Investigation of the molecular genetic causes of non-syndromic primary ovarian ınsufficiency by next generation sequencing analysis

ABSTRACT Objective: The aim of this study is to investigate the molecular genetic causes of non-syndromic primary ovarian insufficiency (POI) cases with the gene panel based on next generation sequencing analysis and to establish the relationship between genotype and phenotype. Subjects and methods: Twenty three cases aged 14-40 years followed up with POI were included. Patients with a karyotype of 46, XX, primary or secondary amenorrhea before the age of 40, with elevated FSH (>40 IU/mL) and low AMH levels (<0.03 ng/mL) were included in the study. Molecular genetic analyzes were performed by the next generation sequencing analysis method targeted with the TruSightTM Exome panel. Results: Median age of the cases was 17.8 (14.0-24.3) years, and 12 (52%) cases admitted before the age of 18. Fifteen (65%) patients had consanguineous parents. In 2 (8.6%) cases, variants detected were in genes that have been previously proven to cause POI. One was homozygous variant in FIGLA gene and the other was homozygous variant in PSMC3IP gene. Heterozygous variants were detected in PROK2, WDR11 and CHD7 associated with hypogonadotropic hypogonadism, but these variants are insufficient to contribute to the POI phenotype. Conclusion: Genetic panels based on next generation sequencing analysis technologies can be used to determine the molecular genetic diagnosis of POI, which has a highly heterogeneous genetic basis.

Silage quality and bacterial diversity of silages inoculated with Listeria monocytogenes and Lacticaseibacillus paracasei_6714

Introduction: King grass (Cenchrus purpureus (Schumach.) Morrone, syn. Pennisetum purpuphoides) and pineapple peel (Ananas comosus) silages are food alternatives for livestock in conditions of feed shortage. Objective: To describe the dynamics of the microbiota present in king grass and pineapple silage during the fermentation process using next generation sequencing (NGS) and to evaluate the protective effect of Lacticaseibacillus paracasei_6714 as a silage inoculum against Listeria monocytogenes. Methods: We used an unrestricted randomized design to characterize the microbiota present in silages made from king grass harvested 70 days after regrowth and pineapple peel. We inoculated mixtures of grass and peel with L. paracasei_6714 or L. monocytogenes, or both, with a non-inoculated treatment as control. The nutritional and fermentative profile was evaluated after 30 days. After 15 and 30 days of fermentation, we used 16S rRNA analysis to determine the dynamics and diversity of the microbiota in the inoculated and control silages. Result: Dry matter content and digestibility did not differ significantly; however, there were differences in crude protein, pH and organic acids. We obtained 4432 amplicon sequence variants of Proteobacteria, Firmicutes, Bacterioidetes, Actinobacteria, Verrucomicrobia, Planctomycetes and Patescibacteria. The relative abundance of each phylum varied depending on the material and fermentation period. Phylum similarity was over 70 % (but not greater than 50 % with Bray-Curtis at the species level). Conclusion: These bacterial communities seem to have an important role during silage fermentation. Proper management of silage processing can reduce or eliminate pathogenic bacteria.

Introducción: Los ensilajes del pasto king grass (Cenchrus purpureus (Schumach.) Morrone, syn. Pennisetum purpuphoides) y cáscaras de piña (Ananas comosus) son alternativas de alimento para ganado en condiciones de escasez alimentaria. Objetivo: Describir las dinámicas de la microbiota presente en los ensilajes de king grass y piña durante el proceso de fermentación usando secuenciación de próxima generación (NGS) y evaluar el efecto de protección de Lacticaseibacillus paracasei_6714 como inoculante de ensilaje ante Listeria monocytogenes. Métodos: Usamos un diseño aleatorio no restringido para caracterizar la microbiota presente en ensilajes de king Grass cosechados 70 días después de rebrote y de cáscaras de piña. Inoculamos mezclas de pasto y cáscara con L. paracasei_6714 o L. monocytogenes, o ambos, con un tratamiento control sin inocular. El perfil nutricional y de fermentación fue evaluado luego de 30 días. Después de 15 y 30 días de fermentación, usamos un análisis de para determinar la dinámicas y diversidad de la microbiota en los ensilajes inoculados y control. Resultados: Los contenidos de materia seca y digestibilidad, no difirieron significativamente; sin embargo, hubo diferencias en proteína cruda, pH y ácidos orgánicos. Obtuvimos 4 432 secuencias variantes de amplicon de Proteobacteria, Firmicutes, Bacterioidetes, Actinobacteria, Verrucomicrobia, Planctomycetes y de Patescibacteria. La abundancia relativa de cada filo vario dependiendo del material y periodo de fermentación. Similitudes de filo fueron mayores al 70 % (pero no mayor que 50 % con Bray-Curtis a nivel de especie). Conclusión: Estas comunidades bacterianas parecen cumplir un papel importante durante la fermentación del ensilaje. Un manejo apropiado del proceso de ensilaje puede reducir o eliminar baterías patogénicas.

