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.

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.

Clinical Characteristics and Diagnostic Methods of Pulmonary Mucormycosis / 中山大学学报(医学科学版)

ObjectiveTo summarize the clinical features and prognosis of pulmonary mucormycosis （PM） in southern China， and to explore the diagnostic value of metagenomic next generation sequencing （mNGS） in PM. MethodsThe clinical manifestations， diagnosis， treatment and prognosis of patients diagnosed with PM in The First Affiliated Hospital of Sun Yat-sen University from January 1， 2019 to January 31， 2022 who had undergone mNGS detection in lung tissue or alveolar lavage fluid were collected retrospectively. A total of 14 patients with PM were included， including 4 patients with confirmed diagnosis and 10 patients with clinical diagnosis. ResultsAll patients had underlying medical conditions， with hematological malignancies and diabetes being the most common. The most common symptoms were fever （n = 10）， cough （n = 9） and shortness of breath （n = 9）. Consolidation was the most common sign of chest CT， followed by mass， mostly with cavity. On laboratory tests， decreased CD4+T lymphocytes， elevated CD8+T lymphocytes， and decreased CD4+/CD8+ ratio， and presentation with pleural effusion indicate poor prognosis. The positive rate of mNGS diagnosis was 78.5%， which was significantly higher than that of histopathology （50%）， fungus rapid fluorescence staining （61.5%） and fungal culture （23.1%） of bronchoalveolar lavage fluid. ConclusionsPulmonary mucormycosis is more likely to occur in patients with underlying diseases or who are immunocompromised. The clinical manifestations lack specificity. The low CD4/CD8 ratio and presentation of pleural effusion on CT imaging indicate poor prognosis of patients. mNGS is a rapid， convenient and sensitive method for the diagnosis of PM， which has advantages in the diagnosis of pulmonary mucormycosis.

Recent Advances in Diagnosis and Treatment Strategies for Multiple Primary Lung Cancer / 中国肺癌杂志

As the utilization of computed tomography in lung cancer screening becomes more prevalent in the post-pandemic era, the incidence of multiple primary lung cancer (MPLC) has surged in various countries and regions. Despite the continued application of advanced histologic and sequencing technologies in this research field, the differentiation between MPLC and intrapulmonary metastasis (IM) remains challenging. In recent years, the specific mechanisms of genetic and environmental factors in MPLC have gradually come to light. Lobectomy still predominates in the treatment of MPLC, but the observation that tumor-specific sublobar resection has not detrimentally impacted survival appears to be a viable option. With the evolution of paradigms, the amalgamated treatment, primarily surgical, is an emerging trend. Among these, stereotactic ablative radiotherapy (SABR) and lung ablation techniques have emerged as efficacious treatments for early unresectable tumors and control of residual lesions. Furthermore, targeted therapies for driver-positive mutations and immunotherapy have demonstrated promising outcomes in the postoperative adjuvant phase. In this manuscript, we intend to provide an overview of the management of MPLC based on the latest discoveries.
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Early diagnostic value of mNGS for 15 cases with critical rickettsial disease in Hainan / 中华检验医学杂志

Objective:To explore the clinical utility of metagenomic next-generation sequencing (mNGS) for patients with critically ill atypical rickettsial infections in the early diagnosis and therapy.Methods:From Jan 2020 to Aug 2022, clinical features, blood biochemical results, imaging data and mNGS results in patients with unexplained critical illnesses were collected and analyzed retrospectively. Fisher's exact test was used to compare the positive rate of mNGS and weil felix reaction.Results:All 15 patients with rickettsial disease had fever, 12 cases had headache, but only 3 had a typical rash or scab of diagnostic significance, 6 had septic shock and all had multi-organ dysfunction; blood mNGS tests were positive in 15 cases, of which 10 had Orientia tsutsugamushi detected in their blood and the remaining five had Rickettsia moschata detected in their blood. The positive rate of mNGS was significantly higher than that of the weil felix reaction (15/15 vs 0, P<0.001). All patients were given doxycycline and other treatments after diagnosis, of which 14 improved and were discharged, and one died 1 week after discharge due to critical condition and abandonment of treatment. Conclusion:mNGS can improve the detection rate of atypical rickettsiae in patients with negative routine test results, which can provide valuable reference basis for early diagnosis and early anti-infection treatment of patients with critical rickettsial disease.

