Application of metagenomic next-generation sequencing in prevention and control of infection in solid organ transplantation / 器官移植

Organ transplantation has become an effective treatment for multiple end-stage diseases. However, the recipients of organ transplantation need to take immunosuppressive drugs for a long time after operation, which leads to low immune function and relatively high incidence of bacterial, viral and fungal infections. Traditional microbial detection methods, such as pathogen culture, immunological detection and polymerase chain reaction, have been widely applied in infection detection, whereas these methods may cause problems, such as long detection time and presumed pathogens. Metagenomic next-generation sequencing has been widely adopted in infection prevention and control in organ transplantation in recent years due to high detection rate and comprehensive detection of pathogen spectrum. In this article, the application of metagenomic next-generation sequencing in the prevention and control of infection in solid organ transplantation was reviewed, aiming to provide reference for the diagnosis and treatment of transplantation-related infection.

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.

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.

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.

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.

Study on the characterization of hepatitis B virus quasi-species based on the long-term cohort study in Long′an County, Guangxi / 中国热带医学

@#Abstract: Objective To clarify the long-term evolution of hepatitis B virus (HBV) quasi-species in HBsAg asymptomatic carriers in Long'an county, Guangxi. Methods ELISA was used to detect serological markers of HBV. Viral loads were measured by real time PCR. HBV DNA was extracted from serum by kits. The whole HBV genome was amplified using nested PCR and amplicons were sequenced by next-generation sequencing (NGS). These sequences from NGS were analyzed by the software like Mega. Results Serum samples were collected from 9 HBsAg asymptomatic carriers in Longan County，Guangxi at 4 different time points in 2004, 2007, 2013, 2019 or 2020. A total of 23 serum samples and 309 full-length gene quasi-species sequences were obtained, with an average amount of (0.18±0.07) G sequencing data for each sample. Genotype of 55.54%(5/9) the studied subjects underwent genotype conversion during the long-term evolution process of HBV quasi-species, and the genotyping results of the phylogenetic tree in the PreS/S region are in perfect agreement with the results of the whole genome analysis; recombinant B/C, I/C were found; the Sn ranged from 0 to 0.37 and the genetic diversity ranged from 0 to 0.11, respectively. A total of 21 special single nucleotide/amino acid mutations (7 in the S region, 2 in the X region, 3 in the PreC region and 9 in the BCP region) and 6 deletion mutations were detected, multiple mutations were found and no drug resistant mutations were found; 77.8%(7/9) of the HBV strains carried by the subjects in 2004 had double mutations at nt1 762(A→T) and 1 764(G→A) and a stop mutation at nt1 896(G→A); HBV mutations can be restored from the mutant type to the wild type and (or) vice versa without antiviral drug pressure, and The evolution rate of HBV genome was 2.03×10-5~3.50×10-3.Conclusion HBV genotype, recombinants, genetic complexity and diversity of HBV quasi-species can change over time during in natural infection. The transformation between HBV mutation type and wild type reduces the value of predicting clinical outcomes by genetic types and related mutations to some extent. The HBV genome evolution rate of asymptomatic carriers of HBsAg in Long'an County is very high.

Ambiguous results of the HLA-DPB1 genotyping results: Based on AllType NGS 11-loci sequencing reagent / 中国输血杂志

【Objective】 To statistically analyze the characteristics of ambiguous results of HLA-DPB1 genotyping given by AllType NGS 11-loci sequencing reagent via two next generation sequencing platforms, i. e. Ion Torrent S5 and Illumina Miseq. 【Methods】 A total of 434 samples from patients or donors were genotyped for HLA-DPB1 locus using AllType NGS 11-loci sequencing reagent from One Lambda company; 336 samples of them were sequenced via the Ion Torrent S5 platform and other 98 samples were sequenced via the Illumina Miseq platform. All 434 samples were genotyped for HLA-DPB1 gene simultaneously using PCR-SSO flow fluorescent bead method. The ambiguous genotypes of HLA-DPB1*13∶01∶01/107∶01 were distinguished by Sanger sequencing. The HLA-DPB1 genotype results by NGS method were assigned by TypeStream Visual professional software, and the ratio of ambiguous combination was calculated by direct count method. 【Results】 Ambiguous results were found in 357 out of 434 samples, accounting for 82.3% (357/434) when HLA-DPB1 allele was assigned to the third field using NGS method. Ambiguous results with 45 types were given in 275 out of 336 samples by the Ion Torrent S5 platform, accounting for 81.8% (275/336) and 82(with 27 types) out of 98 samples by the Illumina Miseq platform, accounting for 83.7% (82/98). All samples were re-genotyped for HLA-DPB1 gene by PCR-SSO, and none HLA-DPB1 allele had been missed by NGS. A total of 43 ambiguous alleles in HLA-DPB1*13∶01∶01/107∶01 involving 41 samples were distinguished by Sanger sequencing; HLA-DPB1*13∶01∶01 were detected in 25 (58.1%, 25/43) and HLA-DPB1*107∶01 in 18 (41.9%, 18/43). 【Conclusion】 There were still a high proportion of HLA-DPB1 ambiguous combinations using the AllType NGS 11-loci sequencing reagent. Sequencing exon 1 of HLA-DPB1 gene by Sanger sequencing can resolve part of the ambiguous results in HLA-DPB1*13∶01∶01/107∶01 alleles.