Effects of environmental stability and phycoperiphyton structure on the macroinvertebrate community of regulated Andean rivers / Efectos de la estabilidad ambiental y estructura del ficoperifiton en la comunidad de macroinvertebrados en ríos andinos regulados

Introduction: The variability in the structure of aquatic communities is frequently attributed to environmental changes; however, in stable environments such as regulated rivers, trophic interactions could be another key environmental factor determining the structure of these communities. These alterations could cause a greater growth of algae, and in turn, changes in the functional groups and in the composition of the macroinvertebrate community favoring the dominance of certain groups of organisms. Objective: To identify the effects of environmental variations and changes in the structure of the phycoperiphyton on the macroinvertebrate community of regulated Andean rivers. Methods: We analyzed environmental and biological data collected in quarterly samples carried out between 2010 and 2018 in two rivers of the Central Andes (Antioquia - Colombia), for a total of 27 samples. Sample collections used standardized methods. Different statistical models were used to establish spatial and temporal patterns of the environmental variables, of the abundance and/or density and diversity of phycoperiphyton and macroinvertebrates, as well as the trophic relationships that exists between them. Results: We found that regulated rivers present relatively little environmental variability. The environmental parameters with the greatest variation were temperature, turbidity, and orthophosphates; these last two were the abiotic variables with the greatest contribution to benthic instability. Conclusion: The presence of scraping and foraging macroinvertebrates was more affected by the stability of the phycoperiphyton density than by environmental variables, showing the importance of trophic interactions in regulated rivers and the bottom up control in these ecosystems.

Introducción: La variabilidad en la estructura de las comunidades acuáticas se atribuye frecuentemente a cambios ambientales, no obstante, en ambientes estables como ríos regulados, las interacciones tróficas podrían ser otro factor ambiental clave determinante de la estructura de estas comunidades. Estas alteraciones podrían provocar un mayor crecimiento de algas y, a su vez, cambios en los grupos funcionales y en la composición de la comunidad de macroinvertebrados favoreciendo la dominancia de determinados grupos de organismos. Objetivo: Identificar el efecto de los cambios ambientales y de la estructura del ficoperifiton sobre la comunidad de macroinvertebrados de ríos Andinos regulados. Métodos: Se analizaron datos ambientales y biológicos recolectados en muestreos trimestrales realizados entre 2010 y 2018 en dos ríos de los Andes Centrales (Antioquia - Colombia), para un total de 27 muestras. La recolección de muestras empleó métodos estandarizados. Se utilizaron diferentes modelos estadísticos para establecer patrones espaciales y temporales de las variables ambientales, de la abundancia y/o densidad y diversidad de ficoperifiton y de los macroinvertebrados, así como las relaciones tróficas que existen entre ellos. Resultados: Se encontró que los ríos regulados presentan relativamente poca variabilidad ambiental. Los parámetros ambientales con mayor variación fueron: temperatura, turbidez y ortofosfatos; las dos últimas variables abióticas fueron las que más aportaron a la inestabilidad bentónica. Conclusión: La presencia de macroinvertebrados raspadores y recolectores fue más afectada por la estabilidad de la densidad del ficoperifiton que por las variables ambientales, evidenciando la importancia de las interacciones tróficas en ríos regulados y el control bottoom up en estos ecosistemas.

Clinicopathological and molecular analyses of uterine carcinosarcomas using next-generation sequencing: A single-center experience

Background: Uterine carcinosarcomas (UCS) constitute 3–4% of all uterine malignancies and 16% of deaths caused due to uterine neoplasms. Aim: In this study, we aimed to perform DNA-based mutation analysis in 12 genes (KRAS, NRAS, EGFR, C-KIT, BRAF, PDGFRA, ALK, ERBB2, ERBB3, ESR1, RAF1, PIK3CA) to determine the molecular subtypes of UCS using next-generation sequencing (NGS) in patients with aggressive UCS and poor prognosis. We aimed to compare the results of our analysis with clinicopathological data to contribute to the development of targeted therapy approaches related to the molecular changes of UCS. Materials and Methods: In this study, we included 12 cases diagnosed with uterine carcinosarcomas and examined the changes in oncogenes that play a role in UCS pathogenesis. For the analysis of mutation, the clinicopathological data were compared with the variations in the DNA-based gene panel consisting of 12 genes and 1237 variants in the UCS using the NGS method. Results: EGFR mutation was found in 91.7% of the cases, mutation in 41.7%, PDGFRA mutation in 25%, KRAS and PIK3CA mutation in 16.7%, and C-KIT mutation in 8.3% of the cases. Although no statistical significance was found between the detected mutation and clinicopathological data, it was concluded that PDGFRA mutation might be associated with advanced-stage disease development. Conclusion: This study's findings regarding different molecular types of UCS and information on oncogenesis of UCS can provide inferences for targeted therapies in the future by identifying targetable mutations representing early oncogenic events and thereby contribute toward further studies on this subject.