Application and consideration of metagenomic next-generation sequencing technology in the diagnosis of insect-borne diseases / 中华检验医学杂志

Insect-borne diseases are serious life-threatening infectious diseases. Rapid and accurate etiological diagnosis are the premise of timely and effective clinical treatments, reducing mortality and sequelae. Laboratory diagnoses of insect-borne diseases mainly focus on targeted serological detection and polymerase chain reaction, which is difficult to detect rare insect borne pathogens. At present, the metagenomic next-generation sequencing (mNGS) technology has moved from scientific research into clinical application. The detection of nucleic acid sequences of all organisms in infected samples by mNGS exhibited significant advantages in the diagnosis and traceability of rare pathogens. But at the same time, mNGS is also suffered with challenges such as background microbial interference, false results caused by database restrictions, pathogen resistance and host immune status information that are urgently needed for clinical treatments. This article systematically summarized applications of mNGS in the diagnosis of insect-borne pathogens and the challenges and difficulties it faces. With the continuous optimization of mNGS in the detection, it will bring new development and innovation to the etiology diagnosis of clinical infectious diseases.

Ureaplasma parvum-induced peritoneal dialysis-associated peritonitis: a case report / 中华肾脏病杂志

Pathogen detection is very important to improve the prognosis of patients with peritoneal dialysis-associated peritonitis. The paper reported a case of peritonitis caused by Ureaplasma parvum diagnosed by metagenomics next-generation sequencing（mNGS）technology. The patient was a middle-aged woman and hospitalized due to abdominal pain and muddy effluent. Anti-infective treatments such as ceftazidime and vancomycin were given but the effect was poor. The result of traditional culture was negative. Ureaplasma parvum was detected by mNGS. After using doxycycline，the patient's inflammation was controlled. It is suggested that mNGS plays an important role in the detection of the pathogens in peritoneal dialysis-associated peritonitis patients with negative culture. Through this case report and literature review，clinical experience is provided for the diagnosis and treatment in such patients.

Etiological diagnostic value of metagenomic next-generation sequencing in peritoneal dialysis-related peritonitis / 中华肾脏病杂志

Objective:To explore the etiological diagnostic value of metagenomic next-generation sequencing (mNGS) in peritoneal dialysis (PD)-related peritonitis.Methods:The study was a retrospective cohort study. The clinical data of patients with PD-related peritonitis who were treated and underwent microbial cultivation and mNGS test at the same time from June 2020 to July 2021 in the Affiliated Drum Tower Hospital, Medical School of Nanjing University were analyzed. The positive rate, detection time and consistency between mNGS test and traditional microbial culture were compared.Results:A total of 18 patients with age of (50.4±15.4) years old and median dialysis time of 34.0 (12.4, 62.0) months were enrolled in the study, including 11 males and 7 females. Pathogenic microorganisms were isolated in 17 patients by mNGS test, with a positive rate of 17/18, which was higher than 13/18 of microbial culture, but the difference was not statistically significant ( P=0.219). Both mNGS test and microbial culture isolated positive pathogenic bacteria in 12 patients, and mNGS test isolated the same types of pathogenic bacteria as microbial cultivation did in 11 patients. In five patients with negative microbial culture, mNGS test also isolated pathogenic microorganisms, including 3 cases of Staphylococcus epidermidis, 1 case of Mycobacterium tuberculosis and 1 case of Ureaplasma urealyticum. In 1 patient, microbial culture isolated pathogenic bacteria ( Escherichia coli) whereas mNGS test did not. The detection time of mNGS was 25.0 (24.0, 27.0) h, which was significantly shorter than 89.0 (72.8, 122.0) h of microbial culture ( Z=3.726, P<0.001). Conclusions:mNGS test can improve the detection rate of pathogenic microorganisms in PD-related peritonitis and greatly shorten the detection time, and has good consistency with microbial culture. mNGS may provide a new approach for pathogen identification of PD-related peritonitis, especially refractory peritonitis.

