DNA (ctDNA) to guide adjuvant treatment decisions in stage II colon cancer. Despite the DYNAMIC trial’s assertion that a ctDNA-guided approach could minimize the use of adjuvant treatment without compromising recurrence-free survival (RFS), we raised concerns regarding the trial’s methodology and the practical implications of its findings in a Debate article. Here, we expand upon these concerns in a response to a correspondence by the authors of the DYNAMIC trial. Main body We dispute the choice of a large non-inferiority margin in the DYNAMIC trial, simply because an 8.5 percentage points decrease in recurrence-free survival could result in significant harm to patients. We challenge the authors’ comparisons of the DYNAMIC trial outcomes with observational studies. Such comparison is subject to selection bias and changes over time that limit their relevance. The prognostic role of ctDNA do not automatically imply that more treatment in patients with ctDNA positivity would improve outcomes, which we highlight. In real-world settings, we anticipate a potential rise in chemotherapy use due to clinicians utilizing ctDNA alongside existing clinicopathologic factors, rather than using ctDNA as an entire replacement. Lastly, a key concern in DYNAMIC was an 350% higher use of oxaliplatin in the ctDNA arm compared with standard management (9.5% versus 2.7%, respectively), which poses a risk for long-term neuropathy. Conclusion We look forward improvements in patient selection in the adjuvant setting, but we maintain our reservations about the DYNAMIC trial and the real-life implementation of its results. As an alternative to exploring de-escalation strategies with large margins non-inferiority trials, we propose that superiority trials in stage II patients could be a more effective strategy.

Abstract Background In the adjuvant setting of cancer treatment, de-escalation strategies have the goal of omitting or minimizing treatment in patients, without compromising outcomes. Historically, eligibility for adjuvant treatment solely relied on the patient’s clinical and tumor’s pathological characteristics. At the turn of the century, based on new biological understanding, molecular-based strategies were tested and sometimes implemented. Main body However, we illustrate how molecularly based de-escalation strategies may paradoxically lead to overtreatment. This may happen when the novel approach is tested in lieu of standard management and may not yield the same results when being implemented in addition to usual practice. In the DYNAMIC trial, adjuvant chemotherapy decision in stage II colon cancer was compared between a circulating tumor DNA (ctDNA)-based approach and the standard care. We show this may result in more patients receiving oxaliplatin-based chemotherapy and may expose a similar proportion of patients to chemotherapy if the novel strategy is implemented in addition to usual practice. The other potential risk is undertreatment. We provide an illustration of early breast cancer, where the decision of adjuvant chemotherapy based on the gene expression signature MammaPrint may lead to inferior outcomes as compared with the clinico-pathologic strategy. This may also happen when non-inferiority designs have large margins. Among solutions, it should be acknowledged that clinico-pathological features, like T4 in colon cancer, may not be abandoned and replaced by novel strategies in real-life practice. Therefore, novel strategies should be tested in addition to standard of care, and not in lieu of. Second, de-escalation trials should focus on the settings where the standard of care has a widespread agreement. This would avoid the risk of testing non-inferiority against an ineffective therapy, which guarantees successes without providing informative data. Conclusion Simply because a molecular test is rational does not mean it can improve patient outcomes. Here, we highlight how molecular test-based strategies may result in either overtreatment or undertreatment. In the rapidly evolving field of medicine, where technological advances may be transformative, our piece highlights scientific pitfalls to be aware of when considering running such trials or before implementing novel strategies in daily practice.

In this review, we present an overview of the main articles published in the field of Helicobacter pylori diagnosis in the past year. Key themes emerging in this area include rapid point-of-care testing and the use of artificial intelligence to enhance the efficacy of existing invasive diagnostic platforms. Studies in the field of endoscopy focussed on improving detection methods to diagnose H. pylori and associated diseases, targeted biopsy sampling, gastric juice analysis by Endofaster and the adoption of artificial intelligence in medical imaging. There have been some studies evaluating staining protocols for histopathology and exciting developments in the field of digital pathology for H. pylori detection and characterisation of inflammatory pathologies. Many articles have addressed the need for rapid, accurate, non-invasive point-of-care tests to analyse saliva or stool for the detection of H. pylori and/or its antimicrobial resistance. Finally, developments and considerations for the molecular detection of H. pylori and antimicrobial resistance using gastric samples are also discussed.

