Enterovirus D68 (EV-D68), a neurotropic respiratory pathogen, poses a considerable clinical threat through its link to pediatric acute flaccid myelitis (AFM) and severe respiratory illness. The possibility of recurrent epidemics, evidenced since the 2014 outbreak, remains a major concern. Genomic determinants of virulence are central to this threat. Sequence variations that affect host–receptor interactions, immune evasion, and replication efficiency serve as critical modifiers of pathogenicity. This article systematically reviews the evidence for specific genomic sites that enhance EV-D68 virulence, focusing on three critical regions: the VP1 receptor-binding site, the 2Apro/TRAF3 cleavage site, and the 3Cpro immunoregulatory region. Mutations in the VP1 receptor-binding site can alter affinity for host receptors such as sialic acid, heparan sulfate, and MFSD6, thereby shaping viral entry and tissue tropism. Alterations in the 2Apro/TRAF3 cleavage site may impair proteolytic cleavage of host TRAF3, attenuating immune evasion and reducing viral pathogenicity. Variations in the 3Cpro region affect its efficiency in cleaving host proteins involved in translation and autophagy, ultimately modulating viral replication and antiviral responses. Finally, we propose that monitoring for mutations in these key virulence determinants, particularly within the surface-exposed VP1, is essential for effective outbreak preparedness.

IntroductionThe persistent circulation of influenza viruses following the COVID-19 pandemic remains a major public health concern, yet local genomic epidemiology in pediatric populations is not well-defined.MethodsIn 2024, we collected 490 influenza antigen-positive specimens from a pediatric hospital in Qidong, eastern China, and performed influenza genome enrichment-based whole-genome sequencing, yielding 425 genomes (86.73%) with at least hemagglutinin (HA) and neuraminidase (NA) segments.ResultsA/H1N1 (45.41%), A/H3N2 (18.35%), and B/Victoria (36.24%) co-circulated, with two activity peaks in January and December. Phylogenetic analysis assigned these viruses to clades 6B.1A.5a.2a (A/H1N1), 3C.2a1b.2a.2a.3a.1 (A/H3N2), and V1A.3a.2 (B/Victoria). Positively selected sites in HA included lineage-specific residues such as position 210 in B/Victoria and positions 187 and 372 in A/H3N2. The oseltamivir resistance marker NA-H275Y was detected in 7/193 (3.65%) A/H1N1 strains, whereas no detected resistance to RNA polymerase inhibitors.DiscussionThese findings highlight the co-circulation patterns, genetic diversity, and antiviral susceptibility profile of influenza viruses in a pediatric population in eastern China, supporting the importance of sustained genomic surveillance to guide local prevention strategies and clinical management.

Enteroviruses (EVs), which include numerous types, are among the most common pathogens causing infections in children. EVs can cause diverse diseases, including herpangina, hand-foot-and-mouth disease, myocarditis, pneumonia, meningitis, encephalitis, and acute flaccid paralysis. Severe cases may result in pediatric mortality, posing substantial threats to child health and public health. The diversity of EV types combined with rapid viral evolution and antigenic variation presents formidable challenges for EV prevention and control, with dominant genotypes being continuously replaced by emerging variants and novel subtypes. EV invades host cells through specific receptors and hijacks cellular machinery for self-replication, engaging in complex interactions with the host while evolving multiple mechanisms to evade host immune responses. Currently, no specific anti-EV agents are available, and treatment remains primarily supportive and symptomatic. Effective prevention and control strategies for EV are limited, and vaccines are currently available only against selected EV serotypes. This review systematically examines EV infections from multiple perspectives, including epidemiology, virology, viral entry mechanisms, virus-host interactions, clinical diagnosis and management, prevention and control measures, with the aim of providing insights and references for research on EV pathogenesis, precision clinical diagnosis and treatment, and the development of antiviral therapies and vaccines.

ABSTRACT Enterovirus D68 (EV-D68) causes severe respiratory diseases and acute flaccid myelitis (AFM), for which no licensed vaccines or therapeutics are currently available. Neutralizing antibodies (nAbs) block infection via binding capsid epitopes. This review summarizes the EV-D68 antigenic structure, nAb mechanisms (receptor blockade and conformational disruption), therapeutic prospects of nAbs, and vaccine progress (inactivated, virus-like particle [VLP], and nucleic acid). Key challenges, like antigenic drift and blood-brain barrier (BBB) penetration, are also highlighted to guide countermeasure development.

