Construction of the Classification Model Using Key Genes Identified Between Benign and Malignant Thyroid Nodules From Comprehensive Transcriptomic Data

Thyroid nodules are present in upto 50% of the population worldwide, and thyroid malignancy occurs in only 5–15% of nodules. Until now, fine-needle biopsy with cytologic evaluation remains the diagnostic choice to determine the risk of malignancy, yet it fails to discriminate as benign or malignant in one-third of cases. In order to improve the diagnostic accuracy and reliability, molecular testing based on transcriptomic data has developed rapidly. However, gene signatures of thyroid nodules identified in a plenty of transcriptomic studies are highly inconsistent and extremely difficult to be applied in clinical application. Therefore, it is highly necessary to identify consistent signatures to discriminate benign or malignant thyroid nodules. In this study, five independent transcriptomic studies were combined to discover the gene signature between benign and malignant thyroid nodules. This combined dataset comprises 150 malignant and 93 benign thyroid samples. Then, there were 279 differentially expressed genes (DEGs) discovered by the feature selection method (Student’s t test and fold change). And the weighted gene co-expression network analysis (WGCNA) was performed to identify the modules of highly co-expressed genes, and 454 genes in the gray module were discovered as the hub genes. The intersection between DEGs by the feature selection method and hub genes in the WGCNA model was identified as the key genes for thyroid nodules. Finally, four key genes (ST3GAL5, NRCAM, MT1F, and PROS1) participated in the pathogenesis of malignant thyroid nodules were validated using an independent dataset. Moreover, a high-performance classification model for discriminating thyroid nodules was constructed using these key genes. All in all, this study might provide a new insight into the key differentiation of benign and malignant thyroid nodules.

Evaluating Pancreatic and Biliary Neoplasms with Small Biopsy-Based Next Generation Sequencing (NGS): Doing More with Less

Pancreatic cancer and cholangiocarcinoma are lethal diseases mainly diagnosed at an inoperable stage. As pancreatobiliary surgical specimens are often unavailable for further molecular testing, this review aimed to highlight the diagnostic, prognostic, and therapeutic impact of next-generation sequencing (NGS) performed on distinct small biopsies, including endoscopic ultrasound fine-needle aspirations and biopsies of pancreatic solid and cystic lesions, biliary duct brushings, and also “liquid biopsies” such as the pancreatic juice, bile, and blood. NGS could clarify indeterminate pancreatic lesions or biliary strictures, for instance by identifying TP53 or SMAD4 mutations indicating high-grade dysplasia or cancer. It could also stratify pancreatic cystic lesions, by distinguishing mucinous from non-mucinous cysts and identifying high-risk cysts that should be excised in surgically fit patients, whereas the combination of cytology, elevated cystic CEA levels and NGS could improve the overall diagnostic accuracy. When NGS is performed on the pancreatic juice, it could stratify high-risk patients under surveillance. On the plasma, it could dynamically monitor the disease course and response to therapy. Notably, the circulating tumor DNA (ctDNA) levels have been associated with staging, grading, and survival. Lastly, NGS has shown potential in identifying potentially actionable molecular alterations. In conclusion, NGS applied on small biopsies could carry significant diagnostic, prognostic, and therapeutic value.

Collaborative workflow between pathologists and deep learning for evaluation of tumor cellularity in lung adenocarcinoma

Owing to the high demand for molecular testing, the reporting of tumor cellularity in cancer samples has become a mandatory task for pathologists. However, the pathological estimation of tumor cellularity is often inaccurate. We developed a collaborative workflow between pathologists and artificial intelligence (AI) models to evaluate tumor cellularity in lung cancer samples and prospectively applied it to routine practice. We also developed a quantitative model that we validated and tested on retrospectively analyzed cases and ran the model prospectively in a collaborative workflow where pathologists could access the AI results and apply adjustments (Adjusted-Score). The Adjusted-Scores were validated by comparing them with the ground truth established by manual annotation of hematoxylin-eosin slides with reference to immunostains with thyroid transcription factor-1 and napsin A. For training, validation, retrospective testing, and prospective application of the model, we used 40, 10, 50, and 151 whole slide images, respectively. The sensitivity and specificity of tumor segmentation were 97% and 87%, and the accuracy of nuclei recognition was 99%. Pathologists altered the initial scores in 87% of the cases after referring to the AI results and found that the scores became more precise after collaborating with AI. For validation of Adjusted-Score, we found the Adjusted-Score was significantly closer to the ground truth than non-AI-aided estimates (p<0.05). Thus, an AI-based model was successfully implemented into the routine practice of pathological investigations. The proposed model for tumor cell counting efficiently supported the pathologists to improve the prediction of tumor cellularity for genetic tests.