Background: Frequent serial monitoring of plasma cytomegalovirus (CMV) viral load caused unnecessary budgets for laboratory testing without changes in treatment. We aimed to implement diagnostic stewardship to limit CMV viral load testing at appropriate intervals. Methods: A quasi-experimental study was performed. To avoid unnecessary plasma CMV viral load testing, the inpatient electronic pop-up reminder was launched in 2021. In cases with plasma CMV viral load testing was ordered in intervals of less than five days, telephone interview and feedback were performed. Pre-post intervention data was compared in terms of clinical and monetary outcomes. The rate of plasma CMV viral load testing performed in intervals of less than five days was compared between 2021 and 2019 using the Poisson regression model. Results: After the protocol implementation, there was a significant decrease in the rate of plasma CMV viral load test orders in intervals of less than five days from 17.5% to 8.0% [incidence rate ratio 0.40, p < 0.001]. There was no statistically significant difference in the incidence of CMV DNAemia and CMV disease (p = 0.407 and 0.602, respectively). As a result, the hospital could save the costs of plasma CMV viral load testing per 1,000 patients performed with intervals of less than five days from 2,646,048.11 to 1,360,062.89 Thai Baht. Conclusions: The diagnostic stewardship program is safe and helpful in reducing unnecessary plasma CMV viral load testing and costs. Key points: Frequent serial plasma CMV viral load testing leads to unnecessary costs and laboratory workload. Diagnostic stewardship to limit repeat CMV viral load tests can reduce unnecessary testing without increasing the incidence of CMV DNAemia and CMV disease. [ABSTRACT FROM AUTHOR]

Background: Nucleic Acid Amplification Test (NAAT) can detect the SARS-CoV-2 at low viral load, but the risk of isolating unnecessarily people who are no longer infectious may preclude its use as screening test. Immunoassay can be an alternative screening tool to detect viral antigens, thanks to their high throughput, feasibility and practicability. In this study, we compare the results obtained by a chemiluminescent immunoassay against the molecular test. Methods: 105 positive NAAT nasopharyngeal swabs (NPS) with threshold cycle (Ct) between 39 and 13, and 15 negative samples were analyzed with two different methods based on chemiluminescent technology (CLIA) targeted to nucleocapsid SARS-Cov-2 antigen: Maglumi (SNIBE diagnostic Shenzen, China), and Liaison XL (DiaSorin s.p.a., Saluggia, Italy). Results: all 15 negative NPS (Ct >40) were confirmed as negative with both CLIA assays; 31 positive NPS out of 105 positive (Ct ≤40) were confirmed positive on Maglumi and Liaison (29.5%). The agreement between the two immunoassays was 100%. Conclusions: as expected, CLIA tests demonstrated to be less sensitive to the viral presence than NAAT. In particular, Maglumi and Liaison can detect a positivity when the virus is detected at a Ct <25. The range between 24 and 25 Ct was a grey area, where NAAT and CLIA were not in perfect agreement. It seems that antigen tests reach satisfactory sensitivities when infected people are more likely to be contagious; this characteristic makes them a useful tool for screening the population, especially during the pandemic [ABSTRACT FROM AUTHOR]

Purpose: Fine-needle aspiration (FNA) biopsy is an effective method to discriminate malignant thyroid nodules but reaches indeterminate results in approximately 30% of cases. Molecular testing can improve the diagnostic accuracy of FNA. This study aimed to investigate the real-life utility of the five-gene panel testing in thyroid FNAs. Methods: 759 thyroid nodules from 740 patients under FNAs were retrospectively enrolled. Gene mutation information and clinical parameters, including age, gender, tumor size, and lymph node metastasis, were respectively recorded. Cytological results were classified based on The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC). We analyzed mutational hotspots in BRAF, KRAS, NRAS, HRAS, and TERT genes from FNA specimens. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were calculated to assess diagnostic performance. Results: We identified 549 five-gene mutations in 759 nodules (72.3%), and the mutation frequency increased from the lower TBSRTC category to the upper category. BRAF.p.V600E showed the highest mutation incidence (71.3%) in the five-gene panel, correlated with the small to medium diameter (p = 0.008, p = 0.012) and high cytological categories (p < 0.001). The sensitivity, specificity, PPV, NPV, and accuracy of the combination of FNA cytology and five-gene detection were 96.83%, 100%, 100%, 42.86%, and 96.90%, respectively. Conclusions: The mutation frequency of the five-gene panel is 72.3% in thyroid FNAs. BRAF.p.V600E has the highest alteration rate, which is closely associated with tumor size and cytological results. The five-gene panel can improve the sensitivity and accuracy of FNA cytology, which may represent a valid adjunct technique in distinguishing thyroid nodules. [ABSTRACT FROM AUTHOR]

