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Endometrial cancer (EC) is a heterogeneous disease with a rising incidence worldwide. The understanding of its molecular pathways has evolved substantially since The Cancer Genome Atlas (TCGA) stratified endometrial cancer into four subgroups regarding molecular features: POLE ultra-mutated, microsatellite instability (MSI) hypermutated, copy-number high with TP53 mutations, and copy-number low with microsatellite stability, also known as nonspecific molecular subtype (NSMP). More recently, the International Federation of Gynecology and Obstetrics (FIGO) updated their staging classification to include information about POLE mutation and p53 status, as the prognosis differs according to these characteristics. Other biomarkers are being identified and their prognostic and predictive role in response to therapies are being evaluated. However, the incorporation of molecular aspects into treatment decision-making is challenging. This review explores the available data and future directions on tailoring treatment based on molecular subtypes, alongside the challenges associated with their testing.

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PURPOSE: To investigate the discordance in sarcoma diagnoses between nonspecialized institutions following revision by dedicated sarcoma pathologists at a reference center in Brazil and the relevance of molecular pathology in this context. METHODS: We conducted a retrospective analysis of sarcoma samples initially analyzed at outside laboratories and subsequently reviewed by two specialized pathologists between January 2014 and December 2020. After obtaining demographic and tumor characteristics, pathology results were matched and classified as complete discordance (CD; benign v malignant, sarcoma v other malignancies), partial concordance (similar diagnosis of connective tumor, but different grade/histological subtype/differentiation), and complete concordance (CC). The concordance for histology or grade, and the role of molecular assessments supporting the diagnosis were also independently determined. Statistical analyses were conducted through the kappa coefficient of agreement and adherence by χ2 test, χ2 test by Person, and Fisher exact test. RESULTS: In total, 197 cases were included, with samples obtained predominately from male patients (57.9%) and localized/primary tumors (86.8%). Following revision, the most frequent final diagnoses were undifferentiated pleomorphic sarcoma (17.8%), well-differentiated/dedifferentiated liposarcoma (8.6%), and leiomyosarcoma (7.6%). CD was found in 13.2%, partial discordance in 45.2%, and CC in 41.6% of reviews (P < .001). We found a concordance for histology or grade of 53.5% (P < .001) and 51.8% (P < .001), respectively. Molecular assessments, comprising next-generation sequencing panels (79.5%) and fluorescent in situ hybridization (20.5%), were performed in 44 (22.3%) cases, with findings classified as of diagnostic relevance in 31.8%. CONCLUSION: In nearly 60% of the cases, the initial sarcoma diagnosis was modified when revised by a reference center and dedicated pathologists, assisted by molecular pathology techniques. These results justify the assembly of referral networks in countries with limited health care resources.

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Histologic separation of injury, T cell-mediated rejection, or antibody-mediated rejection in allograft heart biopsies is difficult. A critical review of publications was performed to evaluate the caveats of using molecular diagnostics (MDX) to distinguish between these entities. Typically, only 1 to 2 fragments of unknown histologic appearance are evaluated. Archetype and molecular classifier analyses use gene lists derived from histologic labels and associated reproducibility issues influence the accuracy of the derived MDX classes. Archetypes A1, A2, and A3 archetypes created by bioinformatics were renamed no rejection, T cell-mediated rejection, and antibody-mediated rejection despite as little as 40% concordance with histologic diagnoses and overlapping archetype scores. Additional archetypes S4 and minor injury were created using arbitrary cutoffs based on visual examination of principal component analysis plots. Therapeutic implications of the numerous discrepancies with histology remain unexplored. Many MDX-derived observations are ambiguous and open to alternate logical explanations. Better molecular methods and more rigorous validation studies are needed to advance the field. Ideally, these methods should analyze all available biopsy fragments to minimize sampling issues. It is also desirable to incorporate spatial transcriptomics into the workflow, so that gene expression data can be directly compared with the underlying histology lesions.

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Os cordomas são tumores ósseos malignos, raros, que ocorrem predominantemente ao longo do esqueleto axial, demonstrando características histológicas e imunofenotípicas reminiscentes da notocorda embrionária. Cordomas de localização extra-axial ou em partes moles são extremamente raros e seu diagnóstico é naturalmente desafiador. Essa dificuldade não se deve apenas à raridade ou topografia incomum, mas também por exibirem semelhanças histo-morfológicas com outras entidades, tais como condrossarcoma mixoide, tumores mioepiteliais de partes moles e até mesmo adenocarcinomas metastáticos. Voltando-se para o desafio dignóstico que representam os cordomas extra-axiais e considerando a relativa carência de conhecimento nesse tema, o objetivo deste estudo foi resumir os principais tópicos para o diagnóstico de tais tumores. Para este trabalho, foi realizada uma revisão de publicaçoes que abordassem aspectos clinico-patológicos, imunohistoquímicos e de patologia molecular dos cordomas axiais e extra-axiais. Para esse propósito, foi utilizado o arquivo de dados PubMed, além de outros textos de reconhecida importância sobre o assunto. As bases histopatológicas dos cordomas axiais já estão estabelecidas há algumas décadas. Entretanto, para a determinação de seus equivalentes extra-axiais é essencial avaliar a expressão do marcador imunohistoquímico braquiúria (brachyury). Tal conhecimento representou um importante fator para o diagnóstico mais preciso e, consequentemente, um tratamento mais eficaz. Somando-se à avaliação histológica de rotina e à realização de um painel imuno-histoquímico adequado, o emprego da patologia molecular pode ser de grande valor para a investigação dessas neoplasias. Apesar disso, a compreensão dos aspectos moleculares dos cordomas extra-axiais ainda é considerada inicial, existindo amplo espaço para evolução