Neuronal Ceroid Lipofuscinosis Type 6 (CLN6) clinical findings and molecular diagnosis: Costa Rica’s experience

Background Commonly known as Batten disease, the neuronal ceroid lipofuscinoses (NCLs) are a genetically heterogeneous group of rare pediatric lysosomal storage disorders characterized by the intracellular accumulation of autofluorescent material (known as lipofuscin), progressive neurodegeneration, and neurological symptoms. In 2002, a disease-causing NCL mutation in the CLN6 gene was identified (c.214G > T) in the Costa Rican population, but the frequency of this mutation among local Batten disease patients remains incompletely characterized, as do clinical and demographic attributes for this rare patient population. Objective To describe the main sociodemographic and clinical characteristics of patients with a clinical diagnosis for Batten Disease treated at the National Children's Hospital in Costa Rica and to characterize via molecular testing their causative mutations. Methods DNA extracted from buccal swabs was used for CLN6 gene sequencing. Participants’ sociodemographic and clinical characteristics were also obtained from their medical records. Results Nine patients with a clinical diagnosis of Batten disease were identified. Genetic sequencing determined the presence of the previously described Costa Rican homozygous mutation in 8 of 9 cases. One patient did not have mutations in the CLN6 gene. In all cases where the Costa Rican CLN6 mutation was present, it was accompanied by a substitution in intron 2. Patients were born in 4 of the 7 Costa Rican provinces, with an average onset of symptoms close to 4 years of age. No parental consanguinity was present in pedigrees. Initial clinical manifestations varied between patients but generally included: gait disturbances, language problems, visual impairment, seizures and psychomotor regression. Cortical and cerebellar atrophy was a constant finding when neuroimaging was performed. Seizure medication was a common element of treatment regimens. Conclusions This investigation supports that the previously characterized c.214G > T mutation is the most common causative NCL mutation in the Costa Rican population. This mutation is geographically widespread among Costa Rican NCL patients and yields a clinical presentation similar to that observed for CLN6 NCL patients in other geographies.

Clinical Symptoms and Outcomes of Severe Pneumonia Caused by Chlamydia psittaci in Southwest China

Here, we aimed to retrospectively analyze the clinical characteristics of 27 patients with severe pneumonia caused by Chlamydia psittaci between January 2019 and April 2021 in southwest China. To this end, we collected data on the exposure history, clinical symptoms, laboratory examination, imaging characteristics, evolution, etiology, treatment, and outcomes to suggest a better diagnosis and prevention system. Our results showed that a metagenomic next-generation sequencing test could provide early diagnosis. All patients were sensitive to quinolones and tetracyclines, and the recovery rate was relatively high. Overall, all patients were in critical condition with moderate to severe acute respiratory distress syndrome and shock. In conclusion, early diagnosis of pneumonia caused by C. psittaci depends on effective molecular testing, and most patients recover after treatment.

Evaluating performance of multiplex real time PCR for the diagnosis of malaria at elimination targeted low transmission settings of Ethiopia