Epidemiological investigation and management of a case of severe Chlamydia psittaci pneumonia / 上海预防医学

ObjectiveTo identify causal factors of a case of severe Chlamydia psittaci pneumonia in Yangpu District and provide a scientific basis for effective prevention and control. MethodsBasic information and epidemiological data of the patient were collected through telephone interviews and field epidemiological surveys. Specimens from the patient， close contacts and the environment were collected for pathogen detection. Metagenomics next-generation sequencing （mNGS） was used to identify unknown pathogens. ResultsA 65-year-old male patient with a history of hypertension and diabetes was admitted to the hospital with symptoms of fatigue， poor appetite for a week， fever and cough for four days. A chest computer tomography （CT） scan showed scattered inflammation in the left lung with infiltration of multiple lobes. Blood gas analysis showed type I respiratory failure. The results of mNGS on the bronchoalveolar lavage fluid of the patient indicated that he was infected with Chlamydia psittaci. Epidemiological investigation showed a clear history of avian exposure， with an incubation period of 30 days. ConclusionThis serious pneumonia is a zoonotic disease caused by Chlamydia psittaci. A clear history of avian exposure and the use of mNGS technology can help in the timely diagnosis of this disease.

Clinical characteristics analysis of Chlamydia psittaci pneumonia detected by metagenomic next-generation sequencing / 中国热带医学

@#Abstract: Objective　To analyze the clinical characteristics of Chlamydia psittaci pneumonia and improve the diagnosis and treatment skills of clinicians on this disease. Methods The clinical data of thirty-nine Chlamydia psittaci pneumonia cases detected by metagenomic next-generation sequencing (mNGS) from September 2020 to January 2022 at the Affiliated Hospital of Southwest Medical University were retrospectively analyzed. Results There was a history of poultry exposure in 89.7%(35 cases) of the patients. The most common clinical manifestations were high fever (92.3%, 36), cough (76.9%,30), muscle soreness (48.7%,19), headache (38.5%,15), etc. Laboratory examinations showed 76.9% of patients had a normal leukocyte count, and 76.9% had decreased lymphocyte count, often accompanied by elevated C-reactive protein (100%), procalcitonin (97.4%), interleukin-6 (95.8%), interleukin-10 (95.8%), alanine aminotransferase (74.4%), and aspartate aminotransferase (84.6%). Univariate analysis indicated that there were statistically significant differences in the levels of aspartate transaminase, blood urea nitrogen, C-reactive protein, and procalcitonin between severe pneumonia patients and non-severe pneumonia patients（P<0.05）. Multivariate logistic regression analysis showed that an elevated blood urea nitrogen (OR=4.899) had guiding significance for predicting the occurrence of severe pneumonia. Bronchoscopy examination showed no abnormalities in 53.6% of the patients. The imaging manifestations of pulmonary lesions were mainly lobar pneumonia (61.5%) and air bronchograms (94.9%). Therapeutically, it was sensitive to tetracyclines, macrocyclic lactones, and fluoroquinolones. A total of 84.6%(33 cases) of the patients were cured and discharged from the hospital at the end of the treatment. Conclusion Chlamydia psittaci pneumonia is a zoonotic disease that can be detected by mNGS. An elevated blood urea nitrogen level has guiding significance for predicting the occurrence of severe pneumonia. Empirically-selected regimens based on doxycycline are effective for the treatment of Chlamydia psittaci pneumonia.

