Improving pulmonary infection diagnosis with metagenomic next-generation sequencing of bronchoalveolar lavage fluid.

Introduction. Inappropriate use of antibiotics and inadequate therapeutic regimens for early-stage pulmonary infections are major contributors to increased prevalence of complications and mortality. Moreover, due to the limitations in sensitivity of conventional testing, there is an urgent need for more diagnostically efficient methods for the detection and characterization of pathogens in pulmonary infections.Hypothesis/Gap Statement. Metagenomic next-generation sequencing (mNGS) can contribute to the diagnosis and management of pulmonary infections.Aim. This study aimed to evaluate the clinical application and value of mNGS in the diagnosis of clinically suspected pulmonary infections by comparing with conventional testing.Methodology. In this study, the diagnosis performance of mNGS was evaluated using bronchoalveolar lavage fluid (BALF) samples from 143 patients with suspected lung infections. First, we conducted a prospective study on 31 patients admitted to Yuebei People's Hospital Affiliated to Shantou University Medical College to investigate the clinical value. Then a retrospective analysis was performed by including more patients (n=112) to reduce the random error. Pathogens were detected by mNGS and conventional methods (culture and PCR). Then, the types and cases of detected pathogens, as well as the specificity and sensitivity, were compared between the two methods. We evaluated the performance of mNGS in detecting bacterial, fungal, viral and mixed infections in BALF. The effect of disease severity in pulmonary infections on the integrity of mNGS pathogen detection was also explored.Results. The mNGS provided an earlier and more comprehensive pathogen profile than conventional testing, which in turn prompted a change in clinical medication, which led to improvement in eight patients (8/31=25.81 %) in the presence of other serious comorbidities. In a retrospective analysis, mNGS was much more sensitive than conventional testing in the diagnosis of pulmonary infections (95.33 % vs. 55.56 %; P<0.001), with a 39.77 % increase in sensitivity. The detection rate of mNGS for mixed infections was significantly higher than that of conventional testing methods for both common and severe pneumonia (48/67=71.64 % vs. 12/52=23.08 %, P<0.001; 44/59=74.58 % vs. 11/59=18.64 %, P<0.0001).Conclusion. The sensitivity of mNGS in the diagnosis of pathogenic microorganisms in pulmonary infections far exceeds that of conventional culture tests. As a complementary method to conventional methods, mNGS can help improve the diagnosis of pulmonary infections. In addition, mNGS pathogen integrity detection rate was similar in common and severe pneumonia. We recommend the prompt use of mNGS when mixed or rare pathogen infections are suspected, especially in immunocompromised individuals and/or critically ill individuals.

Targeted Next-Generation Sequencing for the Diagnosis of Gene Variants in Patients with 46,XY Disorder of Sex Development.

INTRODUCTION: Disorders of sex development (DSDs) are congenital abnormalities in which chromosomal, gonadal, and anatomical sex development are atypical. One of these disorders, 46,XY DSD, is particularly difficult to diagnose and manage because its etiology and clinical phenotypes are highly heterogeneous. METHODS: We used a gene panel containing 141 genes implicated in DSDs to perform targeted next-generation sequencing (NGS) in 50 patients with 46,XY DSD. RESULTS: Gene variants were detected in 23 patients (46%). Among them, 13 patients had previously reported pathogenic or likely pathogenic variants, 9 patients had novel variants, and 1 patient had a previously reported variant of uncertain significance. Three of the novel variants were pathogenic, and the remaining were variants of uncertain significance; therefore, 16 patients had pathogenic or likely pathogenic variants according to ACMG guidelines, and the overall diagnostic rate of 46,XY DSD was 32%. The most common gene variants were SRD5A2 variants, followed by the AR variant. In addition, we analyzed the association between gene variants and clinical phenotypes. Most patients presented with multiple DSD phenotypes (i.e., two or more DSD phenotypes were observed, such as micropenis, hypospadias, and cryptorchidism), but the phenotype with the highest diagnostic rate was micropenis. CONCLUSION: Our results indicate that targeted NGS can effectively detect pathogenic gene variants in patients with 46,XY DSD.