Immunosuppressed patients, transplant recipients, and those with acute or chronic respiratory disease are at increased risk for invasive fungal infections in Argentina. Although the national public system guarantees universal access to health care for all citizens, little is known about the quality of available diagnostic and treatment armamentaria for invasive fungal infections in the country. Between June and August 2022, infectious disease clinicians from each of the 23 provinces and the Autonomous City of Buenos Aires were contacted to describe local access to fungal diagnostic tools and antifungal agents. The information collected included different aspects such as hospital characteristics, patients admitted and wards, access to diagnostic tools, estimated infection incidence, and treatment capacity. Thirty responses were collected from facilities throughout Argentina. Most institutions were governmental (77%). A mycology department was available in 83% of them. Histopathology was available in almost 93% of the sites, while automated methods and galactomannan tests were available in 57%, each; 53% of the sites had access to MALDI-TOF-MS through regional reference laboratories, and PCR was present in 20% of the sites. Susceptibility testing was available in 63% of the laboratories. Candida spp. (24%), Cryptococcus spp. (20%), Aspergillus spp. (18%), and Histoplasma spp. (16%) were described as the main pathogens. Fluconazole was the only antifungal agent available in all institutions. This was followed by amphotericin B deoxycholate (83%) and itraconazole (80%). If an antifungal agent was not available onsite, then 60% of the patients could receive adequate antifungal treatment within the first 48 h upon request. Although there are no significant differences in access to diagnostic and clinical management of invasive fungal infections among the Argentinean centres studied, national awareness-raising initiatives led by policymakers could help to improve their general availability. [ABSTRACT FROM AUTHOR]

Infectious vaginitis due to bacterial vaginosis (BV), vulvovaginal candidiasis (VVC), and Trichomonas vaginalis accounts for a significant proportion of all gynecologic visits in the United States. A prospective multicenter clinical study was conducted to validate the performance of two new in vitro diagnostic transcription-mediated amplification nucleic acid amplification tests (NAATs) for diagnosis of BV, VVC, and trichomoniasis. Patient- and clinician-collected vaginal-swab samples obtained from women with symptoms of vaginitis were tested with the Aptima BV and Aptima Candida/Trichomonas vaginitis (CV/TV) assays. The results were compared to Nugent (plus Amsel for intermediate Nugent) scores for BV, Candida cultures and DNA sequencing for VVC, and a composite of NAAT and culture for T. vaginalis The prevalences of infection were similar for clinician- and patient-collected samples: 49% for BV, 29% for VVC due to the Candida species group, 4% for VVC due to Candida glabrata, and 10% for T. vaginalis Sensitivity and specificity estimates for the investigational tests in clinician-collected samples were 95.0% and 89.6%, respectively, for BV; 91.7% and 94.9% for the Candida species group; 84.7% and 99.1% for C. glabrata; and 96.5% and 95.1% for T. vaginalis Sensitivities and specificities were similar in patient-collected samples. In a secondary analysis, clinicians' diagnoses, in-clinic assessments, and investigational-assay results were compared to gold standard reference methods. Overall, the investigational assays had higher sensitivity and specificity than clinicians' diagnoses and in-clinic assessments, indicating that the investigational assays were more predictive of infection than traditional diagnostic methods. These results provide clinical-efficacy evidence for two in vitro diagnostic NAATs that can detect the main causes of vaginitis.