Enterovirus D68 (EV-D68) is a re-emerging pathogen associated with severe acute flaccid myelitis (AFM) and pneumonia, predominantly in children but also capable of causing severe disease in immunocompromised adults. Seroprevalence data are essential for understanding population immunity, age-specific susceptibility, and viral transmission dynamics; yet, such data for the period after the COVID-19 pandemic are scarce. We conducted a cross-sectional serological survey to characterize the neutralizing antibody profile of EV-D68 among children in Xiamen, China, in 2022. A total of 453 children aged 0–16 years (217 with acute respiratory tract infection [ARTI] and 236 without) were enrolled. Neutralizing antibody (NtAb) titers against the EV-D68 STL strain were measured using a microneutralization assay, seropositivity was defined as a titer ≥1:16. Multivariable regression and analysis of covariance were used to adjust for age and sex. Overall seroprevalence was 96.0% (435/453), exceeding 90.0% in every age subgroup (0–1 y: 100%; 1–3 y: 97.6%; 3–5 y: 92.6%; ≥5 y: 96.2%). The geometric mean titer (GMT) was 76.78 (95% CI: 68.32–85.24). After adjustment, ARTI status was not significantly associated with seropositivity (adjusted OR = 1.42, 95% CI: 0.46–4.41, p = 0.542). In an exploratory comparison, children with ARTI showed nominally higher antibody titers than those without (Mann–Whitney U test, p = 0.003), although the absence of EV-D68-specific PCR testing precludes etiologic attribution of respiratory symptoms to EV-D68 infection. Infants < 1 year showed the highest GMT (86.31), while children aged 1–3 years had the lowest GMT (63.52) and the largest low-titer fraction, indicating a susceptibility gap. Our study revealed EV-D68 circulated endemically in this pediatric population, establishing a high population immune baseline (>90.0%) with distinct age-dependent patterns. These findings provide key reference data for ongoing serosurveillance, outbreak risk assessment, and the evaluation of future vaccines or immunoprophylactic strategies should they become available.

OBJECTIVE:Brainstem encephalitis (BE) can cause sudden death in children. Fewer studies have been conducted on the incidence, clinical manifestations, pathogens and post-infectious sequelae of pediatric infectious BE. METHODS:Pediatric patients diagnosed with BE in our Medical Center from 01 January 2015 to 31 July 2024 were retrospectively reviewed. The clinical data of these children were obtained from the hospital's medical database on 15 August 2024. The number of outpatient and inpatient patients at our Medical Center during that period were provided by the hospital data center. Data analysis was conducted using Excel 2019. RESULTS:A total of twenty-eight cases were diagnosed with BE in our National Children's Medical Center over the past decade. Among them, 57.1% (16/28) cases were diagnosed with infectious BE. The incidence of infectious BE was estimated to be 16 cases per 30 million outpatient visits and 13 cases per 500,000 hospitalized patients. Fever, consciousness disorders and seizures were observed in 75.0% (12/16), 68.8% (11/16) and 62.5% (10/16) of the cases, respectively. Among them, 31.3% (5/16) cases were diagnosed as human enterovirus infections, 12.5% (2/16) cases were confirmed to be influenza B virus infections, while one case each was diagnosed with herpes simplex virus 1 and human herpesvirus 6 infection. The mortality rate during hospitalization was 12.5% (2/16). Among the surviving patients, 50.0% (7/14) of them had follow-up records, 85.7% (6/7) of the survivors suffered from sequelae such as motor disorders. CONCLUSION:Fever, consciousness disorders and seizures were the major clinical manifestations in patients with infectious BE visited our Medical Center. These rare cases exhibited a notably high mortality rate and a significant frequency of long-term complications.

Milk fat globule membrane (MFGM) supplementation of infant formula demonstrates potential efficacy in modulating gut microbiota and metabolic profiles. However, the associated site-specific effects on intestinal microbial composition remain unclear. In this study, we used a neonatal piglet model to investigate the mechanisms associated with the metabolic regulation of supplemental MFGM and characterized the compartment-specific modulatory effects on intestinal microbial communities. A total of 20 piglets were randomly allotted to one of the following three groups: breastfed (BF), standard formula (SF), and MFGM-supplemented formula (EF). These diets were administered until weaning, with subsequent provision of commercial feed until euthanasia. Morphometric, microbial, and serum metabolomic analyses revealed that compared to piglets in the SF group, those in the EF group were characterized by significantly enhanced jejunal villus height (p < 0.05) and reduced cecal Oxalobacter (p < 0.05) and Pasteurella abundances, which were comparable to the levels detected in the BF group. Metabolically, piglets in the SF group demonstrated significantly lower levels of tyrosine, phenylalanine, and β-alanine (p < 0.05) and higher levels of 3-methyl-2-oxovalerate (p < 0.05) than those in BF piglets. In contrast, compared to the SF piglets, EF piglets exhibited significantly elevated levels of betaine (p < 0.05) and lysine. Spearman's correlation analysis revealed significant positive associations between Oxalobacter abundance and creatinine, dimethyl sulfone, phenylalanine, tyrosine, and β-alanine concentrations, with inverse correlations observed for 3-methyl-2-oxovalerate and lysine levels. In conclusion, these findings revealed that MFGM supplementation contributes to maintaining a normal intestinal architecture, modulates site-specific microbiota, and mitigates metabolic disparities between formula-fed and breastfed neonates. Notably, these effects are primarily mediated via choline pathway regulation and competitive inhibition of pathogenic bacteria.