Clinical and epidemiological features analysis of pneumocystis jirovecii pneumonia in kidney transplant recipients / 器官移植

Objective To investigate clinical and epidemiological features of pneumocystis jirovecii pneumonia (PJP) in kidney transplant recipients. Methods Clinical data of 68 kidney transplant recipients admitted from July, 2021 to December, 2021 were collected. All patients were divided into the PJP group (n=11), common pulmonary infection group (n=24) and non-pneumonia group (n=33) according to the status of pulmonary infection. The incidence and treatment of PJP after kidney transplantation were analyzed. Basic characteristics and laboratory parameters of the recipients were compared among all groups. The genotyping and transmission map of PJP patients were evaluated. Results Among 64 kidney transplant recipients, 11 cases were definitely diagnosed with PJP. The most common clinical manifestations included elevated body temperature, and dry cough complicated with progressive dyspnea. Chest CT scan showed diffuse interstitial inflammation and ground glass-like lesions of bilateral lungs in all patients. After diagnosis, all patients were orally given with compound sulfamethoxazole for 3-4 weeks. Two patients received non-invasive ventilator-assisted ventilation due to severe lung infection and dyspnea, and the remaining patients were given with nasal cannula oxygenation. One patient experienced elevated serum creatinine level upon discharge, and developed renal allograft failure. The remaining 10 recipients with PJP obtained normal renal allograft function, and no recipient died. Compared with the non-pneumonia group, the rejection rate was higher, the length of hospital stay was longer, the lymphocyte count was less, the lymphocyte proportion was lower, the levels of C-reactive protein, serum creatinine and lactate dehydrogenase were higher, and the levels of serum albumin was lower and CD4+T cell count was less in the PJP group (all P < 0.05). Compared with common pulmonary infection group, the lymphocyte count was less, the lymphocyte proportion was lower, the CD4+T cell count was less and 1, 3-β-D- glucan (BDG) level was higher in the PJP group (all P < 0.05). No new genotype was detected in 10 of the 12 testing samples. It was considered that PJP mainly depended on two transmission chains and two independent transmission individuals. Conclusions Kidney transplant recipients are prone to pneumocystis jirovecii (PJ) infection due to impaired cellular immune function. The most common clinical manifestations consist of elevated body temperature and dry cough complicated with progressive dyspnea, accompanied by headache and fatigue in partial patients. Chest CT scan shows diffuse interstitial inflammation and ground glass-like lesion of bilateral lungs. PJ may be transmitted through respiratory tract. Small-scale PJP might occur in the follow-up outpatient of kidney transplant recipients. Preventive measures should be delivered in a timely manner.

Research Progress of Lung Cancer Vaccines / 中国肺癌杂志

With the development of medical technology, tumor vaccines as a novel precise immunotherapy approach have gradually received attention in clinical applications. Against the backdrop of the global corona virus disease 2019 (COVID-19) outbreak, vaccine technology has further advanced. Depending on the types of antigens, tumor vaccines can be divided into whole-cell vaccines, peptide vaccines, messenger ribonucleic acid (mRNA) vaccines, recombinant virus vaccines, etc. Although some tumor vaccines have been marketed and achieved certain therapeutic effects, the results of tumor vaccines in clinical trials have been unsatisfactory in the past period. With the maturation of next-generation sequencing (NGS) technology and the continuous development of bioinformatics, dynamic monitoring of the entire process of tumor subpopulation development has become a reality, which has laid a solid foundation for personalized, neoantigen-centered therapeutic tumor vaccines. This article reviews the recent developments of tumor vaccines of different types, starts with lung cancer and summarizes the achievements of tumor vaccines in clinical applications, and provides an outlook for the future development of antigen-centered tumor vaccines.
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Analysis of CSF3R Gene Mutations and Clinical Characteristics in Patients with t(8;21) Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To investigate the occurrence of CSF3R mutation in patients with t(8;21) acute myeloid leukemia (AML) and its correlation with some clinical parameters.@*METHODS@#The clinical and laboratory data of 167 newly diagnosed AML patients with t(8;21) translocation were analyzed retrospectively. High-throughput DNA sequencing technology combined with Sanger sequencing method was used to detect 112 gene mutations. The occurrence of CSF3R gene mutation and its influence on the remission rate after chemotherapy were analyzed.@*RESULTS@#Among 167 patients with t(8;21) AML, 15 patients (9.0%) carried CSF3R mutations, including 6 cases of membrane proximal region mutations and 9 cases of truncation mutations in the cytoplasmic tail. The most common coexisting mutations of CSF3R were KIT (40.0%), TET2 (33.3%), DNMT3A (26.7%), FLT3 (20.0%), CBL (20.0%), IDH1 (13.3%), etc. Compared with the wild type, the CSF3R mutant group had a higher mutation rate of DNA methylation-related genes（P <0.001）. The median peripheral white blood cell (WBC) count of patients with CSF3R gene mutation was 5.80 (3.20-8.56)×109/L at initial diagnosis, which was significantly lower than 8.80 (5.26-19.92)×109/L of the CSF3R wild-type patients (P =0.017). There was no significant difference between the two groups in sex, median age, FAB classification, hemoglobin level, platelet count, etc. (P >0.05). The CR rate of the CSF3R gene mutation group (100%) was significantly higher than that of the wild-type group (86.8%), but the difference was not statistically significant (P >0.05). The CSF3R gene mutation group had a significantly higher CD19 positive rate and a higher -X rate than the wild group (86.7% vs 47.4%, P =0.004; 33.3% vs 13.2%, P =0.037).@*CONCLUSION@#There is a high incidence of CSF3R mutation in t (8;21) AML patients. The clinical characteristics and coexisting mutation genes of CSF3R mutation-positive patients are different from those of wild-type patients.