ObjectiveRecently, in East Azerbaijan province, marketing of top sheep carrying fertility genes became suspected, because some farmers complained about the claim of the seller due to the low performance of such ewes on the farm. With this research motivation, the aim of the present study in the first step is to combine PCR-RFLP and subsequent PCR-Sequencing methods for genetic refinement of the FecB gene locus associated with litter size in the Booroola-Afshari sheep breed. Materials and methodsIn this study, overall 62 ewes and rams of Booroola-Afshari sheep and 30 ewes of Ghezel- Romanov hybrids were obtained from different sheep farms around Tabriz. Then, two-separate phases were designed, after amplification of the 190 bp region of the BMPR15 gene by PCR, two molecular PCR- RFLP methods and direct sequencing were performed according to routine laboratory instructions. Genotype results were recorded and subsequently, using different software such as POPGENE, FinchTV, and MAFFT, the genotypic, allelic, and alignment frequencies of the amplified sequences were performed to confirm the mutation, respectively.ResultsThe results of the present report demonstrated that the source of discrepancy between the identified genotype and the actual genotype can be due to the error of reading the genotype or the seller's fraud and false claim. Also, the combination and both outputs of both molecular techniques, PCR- RFLP and direct sequencing, can minimize the technical error caused by the genotype. In addition, high reproducibility observations confirmed the presence of 8/06% fraud in the FecB gene locus genotype. ConclusionsThe combination of two molecular methods, PCR- RFLP and PCR-sequencing direct sequencing to detect FecB mutations, has shown acceptable efficiencies, and the establishment of government-affiliated genotyping centers for fraud detection can assist regulators in genotyping sheep with fertility genes and may reduce the number of frauds.

PurposeTo evaluate the diagnostic efficacy of CapitalBio Mycobacterium real-time polymerase chain reaction assay (CapitalBio test) in spinal tuberculosis (STB). The value of histopathology combined with the CapitalBio test in diagnosing STB was also assessed.MethodsWe retrospectively analyzed the medical information of suspected STB. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and area under the curve (AUC) of histopathology, CapitalBio test, and histopathology combined with CapitalBio test were calculated to evaluate their diagnostic efficacy compared with a composite reference standard.ResultsA total of 222 suspected STB patients were included in the study. The sensitivity, specificity, PPV, NPV, and AUC of histopathology for STB were recorded to be 62.0, 98.0, 97.4, 68.3%, and 0.80, respectively. The sensitivity, specificity, PPV, NPV, and AUC of the CapitalBio test were 75.2, 98.0, 97.9, 76.7%, and 0.87, respectively, while that of histopathology combined with the CapitalBio test was 81.0, 96.0, 96.1, 80.8%, and 0.89, respectively.ConclusionHistopathology and CapitalBio test exhibited high accuracy and are recommended in diagnosing STB. Histopathology combined with the CapitalBio test might give the best efficacy in STB diagnosis.

Background and Aim: Emergence of drug resistance, especially to second‐line drugs, hampers tuberculosis elimination efforts. The present study aimed to evaluate MTBDRplus and MTBDRsl assays for detecting first‐line and second‐line drug resistance, respectively, in gastrointestinal tuberculosis (GITB). Methods: Thirty ileocecal biopsy specimens, processed in the Department of Microbiology between 2012 and 2022, that showed growth of Mycobacterium tuberculosis on culture were included in the study. DNA, extracted from culture, was subjected to MTBDRplus and MTBDRsl (Hain Lifescience GmbH, Nehren, Germany), following manufacturer's instructions. Their performance was compared against phenotypic drug susceptibility testing (pDST) and gene sequencing. Results: Out of the 30 specimens, 4 (13.33%) were mono‐isoniazid resistant, 4 (13.33%) were multidrug resistant (MDR), 2 (6.67%) were pre‐extensively drug resistant (pre‐XDR), and 2 (6.67%) were mono‐fluoroquinolone resistant. The results were 100% concordant with pDST and gene sequencing. Conclusions: In the wake of growing drug resistance in all forms of extrapulmonary tuberculosis, including GITB, MTBDRplus and MTBDRsl are reliable tools for screening of resistance to both first‐line and second‐line drugs. [ABSTRACT FROM AUTHOR]

Objective: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant syndrome characterized by its clinical variability and complexity in diagnosis and treatment. We performed both clinical and molecular descriptions of four families with MEN1 in a follow-up at a tertiary center in Brası'lia. Methods: From a preliminary review of approximately 500 medical records of patients with pituitary neuroendocrine tumor (PitNET) from the database of the Neuroendocrinology Outpatient Clinic of the University Hospital of Brası'lia, a total of 135 patients met the criteria of at least two affected family members. From this cohort, we have identified 34 families: only four with a phenotype of MEN1 and the other 30 families with the phenotype of familial isolated pituitary adenoma (FIPA). Eleven patients with a clinical diagnosis of MEN1 from these four families were selected. Results: Variants in MEN1 gene were identified in all families. One individual from each family underwent genetic testing using targeted high-throughput sequencing (HTS). All patients had primary hyperparathyroidism (PHPT), and the second most common manifestation was PitNET. One individual had welldifferentiated liposarcoma, which has been previously reported in a single case of MEN1. Three variants previously described in the database and a novel variant in exon 2 have been found. Conclusions: The study allowed the genotypic and phenotypic characterization of families with MEN1 in a follow-up at a tertiary center in Brası'lia. [ABSTRACT FROM AUTHOR]