Implementación asistencial de estudios genómicos aplicados al diagnóstico molecular / Implementation of genomic studies applied to molecular diagnostics

El Hospital Garrahan ha sido pionero en el diagnóstico molecular de patologías pediátricas en Argentina. Los avances tecnológicos de las últimas décadas en el área de la biología molecular, sentaron las bases para la optimización y ampliación del diagnóstico molecular a partir de la secuenciación masiva en paralelo de múltiples genes. El presente trabajo describe el proceso de implementación de los estudios de secuenciación de nueva generación y el desarrollo de la Unidad de Genómica en un hospital público pediátrico de alta complejidad, así como su impacto en las capacidades diagnósticas de enfermedades poco frecuentes de origen genético. La creación del Grupo Interdisciplinario de Estudios Genómicos constituyó la vía institucional para la toma de decisiones que implican la implementación de nuevos estudios genómicos y el establecimiento de prioridades diagnósticas, extendiendo la disponibilidad del diagnóstico molecular a más disciplinas. La Unidad de Genómica trabaja en diseñar las estrategias que permitan la mayor optimización de los recursos con los que cuenta el hospital, teniendo en cuenta el equipamiento disponible, las prioridades establecidas y la frecuencia de las distintas patologías. Se demuestra el salto significativo operado en nuestras capacidades diagnósticas, tanto en la variedad de enfermedades como en el número de genes analizados, habiendo estudiado a la fecha alrededor de 2.000 pacientes, muchos de los cuales ven de este modo finalizada su odisea diagnóstica. Los estudios de NGS se han convertido en una herramienta de la práctica diaria para la atención de un número importante de pacientes de nuestro hospital. Continuaremos trabajando para ampliar su aplicación a la mayor cantidad de patologías, a través de los mecanismos institucionales ya existentes (AU)

The Garrahan Hospital has been a pioneer in the molecular diagnosis of pediatric diseases in Argentina. The technological advances of the last decades in the area of molecular biology have laid the foundations for the optimization and expansion of molecular diagnostics through massive parallel sequencing of multiple genes. This study describes the process of implementation of next-generation sequencing studies and the development of the Genomics Unit in a public pediatric tertiary hospital, and its impact on the capacity to diagnose rare diseases of genetic origin. The creation of the Interdisciplinary Group of Genomic Studies constituted the institutional pathway for decision-making involving the implementation of new genomic studies and the establishment of diagnostic priorities, extending the availability of molecular diagnostics to additional disciplines. The Genomics Unit is working to design strategies that allow for optimization of the resources available to the hospital, taking into account the equipment available, the priorities established, and the frequency of the different diseases. It demonstrates the significant leap in our diagnostic capabilities, both in the variety of diseases and in the number of genes analyzed. To date, around 2,000 patients have been studies, many of whom have thus completed their diagnostic odyssey. NGS studies have become a tool in daily practice for the care of a significant number of patients in our hospital. We will continue working to expand its application to as many diseases as possible, through the existing institutional mechanisms (AU)

Next-generation sequencing-based genetic testing and phenotype correlation in retinitis pigmentosa patients from India

Purpose: Inherited retinal dystrophies (IRD) are a heterogeneous group of retinal diseases leading to progressive loss of photoreceptors through apoptosis. Retinitis pigmentosa (RP) is considered the most common form of IRD. Panel?based testing in RP has proven effective in identifying the causative genetic mutations in 70% and 80% of the patients. This is a retrospective, observational, single?center study of 107 RP patients who had undergone next?generation sequencing?based targeted gene panel testing for IRD genes. These patients were inspected for common phenotypic features to arrive at meaningful genotype–phenotype correlation. Methods: Patients underwent complete ophthalmic examination, and blood was collected from the proband for DNA extraction after documenting the pedigree. Targeted Next Generation Sequencing (NGS) was done by panel?based testing for IRD genes followed by co?segregation analysis wherever applicable. Results: Of the 107 patients, 72 patients had pathogenic mutations. The mean age of onset of symptoms was 14 ± 12 years (range: 5–55). Mean (Best Corrected Visual Acuity) BCVA was 6/48 (0.9 logMAR) (range 0.0–3.0). At presentation, over one?third of eyes had BCVA worse than 6/60 (<1 logMAR). Phenotype analysis with the gene defects showed overlapping features, such as peripheral well?defined chorioretinal atrophic patches in patients with CERKL, PROM1, and RPE65 gene mutations and large macular lesions in patients with RDH12 and CRX gene mutations, respectively. Nummular or clump?like pigmentation was noted in CRB1, TTC8, PDE6A, and PDE6B. Conclusion: NGS?based genetic testing can help clinicians to diagnose RP more accurately, and phenotypic correlations can also help in better patient counselling with respect to prognosis and guidance regarding ongoing newer gene?based therapies.