Species-level respiratory microbiome profiling for etiologic diagnosis of children pneumonia using full length 16S rRNA gene sequencing.

PURPOSE: The study was conducted to analyze the role of respiratory microbiome composition in children pneumonia etiology diagnosis. METHODS: The bronchoalveolar lavage fluid bacterial community between the Mycoplasma pneumoniae pneumonia (MP group, n â€‹= â€‹13) and the pathogen negative pneumonia (N group, n â€‹= â€‹20) children were compared using the full-length 16S rRNA gene sequencing. RESULTS: Distinct bacterial communities were identified in two groups and lower α-diversity was revealed in the MP patients indicating the lower abundance microbiota composition. Dominant bacteria were Mycoplasma and Mycoplasma pneumoniae for MP patients at genus and species levels. Possible pathogens were characterized in 17 out of 20 patients in the N group by detection of higher abundance using the 16S rRNA gene sequencing. CONCLUSIONS: The high taxonomic resolution of full-length 16S rRNA gene sequencing assists in improving characterization of potential pathogens, and more studies are necessary to further evaluate the prognostic roles of specific bacteria in the pathogenicity of pneumonia.

Understanding recurrent pregnancy loss: recent advances on its etiology, clinical diagnosis, and management.

Recurrent pregnancy loss (RPL) has become an important reproductive health issue worldwide. RPL affects about 2%-3% of reproductive-aged women, and makes serious threats to women's physical and mental health. However, the etiology of approximately 50% of RPL cases remains unknown (unexplained RPL), which poses a big challenge for clinical management of these patients. RPL has been widely regarded as a complex disease where its etiology has been attributed to numerous factors. Heretofore, various risk factors for RPL have been identified, such as maternal ages, genetic factors, anatomical structural abnormalities, endocrine dysfunction, prethrombotic state, immunological factors, and infection. More importantly, development and applications of next generation sequencing technology have significantly expanded opportunities to discover chromosomal aberrations and single gene variants responsible for RPL, which provides new insight into its pathogenic mechanisms. Furthermore, based upon patients' diagnostic evaluation and etiologic diagnosis, specific therapeutic recommendations have been established. This review will highlight current understanding and recent advances on RPL, with a special focus on the immunological and genetic etiologies, clinical diagnosis and therapeutic management.

The Epidemiology of Meningitis in Infants under 90 Days of Age in a Large Pediatric Hospital.

BACKGROUND: Meningitis is associated with substantial morbidity and mortality, particularly in the first three months of life. METHODS: We conducted a retrospective review of patients <90 days of age with meningitis at Texas Children's Hospital from 2010-2017. Cases were confirmed using the National Healthcare Safety Network (NHSN) definition of meningitis. RESULTS: Among 694 infants with meningitis, the most common etiology was viral (n = 351; 51%), primarily caused by enterovirus (n = 332; 95%). A quarter of cases were caused by bacterial infections (n = 190; 27%). The most common cause of bacterial meningitis was group B Streptococcus (GBS, n = 60; 32%), followed by Gram-negative rods other than E. coli (n = 40; 21%), and E. coli (n = 37; 19%). The majority of Gram-negative organisms (63%) were resistant to ampicillin, and nearly one-fourth of Gram-negative rods (23%) other than E. coli and 2 (6%) E. coli isolates were resistant to third-generation cephalosporins. Significant risk factors for bacterial meningitis were early preterm birth and the Black race. CONCLUSIONS: Enteroviruses most commonly caused viral meningitis in infants; GBS was the most common bacterial cause despite universal screening and intrapartum prophylaxis. The emergence of MRSA and resistance to third-generation cephalosporins in Gram-negative bacterial meningitis challenges the options for empirical antimicrobial therapy.

[Research progress on etiologic diagnosis of ocular viral diseases].