Molecular and antigen point-of-care tests (POCTs) have augmented our ability to rapidly identify and manage SARS-CoV-2 infection. However, their clinical performance varies among individual studies. The evaluation of the performance of molecular and antigen-based POCTs in confirmed, suspected, or probable COVID-19 cases compared with that of laboratory-based RT-PCR in real-life settings. MEDLINE/PubMed, Scopus, Embase, Web of Science, Cochrane Library, Cochrane COVID-19 study register, and COVID-19 Living Evidence Database from the University of Bern. Peer-reviewed or preprint observational studies or randomized controlled trials that evaluated any type of commercially available antigen and/or molecular POCTs for SARS-CoV-2, including multiplex PCR panels, approved by the United States Food and Drug Administration, with Emergency Use Authorization, and/or marked with Conformitè Europëenne from European Commission/European Union. Close contacts and/or patients with symptomatic and/or asymptomatic confirmed, suspected, or probable COVID-19 infection of any age. Molecular and/or antigen-based SARS-CoV-2 POCTs. Laboratory-based SARS-CoV-2 RT-PCR. Eligible studies were subjected to quality-control and risk-of-bias assessment using the Quality Assessment of Diagnostic Accuracy Studies 2 tool. Summary sensitivities and specificities with their 95% CIs were estimated using a bivariate model. Subgroup analysis was performed when at least three studies informed the outcome. A total of 123 eligible publications (97 and 26 studies assessing antigen-based and molecular POCTs, respectively) were retrieved from 4674 initial records. The pooled sensitivity and specificity for 13 molecular-based POCTs were 92.8% (95% CI, 88.9–95.4%) and 97.6% (95% CI, 96.6–98.3%), respectively. The sensitivity of antigen-based POCTs pooled from 138 individual evaluations was considerably lower than that of molecular POCTs; the pooled sensitivity and specificity rates were 70.6% (95% CI, 67.2–73.8%) and 98.9% (95% CI, 98.5–99.2%), respectively. Further studies are needed to evaluate the performance of molecular and antigen-based POCTs in underrepresented patient subgroups and different respiratory samples. [ABSTRACT FROM AUTHOR]

Lung cancer remains the leading cause of cancer deaths worldwide. International societies have promoted the molecular analysis of MET proto-oncogene, receptor tyrosine kinase (MET) exon 14 skipping for the clinical stratification of non-small cell lung cancer (NSCLC) patients. Different technical approaches are available to detect MET exon 14 skipping in routine practice. Here, the technical performance and reproducibility of testing strategies for MET exon 14 skipping carried out in various centers were evaluated. In this retrospective study, each institution received a set (n = 10) of a customized artificial formalin-fixed paraffin-embedded (FFPE) cell line (Custom METex14 skipping FFPE block) that harbored the MET exon 14 skipping mutation (Seracare Life Sciences, Milford, MA, USA), which was previously validated by the Predictive Molecular Pathology Laboratory at the University of Naples Federico II. Each participating institution managed the reference slides according to their internal routine workflow. MET exon 14 skipping was successfully detected by all participating institutions. Molecular analysis highlighted a median Cq cut off of 29.3 (ranging from 27.1 to 30.7) and 2514 (ranging from 160 to 7526) read counts for real-time polymerase chain reaction (RT-PCR) and NGS-based analyses, respectively. Artificial reference slides were a valid tool to harmonize technical workflows in the evaluation of MET exon 14 skipping molecular alterations in routine practice. [ABSTRACT FROM AUTHOR]