Characterization of third generation cephalosporin-resistant Escherichia coli clinical isolates from Ushuaia, Argentina / Caracterización de aislamientos clínicos de Escherichia coli resistente a cefalosporinas de tercera generación de Ushuaia, Argentina

Abstract Escherichia coli is one of the main human pathogens causing different hospital- and community-acquired infections. During the period from January 2013 to March 2015, 1.96% (32/1632) of E. coli isolates recovered at the Hospital Regional de Ushuaia, Tierra del Fuego province, were resistant to third-generation cephalosporins (TGCs). These isolates were resistant to cefotaxime (91%) and/or ceftazidime (28%). No resistance to carbapenems was detected. Twenty-six isolates were positive for blaCTX-M gene, grouped as CTX-M-1/15 (54%); CTX-M-9/14 (25%); CTX-M-2 (17%); and CTX-M-1/15 plus CTX-M-9/14 (4%). Five TGC-resistant strains were positive for blaCMY gene, while one strain harbored TEM-19 ESBL. Twelve isolates were identified as ST131 E. coli hyperepidemic clone, and one as ST69. Genome sequence analysis of seven blaCTX-M-15 E. coli selected isolates confirm the circulation of ST131, ST617 and ST405 international high-risk clones in the city of Ushuaia.

Resumen Escherichia coli es uno de los principales patógenos humanos causantes de diferentes infecciones de inicio hospitalario y comunitario. Se determinó que el 1,96% (32/1.632) de los aislamientos de E. coli recuperados entre enero de 2013 y marzo de 2015 en el Hospital Regional de Ushuaia, provincia de Tierra del Fuego, fueron resistentes a cefalosporinas de tercera generación (CTG). Estos aislamientos fueron resistentes a cefotaxima (91%) y/o a ceftazidima (28%). No se detectó resistencia a los carbapenemes. Veintiséis aislamientos fueron positivos para el gen blaCTX-M, agrupados como CTX-M-1/15 (54%), CTX-M-9/14 (25%), CTX-M-2 (17%) y CTX-M-1/15 más CTX-M-9/14 (4%). Cinco cepas resistentes a CTG dieron positivo para el gen blaCMY, mientras que un aislamiento presentó la BLEE TEM-19. Doce aislamientos se identificaron como clon hiperepidémico E. coli ST131 y uno como ST69. El análisis de las secuencias del genoma de siete aislamientos seleccionados de E. coli blaCTX-M-15 confirmó la circulación de los clones internacionales de alto riesgo ST131, ST617 y ST405 en la ciudad de Ushuaia.

IMPACT OF EMOTIONAL INTELLIGENCE ON ACADEMIC STRESS: A QUALITATIVE STUDY

Modern technology has become an integral part of the daily lives of the new generation in the fast- changing 21st century. As a result of instant access to digital information and communication technologies via mobile phones and the internet, this generation is called the 'Digital Generation'. Without understanding real love and association with family and friends, they con?ne themselves to the digital world. People with high emotional intelligence (EI) feel a sense of association and belongingness. A person's emotional intelligence is their ability to comprehend and cope with dif?cult and complex situations in life. By overcoming stress, EI can enhance productivity. An analysis of the relationship between Emotional Intelligence and Academic Stress is presented in this paper

Molecular characterization & recombination analysis of complete enterovirus-88 isolated from acute flaccid paralysis cases in India

Background & objectives: Focus on non-polio enteroviruses (NPEVs) causing acute flaccid paralysis (AFP) due to myelitis has increased with the containment of the poliovirus. Enterovirus-B88 (EV-B88) has been associated with the AFP cases in Bangladesh, Ghana, South Africa, Thailand and India. In India, EV-B88 infection was linked to AFP a decade ago; however, to date, no complete genome has been made available. In this study, the complete genome sequence of EV-B88 was identified and reported from two different States (Bihar and Uttar Pradesh) in India using the next-generation sequencing technique. Methods: Virus isolation was performed on the three AFP suspected cases as per the WHO-recommended protocol. Samples showing cytopathic effects in the human Rhabdocarcinoma were labelled as NPEVs. Next-generation sequencing was performed on these NPEVs to identify the aetiological agent. The contiguous sequences (contigs) generated were identified, and reference-based mapping was performed. Results: EV-B88 sequences retrieved in our study were found to be 83 per cent similar to the EV-B88 isolate from Bangladesh in 2001 (strain: BAN01-10398; Accession number: AY843306.1). Recombination analyses of these samples demonstrate recombination events with sequences from echovirus-18 and echovirus-30. Interpretation & conclusions: Recombination events in the EV-B serotypes are known, and this work reconfirms the same for EV-B88 isolates also. This study is a step in increasing the awareness about EV-B88 in India and emphasizes future studies to be conducted in the identification of other types of EV present in India.

ON-CHIP PLANER WAVEGUIDES FOR SUPERCONTINUUM GENERATION IN THE MID-IR REGION OF ELECTROMAGNETIC SPECTRUM: A REVIEW

On-chip planer optical waveguide-based sources for supercontinuum (SC) generation have become highly attractive devices in the twenty-first century. Mid-IR SC sources in the 2-20 ?m wavelength region are advantageously used for gas sensing, high-sensitivity molecular detection, security, and industrial applications. These integrated photonic devices are cost-effective, scalable, and robust, and also offer more flexibility in tailoring the dispersion characteristics relative to other SC generation techniques. This article reviews the evolution of SC sources from fiber-based devices to optical waveguide-based devices and presents a historical as well as recent progress in various types of on-chip optical waveguides with physical mechanisms involved in generating coherent SC sources.