A large number of viruses have been found to be associated with ocular diseases, including human adenovirus, human herpesvirus (HHV), human T lymphotropic virus type-1 (HTLV-1), and newly emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This group of diseases is prone to be misdiagnosed or missed diagnosis, resulting in serious tissue and visual damage. Etiological diagnosis is a powerful auxiliary mean to diagnose the ocular diseases associated with human adenovirus, herpes simplex virus 1 and varicella-zoster virus, and it provides the leading diagnosis evidence of infections with herpes simplex virus 2, Epstein-Barr virus, cytomegalovirus, HHV-6/7, HHV-8, HTLV-1 and SARS-CoV-2. Virus isolation, immunoassay and genetic diagnosis are usually used for etiologic diagnosis. For genetic diagnosis, the PCR technique is the most important approach because of its advantages of rapid detection, convenient operation, high sensitivity and high specificity.

Molecular and non-molecular approaches to etiologic diagnosis of gastroenteritis.

Gastroenteritis is a major cause of mortality and morbidity globally and rapid identification of the causative pathogen is important for appropriate treatment and patient management, implementation of effective infection control measures, reducing hospital length of stay, and reducing overall medical costs. Although stool culture and microscopic examination of diarrheal stool has been the primary method for laboratory diagnosis, culture-independent proteomic and genomic tests are receiving increased attention. Antigen tests for stool pathogens are routinely implemented as rapid and simple analytics whereas molecular tests are now available in various formats from high complexity to waived point-of-care tests. In addition, metagenomic next-generation sequencing stands poised for use as a method for both diagnosis and routine characterization of the gut microbiome in the very near future. Analysis of host biomarkers as indicators of infection status and pathogenesis may also become important for prediction, diagnosis, and monitoring of gastrointestinal infection. Here we review current methods and emerging technologies for the etiologic diagnosis of gastroenteritis in the clinical laboratory. Benefits and limitations of these evolving methods are highlighted.

Application of CT Imaging in the Diagnosis of Cerebral Hemorrhage and Cerebral Infarction Nerve Damage.

OBJECTIVE: This article analyzes computed tomography (CT) angiography and CT perfusion imaging parameters of patients with cerebral hemorrhage and cerebral infarction, and explores its diagnostic value and clinical significance in the diagnosis of cerebral hemorrhage and cerebral infarction. METHODS: This article selected 52 patients with ischemic cerebrovascular disease who were treated in our neurology department from January 2015 to December 2018. Twenty of these patients had transient ischemic attacks, and 32 had neurologic damage. According to the onset time, patients with cerebral infarction were divided into 12 cases in group A (onset time <6 hours) and 20 cases in group B (onset time >6 hours). CT perfusion imaging was performed within 24 hours after the onset of cerebral hemorrhage. Patients immediately underwent CT perfusion imaging in the cerebral infarction group, and recorded the CT perfusion imaging parameters to analyze the nerve damage. RESULTS: The results showed that among the 20 patients with cerebral hemorrhage, 14 cases had anterior circulation cerebral hemorrhage, and 6 cases had posterior circulation cerebral hemorrhage. No lesions were found on CT and magnetic resonance imaging. CT angiography of 20 patients with cerebral hemorrhage showed that 18 patients had vascular lesions. In the cerebral infarction group, 30 cases developed vascular disease. CONCLUSIONS: Studies have confirmed that changes in brain CT perfusion imaging parameters can reflect changes in brain blood perfusion to diagnose nerve damage, and mean transit time and time to peak are the most sensitive during the diagnosis. CT angiography can detect the degree of stenosis and has important clinical value for the etiology of cerebral hemorrhage and cerebral infarction.

Potential utility of targeted Nanopore sequencing for improving etiologic diagnosis of bacterial and fungal respiratory infection.

BACKGROUND: Diversified etiology of lower respiratory tract infection renders diagnosis challenging. The mainstay microbial culture is time-consuming and constrained by variable growth requirements. In this study, we explored the use of Nanopore sequencing as a supplementary tool to alleviate this diagnostic bottleneck. METHODS: We developed a targeted Nanopore method based on amplification of bacterial 16S rRNA gene and fungal internal transcribed spacer region. The performance was compared with routine infectious disease workups on 43 respiratory specimens. RESULTS: Nanopore successfully identified majority of microbes (47/54, 87.04%) and 7 possible pathogens not detected by routine workups, which were attributable to the content of microbiological investigations (n = 5) and negative culture (n = 2). The average sequencing time for first target reads was 7 min (1-43 min) plus 5 h of pre-sequencing preparation. CONCLUSIONS: The Nanopore method described here was rapid, economical and hypothesis-free, which might provide valuable hints to further microbiological follow-up for opportunistic pathogens missed or not detectable by conventional tests.