High-Throughput Sequencing Technology for Detection of Gene Mutations in Myeloid Malignancies and Its Clinical Prognostic Significance / 中国实验血液学杂志

OBJECTIVE@#To detect the gene mutations in patients with myeloid malignancies by high-throughput sequencing and explore the correlation between gene mutations and prognosis.@*METHODS@#A retrospective analysis was performed on 56 patients with myeloid malignancies who were hospitalized in the department of hematology, Peking University International Hospital from January 2020 to May 2021. The genetic mutations of the patients were detected by next-generation sequencing technology, and the correlation between the genetic mutations and prognosis of myeloid malignancies was analyzed.@*RESULTS@#In 56 patients, the number of mutated genes detected in a single patient is 0-9, with a median of 3. Sequencing results showed that the most common mutated genes were RUNX1(21.4%), TET2(17.9%), DNMT3A(17.9%), TP53(14.3%) and ASXL1(14.3%), among which the most common mutations occurred in the signaling pathway-related genes (23.3%) and the transcription factor genes (18.3%). 84% of the patients carried multiple mutated genes (≥2), and correlation analysis showed there were obvious co-occurring mutations between WT1 and FLT3, NPM1 and FLT3-ITD, and MYC and FLT3. TP53 mutation was more common in MDS patients.The overall survival time of patients with NRAS mutation was significantly shortened (P =0.049). The prognosis of patients with TP53 mutation was poor compared with those without TP53 mutation, but the difference wasn't statistically significant (P =0.08).@*CONCLUSION@#The application of next-generation sequencing technology is of great significance in myeloid malignancies, which is helpful to better understand the pathogenesis of the disease, to judge the prognosis and to find possible therapeutic targets.

Application of metagenomic next-generation sequencing of bronchoalveolar lavage fluid in the diagnosis and treatment of refractory pneumonia in children / 中国当代儿科杂志

OBJECTIVES@#To investigate the clinical application of metagenomic next-generation sequencing (mNGS) of bronchoalveolar lavage fluid (BALF) in the etiological diagnosis and treatment of refractory pneumonia (RTP) in children.@*METHODS@#A retrospective analysis was performed on 160 children with RTP who were admitted to the Department of Pediatric Internal Medicine, Maternal and Child Health Hospital of Inner Mongolia Autonomous Region, from January 2020 to March 2023. According to whether mNGS was performed, they were divided into two groups: mNGS (n=80) and traditional testing (n=80). All children received the tests of inflammatory markers and pathogen tests after admission. Traditional pathogenicity tests included microbial culture (sputum specimen collected by suction tube), nucleic acid detection of respiratory pathogens, and serological test (mycoplasma, tuberculosis, and fungi). For the mNGS group, BALF specimens were collected after bronchoscopy and were sent to the laboratory for mNGS and microbial culture. The two groups were analyzed and compared in terms of the detection of pathogens and treatment.@*RESULTS@#Compared with the traditional testing group, the mNGS group had a significantly higher detection rate of pathogens (92% vs 58%, P<0.05), with more types of pathogens and a higher diagnostic rate of mixed infections. Compared with the traditional testing group, the mNGS group had a significantly higher treatment response rate and a significantly lower incidence rate of complications during hospitalization (P<0.05). Treatment was adjusted for 68 children in the mNGS group according to the results of mNGS, with a treatment response rate of 96% (65/68) after adjustment.@*CONCLUSIONS@#Compared with traditional pathogen tests, BALF mNGS can significantly improve the detection rate of pathogens and find some rare pathogens. In clinical practice, when encountering bottlenecks during the diagnosis and treatment of children with RTP, it is advisable to promptly perform the mNGS to identify the pathogens.