Efficacy of bupropion and cognitive behavioral therapy in the treatment of methamphetamine use disorder: a systematic review and meta-analysis

Objectives: We assessed the efficacy of cognitive behavioral therapy and bupropion compared to cognitive behavioral therapy alone for methamphetamine use disorder. Methods: The selection criteria for this systematic review study with meta-analysis were randomized clinical trials on the efficacy of cognitive behavioral therapy and bupropion in the treatment for methamphetamine use disorder (assessed by urine metabolites). The search was conducted in PubMed, PubMed Central, LILACS, SciELO, Cochrane Library, SCOPUS, Google Scholar, Ovid Medline, Clinicaltrials.gov, and the International Clinical Trials Registry Platform. The primary outcome was relapse. Risk of bias was assessed with the RoB 2 tool. The results of each clinical trial were input into an Excel spreadsheet. We performed a meta-analysis using relative risk and a 95%CI. Results: Of the 597 initial articles (498 after removing duplicate records), five were included in the meta-analysis, with an aggregate sample of 539 patients. An overall relative risk of 0.91 (95%CI 0.78-1.05) was estimated for relapse. Conclusion: Our study limitations included publication bias and heterogeneous populations. We found no evidence that cognitive behavioral therapy and bupropion reduced the risk of relapse compared to cognitive behavioral therapy and placebo.

The Data Generation Mechanism: Relationship Between Constructs and Their Indicators / O Mecanismo de Geração de Dados: Relação entre Construtos e seus Indicadores

Abstract The choice of statistical data analysis should be guided by a critical analysis that supports the theoretical relationship between the construct and its indicators. This theoretical article reviews the three main existing psychometric paradigms and their proposals for explaining the relationship between indicators and their constructs. The discussion begins with the standard paradigm that guides the construction and analysis of data in psychology, reflective model. Then, a description of the formative models is performed and finally the Network Analysis as an alternative. The definitions, consequences, and limitations of the use of each measurement model are presented such as a reflection on making decisions about which data generation mechanisms are more appropriate.

Resumo A escolha da análise estatística de dados deveria ser guiada por uma análise crítica que fundamenta a relação teórica entre construto e seus indicadores. Este teórico artigo faz uma revisão dos três principais paradigmas psicométricos e suas propostas de explicação da relação entre os indicadores e seus construtos. A discussão é iniciada com o paradigma padrão que guia a construção e análise de dados na psicologia, os modelos reflexivos. Em seguida, é realizada uma descrição dos modelos formativos e, por fim, a proposta da Análise de Redes como alternativa. São apresentadas as definições, consequências e limitações do uso de cada modelo de medida, bem como uma reflexão na tomada de decisão sobre quais mecanismos de geração de dados são mais apropriados.

A diversidade, as idades, as gerações: das especializações medíocres aos estudos necessários para a Sociologia da Juventude / Diversity, ages, generations: from mediocre specializations to necessary studies for Youth Sociology

Resumo: O objetivo deste artigo é adentrar algumas produções sobre as juventudes e as gerações, tendo como fundamento para análise a decadência ideológica, cunhada por Georg Lukács. Com isso, pretendo afirmar que as bases de algumas especializações contribuem para a disseminação de conceitos pautados por expressões da questão social atribuídas aos sujeitos individualmente, numa espécie de afirmação de essência de sujeito, que carrega determinabilidade, ou seja, estão impregnados de uma ontologia depreciativa do humano.

Abstract: The objective of this article is to enter some productions about youth and generations, having as basis for analysis the ideological decadence, coined by Georg Lukács. With this I intend to affirm that the bases of some specializations contribute to the dissemination of concepts guided by expressions of the social issue attributed to the subjects individually, in a kind of affirmation of subject essence, which carries determinability, i.e., are impregnated with a depreciative ontology of the human.

Clinical and gene mutation characteristics of patients with hereditary ellipsocytosis: nine cases report and literature review / 中华血液学杂志

Objective: To report gene mutations in nine patients with hereditary elliptocytosis (HE) and analyze the characteristics of pathogenic gene mutations in HE. Methods: The clinical and gene mutations of nine patients clinically diagnosed with HE at Institute of Hematology & Blood Diseases Hospital from June 2018 to February 2022 were reported and verified by next-generation sequencing to analyze the relationship between gene mutations and clinical phenotypes. Results: Erythrocyte membrane protein gene mutations were detected among nine patients with HE, including six with SPTA1 mutation, one with SPTB mutation, one with EPB41 mutation, and one with chromosome 20 copy deletion. A total of 11 gene mutation sites were involved, including 6 known mutations and 5 novel mutations. The five novel mutations included SPTA1: c.1247A>C (p. K416T) in exon 9, c.1891delG (p. A631fs*17) in exon 15, E6-E12 Del; SPTB: c.154C>T (p. R52W) ; and EPB41: c.1636A>G (p. I546V) . Three of the six patients with the SPTA1 mutation were SPTA1 exon 9 mutation. Conclusion: SPTA1 is the most common mutant gene in patients with HE.