Etiologic classification of infantile spasms using positron emission/magnetic resonance imaging and the efficacy of adrenocorticotropic hormone therapy.

PURPOSE: The aim of this study was to investigate if the etiologic classification of infantile spasm (IS) using positron emission tomography/magnetic resonance imaging (PET/MR) is feasible. Based on the classified etiologic groups, we further evaluated the efficacy of adrenocorticotropic hormone (ACTH) therapy in different IS groups. MATERIALS AND METHODS: One hundred fifty-five children diagnosed with IS were included in this study. A qualitative assessment of the PET/MR images was performed. The abnormal lesions localized with both MR and PET images were considered to be epileptic foci, and the patients with these lesions were classified into the structural-metabolic group. For the remaining patients, quantitative analyses were further performed on whole-brain T1-weighted (T1WI) and PET images, based on the asymmetry index of bilateral volumes and metabolic quantifications. Patients with asymmetry indices above a certain threshold (15%) were classified into the structural-metabolic group. The patients without positive finding from either qualitative or quantitative analyses were assigned to the unknown etiology group. The efficacy of ACTH therapy was evaluated in the different IS groups. RESULTS: Among the 155 children with IS, 18 genetic cases were first diagnosed by the genetic testing. In the remaining 137 cases, 49 cases were identified with structural-metabolic etiology using qualitative PET/MR assessments. Fifty-two cases were newly diagnosed with quantitative analysis. The remaining 36 cases were classified into the unknown etiology group. The efficacy of ACTH therapy was statistically different for the different etiology groups (p < 0.001). The respective efficacy rates for the genetic, qualitative structural-metabolic, quantitative structural-metabolic, and unknown etiology groups were 27.8% (5/18), 30.61% (15/49), 34.62% (18/52), and 72.22% (26/36), respectively. CONCLUSIONS: The combination of PET and MR provides additional diagnostic information for IS. Quantitative analysis can further improve patient etiologic classifications and the predication of therapy efficacies.

Research progress on etiologic diagnosis of ocular viral diseases / 浙江大学学报·医学版

A large number of viruses have been found to be associated with ocular diseases, including human adenovirus, human herpesvirus (HHV), human T lymphotropic virus type-1 (HTLV-1), and newly emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This group of diseases is prone to be misdiagnosed or missed diagnosis, resulting in serious tissue and visual damage. Etiological diagnosis is a powerful auxiliary mean to diagnose the ocular diseases associated with human adenovirus, herpes simplex virus 1 and varicella-zoster virus, and it provides the leading diagnosis evidence of infections with herpes simplex virus 2, Epstein-Barr virus, cytomegalovirus, HHV-6/7, HHV-8, HTLV-1 and SARS-CoV-2. Virus isolation, immunoassay and genetic diagnosis are usually used for etiologic diagnosis. For genetic diagnosis, the PCR technique is the most important approach because of its advantages of rapid detection, convenient operation, high sensitivity and high specificity.

Potential utility of metagenomic sequencing for improving etiologic diagnosis of infective endocarditis.

Aim: To explore potential utility of metagenomic sequencing for improving etiologic diagnosis of infective endocarditis (IE) caused by fastidious bacteria. Materials & methods: Plasma and heart valves of two patients, who were diagnosed with IE caused by Bartonella quintana and Propionibacterium species, were sequenced by using Illumina MiSeq and Nanopore MinION. Results: For patient 1, B. quintana was detected in the plasma pool collected 4 days before valvular replacement surgery. For patient 2, Propionibacterium sp. oral taxon 193 was detected in the plasma sample collected on hospital day 1. Nearly complete bacterial genomes (>98%) were retrieved from resected heart valves of both patients, enabling detection of antibiotic resistance-associated features. Real-time sequencing of heart valves identified both pathogens within the first 16 min of sequencing runs. Conclusion: Metagenomic sequencing may be a helpful supplement to IE diagnostic workflow, especially when conventional tests fail to yield a diagnosis.