Cross-Platform Application of DNA Methylation Age Estimation Model in Eastern Chinese Han Population / 法医学杂志

OBJECTIVES@#To evaluate the forensic application value of an age estimation model based on DNA methylation in eastern Chinese Han population, and to provide a theoretical basis for exploring age estimation models suitable for different detection platforms.@*METHODS@#According to the 6 age-related methylation sites in the published blood DNA methylation age estimation models of Chinese Han population, the DNA methylation level of 48 samples was detected by pyrosequencing and next-generation sequencing (NGS). After submitting DNA methylation levels to the age estimation model, the DNA methylation ages were predicted and compared with their real ages.@*RESULTS@#The 6 DNA methylation sites in both detection techniques were age-related, with an R2 of 0.85 and a median absolute deviation (MAD) of 4.81 years when using pyrosequencing；with an R2 of 0.84 and MAD of 4.41 years when using NGS.@*CONCLUSIONS@#The blood DNA methylation age estimation model can be used under pyrosequencing and multi-purpose regional methylation enrichment sequencing technology based on NGS and it can accurately estimate the age.

Expert Consensus on Acute Respiratory Failure in Critically Ill Cancer Patients (2023) / 中国医学科学杂志(英文版)

Objective This consensus aims to provide evidence-based recommendations on common questions in the diagnosis and treatment of acute respiratory failure (ARF) for critically ill cancer patients.Methods We developed six clinical questions using the PICO (Population, Intervention, Comparison, and Outcome) principle in diagnosis and treatment for critical ill cancer patients with ARF. Based on literature searching and meta-analyses, recommendations were devised. The GRADE (Grading of Recommendation Assessment, Development and Evaluation) method was applied to each question to reach consensus in the expert panel. Results The panel makes strong recommendations in favor of (1) metagenomic next-generation sequencing (mNGS) tests may aid clinicians in rapid diagnosis in critically ill cancer patients suspected of pulmonary infections; (2) extracorporeal membrane oxygenation (ECMO) therapy should not be used as a routine rescue therapy for acute respiratory distress syndrome in critically ill cancer patients but may benefit highly selected patients after multi-disciplinary consultations; (3) cancer patients who have received immune checkpoint inhibitor therapy have an increased incidence of pneumonitis compared with standard chemotherapy; (4) critically ill cancer patients who are on invasive mechanical ventilation and estimated to be extubated after 14 days may benefit from early tracheotomy; and (5) high-flow nasal oxygen and noninvasive ventilation therapy can be used as a first-line oxygen strategy for critically ill cancer patients with ARFs. A weak recommendation is: (6) for critically ill cancer patients with ARF caused by tumor compression, urgent chemotherapy may be considered as a rescue therapy only in patients determined to be potentially sensitive to the anticancer therapy after multidisciplinary consultations. Conclusions The recommendations based on the available evidence can guide diagnosis and treatment in critically ill cancer patients with acute respiratory failure and improve outcomes.

Application of high-throughput next-generation sequencing in diagnosis and treatment of myeloid hematologic malignancies in the era of precision medicine / 白血病·淋巴瘤

With the wide application of high-throughput next-generation sequencing (NGS) and other molecular genetic detection technologies, researchers have a more and more in-depth understanding of the pathogenesis of hematologic malignancies, especially of the myeloid hematologic malignancies, which makes the diagnosis and treatment of myeloid hematologic malignancies into an era of precision medicine. At the 64th American Society of Hematology (ASH) Annual Meeting in 2022, there were a series of new progresses regarding the application of NGS in the diagnosis and classification, risk stratification, treatment guidance, and minimal residual disease monitoring of myeloid hematologic malignancies. This article focuses on the progress of NGS application in acute myeloid leukemia, myelodysplastic syndromes and myeloproliferative neoplasms.