Clinical application of metagenomic next-generation sequencing in acquired immunodeficiency syndrome patients with pulmonary infections / 中华传染病杂志

Objective:To investigate the pathogen spectrum of acquired immunodeficiency syndrome (AIDS) patients with pulmonary opportunistic infections in the local area, and to evaluate the clinical application of metagenomic next-generation sequencing (mNGS) in these patients.Methods:From January to December 2021, AIDS patients with pulmonary infections admitted to Zhongnan Hospital of Wuhan University were enrolled. Their bronchoalveolar lavage fluid (BALF) was subjected to mNGS and coventional pathogen detection.Routine pathogen detection methods included smear, culture, polymerase chain reaction (PCR), and immunochromatographic colloidal gold. Fisher′s exact probability method was used for statistical analysis.Results:A total of 69 patients were included, and all of them were tested positive for mNGS. Among them, 53 cases (76.8%) were positive for fungi and viruses, 40 cases (58.0%) were positive for bacteria (excluding Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM)), six cases were positive for MTB, 11 cases were positive for NTM, and seven cases were positive for other pathogens. Mixed infections with two or more pathogens were found in 89.9%(62/69) of the patients. Among the conventional pathogen detections of BALF, 79.7%(55/69) of the patients were positive for pathogens, including 42 cases positive for Pneumocystis jirovecii PCR, 16 cases positive for BALF culture, nine cases positive for MTB PCR, and five cases positive for Cryptococcus antigen. The total detection rate of mNGS was 100.0%(69/69), which was higher than that of the conventional pathogen detection rate of 79.7%(55/69), and the difference was statistically significant (Fisher′s exact probability method, P<0.001). The specificity of mNGS detection was 88.4%. Combining clinical and two detection methods, the top five pathogens were Pneumocystis jirovecii (62.3%(43/69)), Candida (29.0%(20/69)), MTB (20.3%(14/69)), NTM and Talaromyces marneffei (15.9%(11/69), each). Fifty-three patients (76.8%) had co-infection with virus. Conclusions:The main cause of pulmonary infection in AIDS patients in this area is mixed infection, and Pneumocystis jirovecii is the most common pathogen. mNGS could significantly improve the pathogen detection rate in AIDS patients with pulmonary infections.

Etiological diagnosis value of metagenomic next generation sequencing in the patients with spinal infection / 中华传染病杂志

Objective:To investigate the efficacy of metagenomic next generation sequencing (mNGS) in the etiological diagnosis of patients with spinal infection, so as to provide reference for timely diagnosis and treatment.Methods:A total of 40 patients with suspected spinal infection admitted to the Department of Infectious Diseases in Henan Provincial People′s Hospital from January 2020 to July 2022 were included. The results of tissue culture, histopathological examination and tissue mNGS detection were analyzed retrospectively. According to the clinical diagnose, the patients were divided into the spinal infection group (28 cases) and the non-spinal infection group (12 cases). The positive rate, sensitivity and specificity of mNGS and tissue culture in the pathogen detection of patients with spinal infection were compared. McNemar test was used for statistical analysis.Results:There were 23 males and 17 females in 40 patients. The positive rate of mNGS was higher than that of tissue culture (75.0%(30/40) vs 12.5%(5/40)), and the difference was statistically significant ( χ2=0.08, P<0.001). Based on clinical diagnostic criteria, the sensitivity of mNGS in the diagnosis of spinal infection was higher than that of tissue culture (82.1% vs 17.9%), with a statistically significant difference ( χ2=0.02, P<0.001), while the specificity compared to the tissue culture (33.3% vs 100.0%), the difference was not statistically significant ( P>0.05). Conclusions:mNGS has a high pathogen detection rate and sensitivity in the etiological diagnosis of patients with spinal infection, which could provide clinical guidance for the diagnosis and treatment of patients with spinal infection.

Application of metagenomic next-generation sequencing in the detection of pathogenic bacteria in brain abscesses / 中国综合临床