Transmission Electron Microscopy Improves the Diagnostic Sensitivity in Nonbacterial Etiology of Severe Pneumonia: A Retrospective Study.

BACKGROUND: Severe pneumonia is responsible for great mortality and morbidity worldwide, and early-applied effective anti-infective therapy can improve the prognosis of patients. However, identification of infectious agents in severe pneumonia remains a major challenge so far. In this study, the potential utility of transmission electron microscopy (TEM) in detecting nonbacterial pathogens in patients with severe pneumonia was retrospectively evaluated. MATERIALS AND METHODS: A total of 106 patients diagnosed with severe pneumonia at our hospital from September 2015 to December 2017 were included, and their baseline clinical characteristics were collected. Nonbacterial infectious agents detected by TEM in bronchoalveolar lavage fluid (BALF) and serological tests were summarized. The detection rates were further compared between TEM and serological tests. RESULTS: BALF examination under the transmission electron microscope revealed 24 viruses, 16 mycoplasmas, 18 chlamydia, 2 fungi and 74 bacteria in 99 samples, among which 61 samples were mixed infections. The combined use of serological tests and TEM significantly improved the detection rate of nonbacterial infectious agents in patients with severe pneumonia. CONCLUSIONS: Our data support that implementation of TEM could improve the sensitivity for detecting viruses, atypical pathogens and mixed infections in BALF from patient of severe pneumonia. Therefore, TEM may be used as an auxiliary diagnostic method of other microbiological tests in severe pneumonia.

Úlceras corneales bacterianas: actualización terapéutica / Bacterial corneal ulcers: update on treatment

Las úlceras corneales constituyen un problema de salud visual frecuente en las consultas de Oftalmología. En ocasiones, su causa puede ser imprecisa, y las consecuencias de un error en el diagnóstico o en el tratamiento pueden ser graves. Esta entidad reviste gran importancia por su repercusión en la visión y las secuelas que suelen acompañar la recuperación del paciente, por lo que realizamos una búsqueda actualizada de los últimos diez años de diversos artículos publicados para profundizar en el diagnóstico clínico-etiológico y en el tratamiento(AU)

Corneal ulcers represent a frequent visual health problem in ophthalmology. The cause may be inaccurate sometimes and the consequences of an error in diagnosis or treatment can be severe. This feature is of great importance because of its impact on the vision and the sequelae that may accompany the patient's recovery; therefore, we made a literature review of several articles for the last ten years to delve into the clinical and etiological diagnosis and into treatment(AU)

Úlceras corneales bacterianas: actualización terapéutica / Bacterial corneal ulcers: update on treatment

Las úlceras corneales constituyen un problema de salud visual frecuente en las consultas de Oftalmología. En ocasiones, su causa puede ser imprecisa, y las consecuencias de un error en el diagnóstico o en el tratamiento pueden ser graves. Esta entidad reviste gran importancia por su repercusión en la visión y las secuelas que suelen acompañar la recuperación del paciente, por lo que realizamos una búsqueda actualizada de los últimos diez años de diversos artículos publicados para profundizar en el diagnóstico clínico-etiológico y en el tratamiento(AU)

Corneal ulcers represent a frequent visual health problem in ophthalmology. The cause may be inaccurate sometimes and the consequences of an error in diagnosis or treatment can be severe. This feature is of great importance because of its impact on the vision and the sequelae that may accompany the patient's recovery; therefore, we made a literature review of several articles for the last ten years to delve into the clinical and etiological diagnosis and into treatment(AU)

Multidisciplinary Diagnostic Approach and Etiologic Evaluation of Patients with Developmental Disorders / 대한소아신경학회지