Ebola is a highly pathogenic virus, which in humans reaches a mortality rate above 50%. Due to a lack of laboratories in territories where Ebola viruses are endemic and the limited number of surveillance programmes, tests for the confirmation of suspected cases of Ebola are often performed in Reference Laboratories. While this provides guarantees regarding the accuracy of results, the shipment of samples to a centralized facility where the diagnostic test can be performed and the time required to achieve the results takes several days, which increases costs and entails delays in the isolation of positive subjects and therapeutic intervention with negative consequences both for patients and the community. Molecular tests have been the most frequently used tool in Ebola diagnosis in recent outbreaks. One of the most commonly used molecular tests is the Real-Star Altona, which targets a conserved area of the L gene. This assay showed different sensitivities depending on the Ebola virus: 471 copies/mL (EBOV) and 2871 copies/ml (SUDAN virus). The Cepheid system also showed good sensitivity (232 copies/mL). The LAMP platform is very promising because, being an isothermal reaction, it does not require high-precision instrumentation and can be considered a Point of Care (PoC) tool. Its analytical sensitivity is 1 copy/reaction. However, since data from real life studies are not yet available, it is premature to give any indications on its feasibility. Moreover, in November 2014, the WHO recommended the development of rapid diagnostic tests (RDT) according to ASSURED criteria. Several RDT assays have since been produced, most of which are rapid tests based on the search for antibody anti-Ebola viral proteins with immunochromatographic methods. Several viral antigens are used for this purpose: VP40, NP and GP. These assays show different sensitivities according to the protein used: VP40 57.4–93.1%, GP 53–88.9% and 85% for NP compared to reference molecular assays. From these results, it can be deduced that no RDT reaches the 99% sensitivity recommended by the WHO and therefore any RDT negative results in suspected cases should be confirmed with a molecular test.

We performed a prospective study to determine if the pretest probability of a positive loop-mediated isothermal amplification test is greater when there are more signs and symptoms of GAS pharyngitis. Patients were enrolled if a clinician obtained a GAS RADT. The McIsaac score was calculated. The prevalence of positive LAMP and RADT results increased as the McIsaac score increased. The calculated sensitivity of LAMP was superior to RADT. [ABSTRACT FROM AUTHOR]

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In the Redclaw Crayfish, Cherax quadricarinatus, males grow larger and faster than females, and interest has focused on monosex breeding techniques for the production of males only. The adult male and female sexual characteristics of Cherax quadricarinatus are easily distinguished but cannot be determined in the embryo and juvenile stages. Because there is no DNA marker for the identification of sex in Cherax quadricarinatus, we sequenced its juvenile transcriptome in order to produce a reliable transcript to determine sex and to investigate its sex differentiation mechanisms at the molecular level. In this study, a female sex‐linked expression gene CqLRPPRC (Leucine‐rich PPR‐motif‐containing) of Cherax quadricarinatus was found in the transcriptome. Using semi‐quantitative RT‐PCR analysis, we found that the expression of the LRPPRC (leucine‐rich pentatricopeptide repeat cassette) gene was greater in the ovaries of females than in the testes of males, from the early juvenile stages onwards. Furthermore, in situ hybridization showed that the expression of its sexual dimorphism may be focused in the gonads. The results of an RNA interference treatment showed that a knockdown of CqLRPPRC up‐regulated the expression of vitellogenin (Vtg). The LRPPRC gene, and characteristic of shrimp, are reported here for the first time, and an assay of this gene can provide a molecular test to determine the sex of Cherax quadricarinatus in its different developmental stages, especially those during which no obvious sexual differences are visible. [ABSTRACT FROM AUTHOR]

The present study examined and reported Hepatocystis sp. infection in wild-caught vervet monkeys imported from Tanzania into the Razi vaccine and serum research institute (RVSRI). Polymerase chain reaction (PCR) targeting of 18S rRNA gene, followed by sequencing, Basic Local Alignment Search Tool (BLAST), and phylogenetic studies revealed that 82.8% of the imported monkeys were infected with Hepatocystis. Nevertheless, as illustrated by a routine parasitological examination of blood smears and histopathological examination of liver collected samples, the rates of Hepatocystis infection were obtained at 33.9% and 38.1%, respectively. Two isolated 18S rRNA gene sequences of Hepatocystis sp. from Tanzanian vervet monkeys were registered under the accession numbers OM281567 and OM281564 in GenBank. Although Hepatocystis infections do not cause clinical disease, they may interfere with the research data. The results of the current study pointed out that after proper nutrition and implementation of good physical environmental conditions for 3-4 months, the imported monkeys obviously gained weight and most of their hematological parameters, even in the presence of the parasite, returned to the normal levels and the experimental monkeys would be ready for use in studies. [ABSTRACT FROM AUTHOR]