Chordomas are rare malignant tumors that occur predominantly along the axial skeleton, demonstrating histological and immunophenotypic features reminiscent of the embryonic notochord. Extra-axial or soft tissue chordomas are extremely rare and their diagnosis is naturally challenging. This difficulty is not only due to their rarity or unusual topography, but also because they exhibit histo-morphological similarities with other entities, such as myxoid chondrosarcoma, soft tissue myoepithelial tumors and even metastatic adenocarcinomas. Regarding the diagnostic challenge that extra-axial chordomas represent and considering the relative lack of knowledge on this matter, the objective of this study was to summarize the main points for the diagnosis of these tumors. For this paper, a review of publications that addressed clinicopathological, immunohistochemical and molecular pathology aspects of axial and extra-axial chordomas was carried out. For this purpose, the PubMed database was used, along with other texts of recognized importance on the subject. The histopathological basis of axial chordomas has been established for some decades. However, to distinguish their extra-axial counterparts it is essential to evaluate the expression of the immunohistochemical marker brachyury. Such knowledge represented an important factor for a more accurate diagnosis and, consequently, more effective treatment. In addition to performing an appropriate immunohistochemical panel and the routine histological evaluation, the use of molecular pathology can be of great value for the assessment of these neoplasms. Despite this, the understanding of the molecular aspects of extra-axial chordomas is still considered initial, and there is room for improvement in this field

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Chlamydophila pneumoniae is a cause of community-acquired pneumonia (CAP) and responsible for 1-2% of cases in paediatric patients. In Mexico, information on this microorganism is limited. The aim of this study was to detect C. pneumoniae using two genomic targets in a real-time PCR and IgM/IgG serology assays in paediatric patients with CAP at a tertiary care hospital in Mexico City and to describe their clinical characteristics, radiological features, and outcomes. A total of 154 hospitalized patients with diagnosis of CAP were included. Detection of C. pneumoniae was performed by real-time PCR of the pst and arg genes. Complete blood cell count, C-reactive protein measurement and IgM and IgG detection were performed. Clinical-epidemiological and radiological data from the patients were collected. C. pneumoniae was detected in 25 patients (16%), of whom 88% had underlying disease (P = 0.014). Forty-eight percent of the cases occurred in spring, 36% in girls, and 40% in children older than 6 years. All patients had cough, and 88% had fever. Interstitial pattern on chest-X-ray was the most frequent (68%), consolidation was observed in 32% (P = 0.002). IgM was positive in 7% and IgG in 28.6%. Thirty-six percent presented complications. Four percent died. A high proportion showed co-infection with Mycoplasma pneumoniae (64%). This is the first clinical report of C. pneumoniae as a cause of CAP in Mexican paediatric patients, using two genomic target strategy and serology. We found a frequency of 16.2% with predominance in children under 6 years of age. In addition; cough and fever were the most common symptoms. Early detection of this pathogen allows timely initiation of specific antimicrobial therapy to reduce development of complications. This study is one of the few to describe the presence of C. pneumoniae in patients with underlying diseases.

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SARS-CoV-2, a novel coronavirus within the Coronaviridae family, is the causative agent behind the respiratory ailment referred to as COVID-19. Operating on a global scale, COVID-19 has led to a substantial number of fatalities, exerting profound effects on both public health and the global economy. The most frequently reported symptoms encompass fever, cough, muscle or body aches, loss of taste or smell, headaches, and fatigue. Furthermore, a subset of individuals may manifest more severe symptoms, including those consistent with viral pneumonitis, which can be so profound as to result in fatalities. Consequently, this situation has spurred the rapid advancement of disease diagnostic technologies worldwide. Predominantly employed in diagnosing COVID-19, the real-time quantitative reverse transcription PCR has been the foremost diagnostic method, effectively detecting SARS-CoV-2 viral RNA. As the pandemic has evolved, antigen and serological tests have emerged as valuable diagnostic tools. Antigen tests pinpoint specific viral proteins of SARS-CoV-2, offering swift results, while serological tests identify the presence of antibodies in blood samples. Additionally, there have been notable strides in sample collection methods, notably with the introduction of saliva-based tests, presenting a non-invasive substitute to nasopharyngeal swabs. Given the ongoing mutations in SARS-CoV-2, there has been a continuous need for genomic surveillance, encompassing full genome sequencing and the identification of new variants through Illumina technology and, more recently, nanopore metagenomic sequencing (SMTN). Consequently, while diagnostic testing methods for COVID-19 have experienced remarkable progress, no test is flawless, and there exist limitations with each technique, including sensitivity, specificity, sample collection, and the minimum viral load necessary for accurate detection. These aspects are comprehensively addressed within this current review.