PURPOSE: The prevalence of developmental delay is 5-10% of the total pediatric population and early diagnosis and proper intervention are essential but a challenge for clinicians. We performed this study for several purposes. First is to know the distribution & characteristics of developmental disorders in Korea. Second is to identify all possible causes of these disorders through a multidisciplinary diagnostic approach, and thus to find out the clinical variables that are helpful in finding the etiology. And finally to develop a useful protocol that eliminates the cost of unnecessary tests and raises the diagnostic rate of the cause. METHODS: 518 patients(M 349, F 169) were studied who visited Ilsan Hospital Developmental Disorder Clinic(DDC) for the evaluation of developmental delay from April 2001 to Jan 2005. RESULTS: The mean age was 51.5+/-32.9 months, ranging from 2 months to 16.0 years of age with a majority of the preschool children(<6 yr)(79.3%). Phenomenological diagnosis consisted of 133 cases of mental retardation, 122 cases of autistic disorders, 101 cases of delayed language disorders, 27 cases of cerebral palsies, and 91 cases labeled as simple developmental delay requiring follow up due to age less than 2 years of age. Etiologic diagnosis was obtained in 119 cases(22.9%) out the 518 cases of developmental delays. 37 cases of chromosomal anomalies, 23 cases of Periventricular leukomalacia and hypoxic ischemic encephalopathy, 21 cases of syndromes, 7 cases of malformation of cortical development, 4 cases of myopathies, 4 cases of neuropathies, and 4 cases of cerebral infarctions were found. Among the clinical variables, low birth weight, facial dysmorphism, hypotonia, focal neurologic signs, and abnormalities in MRI, chromosome, EEG and EMG studies contributed to the yields of etiologic diagnosis significantly. CONCLUSION: Possible etiology was determined in about 23% of the subjects. The most important part of the assessment for the identification of etiology is thorough history taking, physical and neurologic examination. Neuroimaging study is useful in case of micro or macrocephaly, focal neurologic signs. Genetic studies increasingly produce a yield, when there is family history of inherited disorder and there are dysmorphic features. Routine metabolic screening test has limited utility. Development of a useful screening protocol adequate for Korean situation is required.

Clinical Investigation of Primary Amenorrhea / 대한산부인과학회잡지

OBJECTIVE: To assess the etiologic diagnosis of primary amenorrhea and review the clinical significance in management of primary amenorrhea. METHODS: To make the accurate etiologic diagnosis of primary amenorrhea, karyotype, hormone study (TSH, Prolactin, LH, FSH, Estradiol, Testosterone, DHEA-S), various imaging techniques were performed in total 57 patients. And additional GnRH stimulation test and bone densitometry were also performed in group of hypogonadotropic amenorrhea with normal sella and brain imaging for discriminating the hypothalamic and pituitary cause and in patients with low estrogenic state for identifying the risk of osteoporosis, respectively, then reviewed as to clinical significance according to etiologic classification, karyotypical abnormalities, and risk of osteoporosis in low estrogenic group. RESULTS: The range of age at diagnosis was from 13 to 34 years, most commonly, 17-18 years, 26.3%. The most common causes of primary amenorrhea was 46,XX ovarian failure and hypothalamic failure, 19.3% and 19.3%, respectively. The next common causes were genetic disorder related with Turner syndrome (17.5%), M llerian agenesis (12.3%), complete androgen insensitivity syndrome (10.5%), orderly. In cytogenetic study, 19 patients (34%) showed abnormal karyotype, of abnormal karyotypes, Turner genotype was most common (52%), and 46,XY was second most common (31.5%). Almost all patients with low estrogenic state showed osteopenia or osteoporosis. CONCLUSION: The most common causes of primary amenorrhea were 46,XX gonadal failure, hypothalamic failure, Turner syndrome. These all patients were at high risk of osteoporosis or osteopenia.

Value of Bronchoscopy and Bronchoalveolar Lavage in Etiologic Diagnosis of Immunocompromised Patients with Pneumonia / 中华医院感染学杂志

OBJECTIVE To investigate the value of fibrobronchoscopy and bronchoalveolar lavage in etiologic diagnosis of pneumonia in immunocompromised patients.METHODS The clinical document and results of fibrobronchoscopy and bronchoalveolar lavage in 36 immunocompromised patients with pneumonia were retrospectively analyzed,whose conditions were mainly after organ transplantation and hematologic neoplasia.RESULTS Through fibrobronchoscopy and(or) bronchoalveolar lavage,22 cases(61.1%) were etiologically diagnosed.In 19 cases taking cytomegalovirus(CMV) quantitative PCR test of both peripheral blood and BALF,the positive rate of blood and BALF was 14.3% and 42.9%,respectively(P