Objective:To analyze the application value of metagenomic next-generation sequencing (mNGS) in the detection of pathogenic bacteria in brain abscesses.Methods:The data of patients with brain abscess in Tianjin Huanhu Hospital from January 2019 to December 2021 were retrospectively analyzed. All patients underwent stereotaxic abscess puncture and drainage. According to the different methods of pathogen detection, they were divided into bacterial culture group (bacterial culture only) and mNGS group (bacterial culture with mNGS). The clinical symptoms, abscess site, bacterial culture and mNGS results, antibiotic application protocol and prognosis of the patients were analyzed. The bacterial detection results of the two groups were analyzed, and the antibiotic application and prognosis were compared. χ 2 test, exact probability method and Mann Whitney test were used to compare the difference between the two groups. Results:A total of 43 patients with brain abscess were enrolled, including 21 cases in bacterial culture group and 22 cases in mNGS group. The positive rate of bacteria culture group was 42.9% (9/21), the positive rate of bacteria culture group was 45.5% (10/22), and the positive rate of mNGS detection was 100% (22/22). Only 3 cases in the bacterial culture group could have a clear bacterial source, while 17 cases in the mNGS group could have a clear bacterial source according to the bacterial results, showing a significant statistical difference between the two groups (χ 2=19.69, P<0.001). The return time of bacterial culture was 7.0 (4.0,7.0) days, and the average return time of mNGS was 1.5 (1.5,1.5) days, the difference of bacterial return time between the two groups was statistically significant ( Z=0.00, P<0.001). The cost of antibiotic use in bacterial culture group was 24.00 (5.60,31.00) thousands yuan, and the cost of antibiotic use in mNGS group was 12.00 (2.10, 20.00) thousands yuan, and there was significant statistical difference between them ( Z=5.22, P=0.026). Conclusions:MNGS can quickly and accurately identify the types and sources of brain abscess pathogens, guide the clinical application of antibiotics more targeted, reduce the cost of antibiotic use, and is an effective method for the detection of brain abscess pathogenic bacteria.

Rapid evaluation of the early pathogen of severe Chlamydophila psittaci pneumonia by diagnostic bronchoscopy / 中华危重病急救医学

Objective:To explore the rapid evaluation of the early pathogen of severe Chlamydophila psittaci pneumonia by bedside diagnostic bronchoscopy, so as to start effective anti-infection treatment before the results of macrogenome next generation sequencing (mNGS) test. Methods:The clinical data of three patients with severe Chlamydophila psittaci pneumonia who were successfully treated in the First Affiliated Hospital of Xinjiang Medical University, the First People's Hospital of Aksu District, and the First Division Hospital of Xinjiang Production and Construction Corps from October 2020 to June 2021 were retrospectively analyzed, including the rapid assessment of early pathogens by bedside diagnostic bronchoscopy and the use of antibiotics to start anti-infection treatment. These patients were successfully treated. Results:The three patients were male, aged 63, 45 and 58 years old, respectively. Before the onset of the penumonia, they had a clear medical history of bird exposure. The clinical manifestations mainly included fever, dry cough, shortness of breath and dyspnea. One case had abdominal pain and lethargy. The results of laboratory examination indicated that the peripheral blood white blood cell count (WBC) of two patients were high [(10.2-11.9)×10 9/L], the percentage of neutrophils increased (85.2%-94.6%) and the percentage of lymphocytes decreased (3.2%-7.7%) in all 3 patients after admission to hospital and entering into intensive care unit (ICU). The procalcitonin (PCT) of 3 patients increased after admission, and still increased when entering ICU (0.3-4.8 ng/L), so did C-reactive protein (CRP, 58.0-162.0 mg/L) and erythrocyte sedimentation rate (ESR, 36.0-90.0 mm/1 h). After admission, serum alanine transaminase (ALT) increased in 2 cases (136.7 U/L, 220.5 U/L), so did aspartate transaminase (AST) in 2 cases (249.6 U/L, 164.2 U/L). ALT (162.2-267.9 U/L) and AST (189.8-223.2 U/L) increased in 3 patients when they entered ICU. The level of serum creatinine (SCr) of 3 patients were normal after admission and entering ICU. The chest computed tomography (CT) findings of 3 patients were acute interstitial pneumonia, bronchopneumonia and lung consolidation, of which 2 cases were accompanied by a small amount of pleural effusion, and 1 case was accompanied by more regular small air sacs. Multiple lung lobes were involved, but mainly one lung lobe. The oxygenation index (PaO 2/FiO 2) of the 3 patients admitting to ICU were 100.0, 57.5 and 105.4 mmHg (1 mmHg ≈ 0.133 kPa), respectively, which met with the diagnostic criteria of moderate and severe acute respiratory distress syndrome (ARDS). All three patients received endotracheal intubation and mechanical ventilation. Under the bedside bronchoscope, the bronchial mucosa of 3 patients were obviously congested and edematous, without purulent secretion, and there was 1 case with mucosal hemorrhage. Three patients underwent bedside diagnostic bronchoscopy, and the evaluation result of the pathogen was that it might be atypical pathogen infection, so they were given moxifloxacin, cisromet and doxycycline intravenously, respectively, and combined with carbapenem antibiotics intravenously. After 3 days, the detection results of mNGS in bronchoalveolar lavage fluid (BALF) showed that only Chlamydia psittaci was infected. At this time, the condition was significantly improved, and PaO 2/FiO 2 was significantly increased. Therefore, the antibiotic treatment scheme remained unchanged, and mNGS only served to verify the initial diagnosis. Two patients were extubated on the 7th and 12th day of admission to the ICU, respectively, while one patient was extubated on the 16th day of admission to the ICU due to nosocomial infection. All 3 patients were transferred to the respiratory ward after the condition was stable. Conclusion:The bedside diagnostic bronchoscopy based on clinical characteristics is conducive to not only the rapid assessment of the early pathogens of severe Chlamydia psittaci pneumonia, but also effective anti-infection treatment before the returning of mNGS test results, which can make up for the lag and uncertainty of the mNGS test results.