Diagnosis of a case of leptomeningeal metastasis from endometrial carcinoma by next-generation sequencing and cerebrospinal fluid cytology / 中华神经科杂志

Endometrial carcinoma is one of common malignant tumors in female reproductive system, but it is extremely rare in leptomeningeal metastasis. The clinical manifestations and signs of meningeal carcinomatosis are complex and not specific. It is difficult to get a precise diagnosis early, and it has high rate of misdiagnosis and missed diagnosis. Accurate diagnosis and treatment of a case of leptomeningeal metastasis from endometrial carcinoma by next-generation sequencing and cerebrospinal fluid cytology are reported. The patient is an elderly female with a history of endometrial cancer. The main manifestations were fever, headache and dizziness; cerebrospinal fluid cytology showed tumor cells; AKT1 gene and TP53 gene were detected in endometrial carcinoma tissue, plasma and cerebrospinal fluid by next-generation sequencing. After treatment with intrathecal chemotherapy, immunotherapy combined with anti angiogenesis, the patient′s condition still progressed gradually and died finally. The purpose of this case report is to raise clinical awareness of recognition and treatment in early meningeal metastasis of endometrial carcinoma.

Urine virology and clinical characteristics of female patients with overactive bladder / 中华泌尿外科杂志

Objective:To investigate the urinary virology and clinical characteristics of female overactive bladder (OAB) patients.Methods:Catheterized urine samples were collected from 55 women with OAB and 18 control individuals between January 2021 and August 2021. Inclusion criteria were: female with age>18, diagnosed as OAB, OABSS total score≥3 and item Urgency score≥2, informed consent signed. Exclusion criteria were: Urine culture positive, urinary catheter indwelling status, antibiotic usage in recent 30 days, other disease leading to OAB-like symptoms, pelvic organ prolapse and current pregnancy, immunosuppressive therapy or status. Clinical characteristic and history were collected. OAB symptoms were assessed via both OABSS (overactive bladder symptom score) and OAB-V8 (8-item overactive bladder questionnaire). The urine specimens were analyzed using mNGS for identifying viral infections. The correlation between the disease and JC virus infection was analyzed by t test, chi-square test, binary logistic regression analysis and Spearman correlation matrix, and the Nomogram map for predicting the risk of viral infection was constructed. Results:In total, 55 women with OAB and 18 healthy controls were recruited in the study. There are significant difference in terms of UTI history, pelvic surgery history and the habit of holding urine [60.0%(n=33)to 16.7%(n=3), P=0.002; 43.6%(n=24)to 0.0%(n=0), P<0.01; 36.4%( n=20)to 5.6%( n=1), P=0.015]. Based on mNGS results, OAB patients were identified with more positive viral infection [47.3%(n=26)to 33.3%(n=6)] and more JC virus infection. In the OAB group, subtype 7B of JCV ( n=8) was identified, while in the control group, subtype 7A(n=2) was identified. Pairwise Spearman correlation analysis indicated high correlations between viral infection and OABSS ( r=0.58), age and pausimenia ( r=0.68), hypertension and age ( r=0.53), respectively. Estimates from binary logistic regression model indicated risk factors for virus infection in OAB patients including age ( OR=1.99, 95% CI 0.02-2.61), holding urine habit( OR=2.16, 95% CI 0.18-3.85) and pelvic surgery ( OR=2.53, 95% CI 0.54-4.27). Conclusions:Urinary viral infections appear to be associated with more severe OAB symptoms and JC virus may be a potential therapeutic target for OAB.

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.