Introduction: Sclerosing pneumocytoma (SP) is a rare benign tumor and a potential diagnostic pitfall. Our aim was to review the cytologic features of our surgically diagnosed SP cases including the clinical, immunohistochemical and available molecular findings., Materials and Methods: A computerized search from 2013 to 2020 for surgical cases with corresponding cytology specimens diagnosed as SP was performed. The clinical data, cytology, and surgical specimens were collated for analysis., Results: Six cytology specimens were collected. All were female (mean age = 35). Three have incidental lung nodules and three with cough. Cytologic findings showed variable architectural pattern (papillary, solid, singly scattered, acinar/rosette-like) and cellular heterogeneity (surface, stromal, epithelioid, plasmacytoid cells). Atypia was identified in 4/6 cases. The original cytology diagnoses were negative = 1, SP = 2 and adenocarcinoma = 3. The latter diagnoses were amended to SP after review of the surgical specimens. The three false positive cases on review have cytologic features mimicking adenocarcinoma. Immunohistochemical stains showed tumor cells (surface and stromal) were positive for TTF-1, and EMA with only the surface cells positive for pancytokeratin and Napsin A. Though two cases sent for molecular testing were negative for AKT1 or CTNNB1 exon 3 mutation, our panel did not evaluate AKT1 exon 4., Conclusions: SP is a diagnostic pitfall with 50% initially misdiagnosed as adenocarcinoma. Integrating the clinical/radiologic findings, cytologic features, and performance of immunohistochemistry on cell block are helpful in avoiding misdiagnosis. Molecular testing for recurrent mutations, if present, could be helpful for diagnosis and possible therapy options. However, routinely used molecular testing may not always capture relevant molecular markers for SP., (© 2024 The Author(s). Diagnostic Cytopathology published by Wiley Periodicals LLC.)

Objectives: The rising rates of Helicobacter pylori antimicrobial resistance highlight the importance of better understanding its epidemiology in the pediatric population. We aim to study the epidemiological factors associated with antimicrobial resistance in a cohort of US children with H. pylori infection., Methods: A retrospective cohort study of patients in the New England region of the United States between January 15, 2015 and October 15, 2022, with a first-time diagnosis of H. pylori on gastric biopsy and who had antimicrobial resistance data available. Descriptive statistics and logistic regression models were used to determine associations between the patient's demographics, clinical factors, endoscopic findings, and antimicrobial resistance., Results: Out of 273 patients (46% male, median 12.8 years), 118 (43.2%) were resistant to at least one antimicrobial. Clarithromycin resistance (24.5%) was the highest, followed by metronidazole (21.6%), fluoroquinolones (9.9%), rifabutin (3.3%), and amoxicillin (2.6%). Clarithromycin resistance was higher in Asians compared to white individuals (odds ratio [OR]: 4.68, 95% confidence interval [CI]: [2.01-10.89], p < 0.001), and lower in black compared to white individuals (OR: 0.23, 95% CI: [0.08-0.69], p = 0.01). Antimicrobial resistance to clarithromycin and metronidazole were similar among Hispanics. Asian individuals were more likely to demonstrate one or more antimicrobial resistances compared to whites (OR: 3.66, 95% CI: [1.50-8.89], p < 0.001). Compared to individuals from higher household incomes (>$100,000), those from less than $30,000 household incomes had a higher risk of metronidazole resistance (OR: 7.94, 95% CI: [1.83-34.36], p = 0.01)., Conclusion: Our study provides novel information concerning the association of H. pylori antimicrobial resistance to race, socioeconomic status, and age in the pediatric population., (© 2024 European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition.)