Analysis of the genome and transmission characteristics of an aggregated novel coronavirus infection based on metagenomic next-generation sequencing / 中华地方病学杂志

Objective:To analyze the viral genome sequence of novel coronavirus infected persons in Baotou City, understand the mutation characteristics of novel coronavirus genome in the process of transmission among cases, and explore the transmission rule of novel coronavirus in the clustered populations.Methods:Nine throat swabs samples (No. 1 - 7, No. 9, and No. 10), two sputum samples (No. 8, No. 11, and No. 11 sample was from No. 10 case), and one surface smear sample (No.12, and No. 12 sample was from No. 10 case) were collected from 10 confirmed cases of novel coronavirus infection in Baotou City from January 25 to February 21, 2020. Samples 1 and 3 were from single cases, and the rest were from clustered cases. The virus genome was sequenced by metagenomic next-generation sequencing (mNGS), and single nucleotide polymorphism (SNP) mutation sites were screened by comparing with NC_045512, a reference strain of novel coronavirus. Combined with relevant epidemiological information, gene mutation, virus typing, and evolutionary traceability analysis were carried out.Results:The results of viral genome mNGS showed that 76 SNP mutation sites were detected in 12 samples compared with the reference strain NC_045512, including 3 (3.95%) transitions and 73 (96.05%) reversals. There were 19 (25.00%) synonymous mutations and 57 (75.00%) non-synonymous mutations. The analysis of nucleotide and amino acid variation sites showed that mutations were found at five sites (T2821C, C6548T, T16464C, G16858A and T251C) in all the clustered cases (cases 2, 4 - 10). In the single cases, sample 1 had mutations at C9245T and A15340T, and sample 3 had mutation at C13T. The virus typing analysis showed that the samples 1 and 3 belonged to the L type of novel coronavirus, while the rest belonged to the S type of novel coronavirus. The results of genomic evolutionary relationship analysis showed that all the samples could be divided into two branches. The branches of sample 1 and 3 belonged to single cases, and the rest belonged to family clustered cases.Conclusion:The genomic characteristics of the clustered cases of novel coronavirus infection in Baotou City are basically consistent with the epidemiological investigation results, and the transmission of the virus is mainly related to close contact and family gathering.

Diagnosis of Pneumocystis jirovecii pneumonia by cytological examination of bronchoalveolar lavage fluid combined with metagenomic next-generation sequencing / 中国热带医学

@#Abstract：Objective To investigate the morphological features of the Pneumocystis jirovecii, in order to facilitate early detection and rapid diagnosis of this rare pathogen from a morphology point of view by laboratory technicians. By analyzing the laboratory features and application value of different pathogen detection methods in the diagnosis of Pneumocystis jirovecii pneumonia, we aim to provide the most reliable diagnostic basis for rapid diagnosis of Pneumocystis jirovecii pneumonia.Methods A retrospective analysis was conducted on the test results of bronchoalveolar lavage fluid samples from a comprehensive hospital in Zhangqiu District, Jinan City, Shandong Province, and a hospital in Changde City from April 2022 to October 2022. Five confirmed cases of Pneumocystis jirovecii pneumonia were detected. Its clinical manifestations, laboratory results, and morphological characteristics of pathogens under different stains were analyzed to discuss the advantages and disadvantages of different detection methods. Results Cytological examination of bronchoalveolar lavage fluid found the trophozoites and cysts of Pneumocystis jirovecii by Wright's-Giemsa staining in 4 cases (80%), and the cysts of Pneumocystis jirovecii by Silver hexamine staining in 4 cases (80%), while the metagenomic next-generation sequencing confirmed all the 5 positive results. All 5 patients had different degrees of reduction in the absolute count of peripheral blood lymphocytes, and the serum lactic dehydrogenase and (1-3)-β-D-Glucan were increased. Among the 5 patients in this study, 4 were treated with sulfamethoxazole combined with caspofungin, and 1 was treated with sulfamethoxazole. Three patients were cured and discharged from hospital after treatment, but two died. Conclusions The method of Wright's-Giemsa staining for the cytological examination of bronchoalveolar lavage fluid to find Pneumocystis jirovecii has the unique and irreplaceable advantages as silver staining. Metagenomic next-generation sequencing can further increase the positive detection rate of Pneumocystis jirovecii. The combination of cytological examination of bronchoalveolar lavage fluid with metagenomic nextgeneration sequencing is a powerful diagnostic method for rapid diagnosis of Pneumocystis jirovecii pneumonia, which can diagnose accurately and reduce missed diagnosis.