Background Malaria incidence has declined in Ethiopia in the past 10 years. Current malaria diagnostic tests, including light microscopy and rapid antigen-detecting diagnostic tests (RDTs) cannot reliably detect low-density infections. Studies have shown that nucleic acid amplification tests are highly sensitive and specific in detecting malaria infection. This study took place with the aim of evaluating the performance of multiplex real time PCR for the diagnosis of malaria using patient samples collected from health facilities located at malaria elimination targeted low transmission settings in Ethiopia. Methods A health facility-based, cross-sectional survey was conducted in selected malaria sentinel sites. Malaria-suspected febrile outpatients referred to laboratory for malaria testing between December 2019 and March 2020 was enrolled into this study. Sociodemographic information and capillary blood samples were collected from the study participants and tested at spot with RDTs. Additionally, five circles of dry blood spot (DBS) samples on Whatman filter paper and thick and thin smear were prepared for molecular testing and microscopic examination, respectively. Multiplex real time PCR assay was performed at Ethiopian Public Health Institute (EPHI) malaria laboratory. The performance of multiplex real time PCR assay, microscopy and RDT for the diagnosis of malaria was compared and evaluated against each other. Results Out of 271 blood samples, multiplex real time PCR identified 69 malaria cases as Plasmodium falciparum infection, 16 as Plasmodium vivax and 3 as mixed infections. Of the total samples, light microscopy detected 33 as P. falciparum, 18 as P. vivax, and RDT detected 43 as P. falciparum, 17 as P. vivax, and one mixed infection. Using light microscopy as reference test, the sensitivity and specificity of multiplex real time PCR were 100% (95% CI (93–100)) and 83.2% (95% CI (77.6–87.9)), respectively. Using multiplex real time PCR as a reference, light microscopy and RDT had sensitivity of 58% (95% CI 46.9–68.4) and 67% (95% CI 56.2–76.7); and 100% (95% CI 98–100) and 98.9% (95% CI 96–99.9), respectively. Substantial level of agreement was reported between microscopy and multiplex real time PCR results with kappa value of 0.65. Conclusions Multiplex real-time PCR had an advanced performance in parasite detection and species identification on febrile patients’ samples than did microscopy and RDT in low malaria transmission settings. It is highly sensitive malaria diagnostic method that can be used in malaria elimination programme, particularly for community based epidemiological samples. Although microscopy and RDT had reduced performance when compared to multiplex real time PCR, still had an acceptable performance in diagnosis of malaria cases on patient samples at clinical facilities.

Targeted Therapies for Lung Cancer Patients With Oncogenic Driver Molecular Alterations

Lung cancer has traditionally been classified by histology. However, a greater understanding of disease biology and the identification of oncogenic driver alterations has dramatically altered the therapeutic landscape. Consequently, the new classification paradigm of non–small-cell lung cancer is further characterized by molecularly defined subsets actionable with targeted therapies and the treatment landscape is becoming increasingly complex. This review encompasses the current standards of care for targeted therapies in lung cancer with driver molecular alterations. Targeted therapies for EGFR exon 19 deletion and L858R mutations, and ALK and ROS1 rearrangements are well established. However, there is an expanding list of approved targeted therapies including for BRAF V600E, EGFR exon 20 insertion, and KRAS G12C mutations, MET exon 14 alterations, and NTRK and RET rearrangements. In addition, there are numerous other oncogenic drivers, such as HER2 exon 20 insertion mutations, for which there are emerging efficacy data for targeted therapies. The importance of diagnostic molecular testing, intracranial efficacy of novel therapies, the optimal sequencing of therapies, role for targeted therapies in early-stage disease, and future directions for precision oncology approaches to understand tumor evolution and therapeutic resistance are also discussed.

Bagaza virus and Plasmodium spp. coinfection in red-legged partridges ( Alectoris rufa ), in southern Spain 2019

Flaviviruses West Nile (WNV), Usutu (USUV) and Bagaza (BAGV) virus and avian malaria parasites are vector borne pathogens that circulate naturally between avian and mosquito hosts. WNV and USUV and potentially also BAGV constitute zoonoses. Temporal and spatial co-circulation and co-infection with Plasmodium spp., and West Nile virus has been documented in birds and mosquito vectors, and fatally USUV infected passerines coinfected with Plasmodium spp. had more severe lesions. Also, WNV, USUV and BAGV have been found to co-circulate. Yet little is known about the interaction of BAGV and malaria parasites during consecutive or co-infections of avian hosts. Here we report mortality of free-living red-legged partridges in a hunting estate in Southern Spain due to coinfection with BAGV and Plasmodium spp. The outbreak occurred in the area where BAGV first emerged in Europe in 2010 and where co-circulation of BAGV, USUV and WNV was confirmed in 2011 and 2013. Partridges were found dead in early October 2019. Birds had mottled locally pale pectoral muscles, enlarged, congestive greenish-black tinged livers and enlarged kidneys. Microscopically congestion and predominantly mononuclear inflammatory infiltrates were evident and Plasmodium phanerozoites were present in the liver, spleen, kidneys, muscle and skin. Molecular testing and sequencing detected Plasmodium spp. and BAGV in different tissues of the partridges, and immunohistochemistry confirmed the presence and co-localization of both pathogens in the liver and spleen. Although Plasmodium spp. are known to be highly prevalent in red-legged partridges, this is the first account of mortality caused by co-infection with BAGV and Plasmodium sp. Due to the importance of the red-legged partridge in the ecosystem of the Iberian Peninsula and as driver of regional economy such mortalities are of concern. Also, they may reflect climate change related changes in host, vector and pathogen ecology and interactions that could emerge similarly in other pathogens.