Context: BRAF V600E mutation is the most common genetic driver of papillary thyroid cancer (PTC), where it is found with various allele frequency (AF), reflecting the proportion of cells carrying the mutant and wild-type gene alleles., Objective: To determine whether BRAF V600E AF can improve prognostication and inform initial surgical management of PTC., Design: Retrospective cohort study (2016-2019)., Setting: UCLA health., Patients: Consecutive patients with Bethesda V/VI nodules and isolated BRAF V600E mutation who underwent surgery with histopathology showing PTC., Interventions: Blinded ThyroSeq v3 molecular analysis after completion of initial management and follow-up., Main Outcomes Measures: Aggressive histopathology and cancer persistence/recurrence., Results: Of 73 patients, the median BRAF V600E AF was 25.5% (IQR, 16.7-34.3%). Higher median AF was seen in patients classified as American Thyroid Association (ATA) high-risk (37%) vs. intermediate-risk (25.3%, p<0.01) and low-risk (24.7%, p<0.01), largely attributed to higher AF in patients with gross extrathyroidal extension (ETE) (40.1% vs. 25.2% without gross ETE, p=0.02). No differences in AF were observed on the basis of lymph node positivity or presence of aggressive variants of PTC. A higher BRAF V600E AF was also found in patients with tumors ≥2cm vs. <2cm (median 32.0% vs. 24.4%, p<0.01). Over 4.1 years of follow-up, disease persistence/recurrence was found in 7 patients (9.4%) and was associated with higher median AF than those without recurrence (35.3% vs. 25.2%, p=0.02). Higher AF was associated with poorer recurrence-free survival (AF≥35%, HR 7.40, CI 1.4-38.1)., Conclusions: Higher AF was associated with gross ETE and increased recurrence risk. This may inform initial management in patients with PTC harboring an isolated BRAF V600E mutation., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com. See the journal About page for additional terms.)

Abstract Background Asymptomatic Leishmania infection may play an important role in the transmission of the parasite in endemic areas. At present there is no consensus on the definition of asymptomatic Leishmania infection, nor is there a safe and accessible gold standard test for its identification. Methods This paper presents a scoping review to summarize definitions of asymptomatic Leishmania infection found in the literature, as well as to detail the approach (molecular, serological, cellular, and/or parasitological tests) used by researchers to identify this asymptomatic population. A scoping review of published and gray literature related to asymptomatic Leishmania infection was conducted; retrieved citations were screened based on predefined eligibility criteria, and relevant data items were extracted from eligible articles. The analysis is descriptive and is presented using tables, figures, and thematic narrative synthesis. Results We conducted a screening of 3008 articles, of which 175 were selected for the full review. Of these articles, we selected 106 that met the inclusion criteria. These articles were published between 1991 and 2021, and in the last 5 years, up to 38 articles were reported. Most of the studies were conducted in Brazil (26%), Spain (14%), India (12%), Bangladesh (10%), and Ethiopia (7%). Of the studies, 84.9% were conducted in the immunocompetent population, while 15.1% were conducted in the immunosuppressed population (HIV, immunosuppressive drugs, and organ transplantation population). We report 14 different techniques and 10 strategies employed by researchers to define asymptomatic Leishmania infection in an endemic area. Conclusions The definition of asymptomatic Leishmania infection is not unified across the literature, but often includes the following criteria: residence (or extended stay) in a Leishmania-endemic area, no reported signs/symptoms compatible with leishmaniasis, and positive on a combination of serological, molecular, cellular, and/or parasitological tests. Caution is recommended when comparing results of different studies on the subject of asymptomatic infections, as the reported prevalence cannot be confidently compared between areas due to the wide variety of tests employed by research groups. More research on the importance of asymptomatic immunosuppressed and immunocompetent Leishmania-positive populations in leishmaniasis epidemiology is required. Graphical Abstract

ABSTRACT: Introduction: In the coronavirus disease 2019 era, debate around the risk of contagion in school is intense in Italy. The Department of Welfare and Health of Florence promoted a screening campaign with rapid antigen tests for all students and school personnel. The aim of this study was to assess the circulation of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in the school setting by means of mass screening in every primary and middle school in Florence. Methods: All students and school personnel at primary and middle schools in Florence were asked to take part. The campaign started on 16 November 2020 and was completed on 12 February 2021. If a subject had a positive result on rapid antigen testing, a molecular test was performed to confirm the result. Results: In total, 18,414 subjects were tested: 15,233 students (82.7%) and 3181 school personnel (17.3%). The rapid antigen test gave a positive result in 27 cases (0.15%). Of these, only 14 tests were confirmed to be positive on molecular testing. These results show a very low number of cases of SARS-CoV-2 among the study subjects (0.08%). Conclusions: These results show that the spread of SARS-CoV-2 in the school setting was low in Florence during the screening period.