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Chagas disease (CD), caused by the protozoan Trypanosoma cruzi (T. cruzi), affects millions of people worldwide. Polymerase Chain Reaction (PCR) and real-time quantitative PCR (qPCR) have been used as tools to monitor parasitic levels in the bloodstream of individuals exposed to infection, thus enabling the monitoring of relapses and the effectiveness of therapy, for example. The aim of this study was to evaluate the TcSAT-IAM system, developed by our research group, on samples from patients with suspected Chagas disease infection. Initially, primer systems were developed for the detection of the nuclear DNA (SAT-DNA) from T. cruzi (TcSAT-IAM). The Cruzi system, predicted in the literature, and TcSAT-IAM were then evaluated in relation to their analytical sensitivity, specificity and efficiency. Afterwards, the applicability of the qPCR technique using both systems (separately) for the diagnosis of acute CD was evaluated in samples from 77 individuals exposed to the outbreak that occurred in Pernambuco-Brazil, relating the results obtained to those of the classical diagnostic methods recommended for this stage of the infection. TcSAT-IAM and Cruzi had a detection limit of 1 fg of target DNA (0,003 parasites). Thirty-eight cases were recorded, 28 by laboratory criteria and 10 by clinical and epidemiological criteria. Blood samples from 77 subjects were submitted to qPCR by both systems, reaching an agreement of 89.61% between them. After analyzes between systems and diagnostic criteria, the TcSAT-IAM showed sensitivity and specificity of 52.36% (CI 37.26-67.52) and 92.31% (CI 79.68-97.35), respectively, accuracy of 72.73% and moderate agreement. The TcSAT-IAM showed an accuracy of 72.58% and 75% in relation to parasitological and serological tests (IgM anti-T. cruzi), respectively. Therefore, quantitative PCR should be incorporated into the diagnosis of suspected acute cases of Chagas disease.

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BACKGROUND: The etiological diagnosis of community-acquired pneumonia (CAP) is still a challenge. We compared the conventional culture method and real-time polymerase chain reaction (RT-PCR) for the identification of Streptococcus pneumoniae in severe pediatric CAP. METHODS: A retrospective hospital-based study was conducted. From 2012 to 2018, we have selected patients who had peripheral blood and/or pleural fluid collected for etiological investigation by RT-PCR. RESULTS: We included 113 children (median age: 3 years; interquartile range 1-6 years). RT-PCR increased the detection rate of S. pneumoniae by 6.5 times using blood samples and eight times using pleural fluid samples. Patients subjected to RT-PCR showed more prolonged hospitalization (p = 0.006), fewer comorbidities (p = 0.03), presence of pleural effusion (p = 0.001), presence of young forms of leukocytes (p = 0.001) and radiograph with characteristics of pneumonia (p = 0.002). The presence of pleural effusion [odds ratio (OR) = 14.7, 95% confidence interval (CI) 1.6-133.9; p = 0.01] and young forms of leukocytes (OR = 8.9, 95% CI 0.9-84.4; p = 0.05) were risk factors for positive RT-PCR pneumococcal when multivariate analysis was performed. CONCLUSIONS: RT-PCR is a reliable method for diagnosing severe CAP using sterile materials and a potentially applicable method in patients with clinical, radiological and non-specific laboratory characteristics of lower respiratory tract infection, especially in complicated cases with pleural effusion.

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BACKGROUND: In the current COVID-19 pandemic, early and rapid diagnosis of potentially infected and contagious individuals enables containment of the disease through quarantine and contact tracing. The rapid global expansion of these diagnostic testing services raises questions concerning the current state of the art with regard to standardization of testing and quality assessment practices. The aim of this study was to provide a global overview of the test methods, laboratory procedures and quality assessment practices used for SARS-CoV-2 diagnostics. METHODS: The Molecular Diagnostics Committee of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC C-MD) initiated a survey among international laboratories performing molecular genetic detection of SARS-CoV-2. Questions on quality assurance, variant testing, sequencing and the transmission of findings were included in the survey. RESULTS: A total of 273 laboratories from 49 countries participated in the survey. The majority of the participating laboratories (92.2%) use reverse transcriptase polymerase chain reaction (RT-PCR). The majority of participating laboratories do not conduct testing to identify SARS CoV-2 variants. Participation in external quality assessment programs was reported by the majority of laboratories, however, 33.2% of the laboratories reported not participating in external quality assurance programmes. CONCLUSIONS: Based on the survey, molecular diagnostic methods for SARS-CoV-2 detection are clearly not standardized across different countries and laboratories. The survey found an array of responses in regard to sample preparation, collection, processing and reporting of results. This work suggests quality assurance is insufficiently performed by diagnostic laboratories conducting SARS-CoV-2 testing.

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