HSP110 as a Diagnostic but Not a Prognostic Biomarker in Colorectal Cancer With Microsatellite Instability

Determination of microsatellite instability (MSI) using molecular test and deficient mismatch repair (dMMR) using immunohistochemistry (IHC) has major implications on colorectal cancer (CRC) management. The HSP110 T17 microsatellite has been reported to be more monomorphic than the common markers used for MSI determination. Large deletion of HSP110 T17 has been associated with efficacy of adjuvant chemotherapy in dMMR/MSI CRCs. The aim of this study was to evaluate the interest of HSP110 deletion/expression as a diagnostic tool of dMMR/MSI CRCs and a predictive tool of adjuvant chemotherapy efficacy. All patients with MSI CRC classified by molecular testing were included in this multicenter prospective cohort (n = 381). IHC of the 4 MMR proteins was carried out. HSP110 expression was carried out by IHC (n = 343), and the size of HSP110 T17 deletion was determined by PCR (n = 327). In the 293 MSI CRCs with both tests, a strong correlation was found between the expression of HSP110 protein and the size of HSP110 T17 deletion. Only 5.8% of MSI CRCs had no HSP110 T17 deletion (n = 19/327). HSP110 T17 deletion helped to re-classify 4 of the 9 pMMR/MSI discordance cases as pMMR/MSS cases. We did not observe any correlation between HSP110 expression or HSP110 T17 deletion size with time to recurrence in patients with stage II and III CRC, treated with or without adjuvant chemotherapy. HSP110 is neither a robust prognosis marker nor a predictor tool of adjuvant chemotherapy efficacy in dMMR/MSI CRC. However, HSP110 T17 is an interesting marker, which may be combined with the other pentaplex markers to identify discordant cases between MMR IHC and MSI.

The Diagnostic Accuracy of Syndromic Management for Genital Ulcer Disease: A Systematic Review and Meta-Analysis

Objectives: Genital Ulcer Disease (GUD) carries a significant disease burden globally. With limited access to diagnostics, the 2001 World Health Organization (WHO) sexually transmitted illnesses (STI) guidelines proposed a syndromic management algorithm that required a clinical decision to determine the management of GUD. We assessed the diagnostic accuracy of this algorithm.Methods: We conducted a systematic review (Prospero: CRD42020153294) using eight databases for publications between 1995 and January 2021 that reported primary data on the diagnostic accuracy of clinical diagnosis to identify aetiological agents of GUD. Titles and abstracts were independently assessed for eligibility, and data were extracted from full texts for sensitivity/specificity. A hierarchical logistic regression model was used to derive pooled sensitivity and specificity. We used GRADE to evaluate the certainty of evidence.Results: Of 24,857 articles, 151 full texts were examined and 29 included in the analysis. The majority were from middle-income countries [(14/29 (48%) lower middle, 10/29 (34%) upper middle)]. We pooled studies where molecular testing was using to confirm the aetiology of GUD: 9 studies (12 estimates) for herpes, 4 studies (7 estimates) for syphilis, and 7 studies (10 estimates) for chancroid. The pooled sensitivity and specificity of GUD for the detection of herpes was 43.5% [95% confidence interval (CI): 26.2–62.4], and 88.0% (95% CI: 67.0–96.3), respectively (high certainty evidence); and for syphilis were 52.8% (95% CI: 23.0–80.7), and 72.1% (95% CI: 28.0–94.5) (moderate certainty evidence); and for chancroid were 71.9% (95% CI: 45.9–88.5) and 53.1% (95% CI: 36.6–68.9) (moderate certainty evidence), respectively.Conclusion: Algorithms requiring a clinical diagnosis to determine and treat the aetiology of GUD have poor sensitivities for syphilis and herpes simplex virus, resulting in significant numbers of missed cases. There is an urgent need to improve access to affordable and efficient diagnostics (e.g., point-of-care tests) to be incorporated into GUD algorithms to better guide appropriate management.Systematic Review Registration: PROSPERO, identifier: CRD42020153294.