Enterovirus D68 (EV-D68) is an emerging pathogen that causes severe respiratory infections in children worldwide and has the potential to re-outbreak. The aim of this study was to assess EV-D68 detection and genetic variability in pediatric patients with community-acquired pneumonia (CAP) during and after the COVID-19 pandemic in China. Respiratory samples, including bronchoalveolar lavage fluid (BALF), were collected from hospitalized children with CAP in Shanghai, Shandong, Fujian, Zhejiang and Jiangsu province between January 1, 2021 and December 31, 2024. Samples were screened for EV-D68 using real-time RT-PCR, and clinical characteristics of positive cases and virus strain sequences were analyzed. Of 2,842 cases, 0.7% (21/2,842) positive with EV-D68. The median age of these patients was 5.0 years (IQR: 3.6-8.0 years), with 42.9% (9/21) presented severe CAP. Of the 21 identified virus strains, 15 belonged to subclade B3 and 6 to subclade D3. In 2024, a total of 14 strains were detected, including 6 from the newly emerged subclade D3. Seven strains, 5 of which were subclade D3, were found in BALF samples from severe CAP patients. One child with severe CAP exhibited subclade D3 EV-D68 viremia. Notable mutations in the VP1 protein included H99D, K168E, and E169K. Our research showed that the EV-D68 continued to spread among children in eastern China before and after the COVID-19 pandemic. From 2021 to 2024, subclades B3 and D3 predominated, with increase detections in 2024 and association with pediatric severe CAP.

To investigate the epidemiological characteristics of pediatric respiratory pathogens in Guangzhou, China, from 2018 to 2023 in the context of the COVID-19 Pandemic and to evaluate the impact of non-pharmaceutical interventions (NPIs) on the transmission dynamics and seasonal patterns of respiratory pathogens. A retrospective analysis was conducted at Guangzhou Women and Children’s Medical Center between January 2018 and December 2023. Pediatric patients who underwent the respiratory pathogens tests were enrolled in the study and divided into four groups by age: Infant group, Toddler group, Preschool group, and School-age group. The nasopharyngeal swab or bronchoalveolar lavage fluid (BALF) samples were collected for real-time fluorescence quantitative polymerase chain reaction (qPCR) test of respiratory pathogens. Ten common respiratory pathogens, including respiratory syncytial virus (RSV), Mycoplasma pneumoniae (MP) and Influenza A virus (FluA), were detected. In addition, the study period was divided into three phases: Pre-COVID-19 (2018–2019), COVID-19 (2020–2022), and Post-COVID-19 (2023). Detection rates, distribution patterns, and seasonal variations of respiratory pathogens were analyzed between different phases and different age groups. This study included 317,828 pediatric patients (median age: 3.4 years, IQR: 1.3-6.0), from whom 1,160,764 respiratory pathogen tests were conducted. The overall pathogen detection rate was 8.02

Enterovirus D68 (EV-D68) is an emerging pathogen that has caused outbreaks of severe respiratory disease worldwide, especially in children. We aim to investigate the prevalence and genetic characteristics of EV-D68 in children from Shanghai. Nasopharyngeal swab or bronchoalveolar lavage fluid samples collected from children hospitalized with community-acquired pneumonia were screened for EV-D68. Nine of 3997 samples were EV-D68-positive. Seven of nine positive samples were sequenced and submitted to GenBank. Based on partial polyprotein gene (3D) or complete sequence analysis, we found the seven strains belong to different clades and subclades, including three D1 (detected in 2013 and 2014), one D2 (2013), one D3 (2019), and two B3 (2014 and 2018). Overall, we show different clades and subclades of EV-D68 spread with low positive rates (0.2%) among children in Shanghai between 2013 and 2020. Amino acid mutations were found in the epitopes of the VP1 BC and DE loops and C-terminus; similarity analysis provided evidence for recombination as an important mechanism of genomic diversification. Both single nucleotide mutations and recombination play a role in evolution of EV-D68. Genetic instability within these clinical strains may indicate large outbreaks could occur following cumulative mutations.

Enterovirus D68 (EV-D68) infection causes severe acute respiratory infection and severe neurological complications, such as acute flaccid myelitis (AFM), in children. However, although EV-D68 has pandemic potential, no effective drugs or vaccines are currently clinically available. Furthermore, EV-D68 infection-induced inflammatory response and cell death are not fully understood. In this study, we demonstrated that several inflammatory cytokines were upregulated in a multiplicity of infection (MOI) dependent manner in EV-D68-infected human rhabdomyosarcoma (RD) cells. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) confirmed that tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), C-C motif chemokine ligand-5 (CCL-5), and CXC motif chemokine ligand-5 (CXCL-5) mRNA levels were highly upregulated after EV-D68 infection. IL-1β processing and maturation mediated by caspase-8 was inhibited by the caspase-8 inhibitor Z-IETD-FMK. EV-D68 infection activates caspase-8 to mediate IL-1β maturation and secretion. Additionally, EV-D68 activated cell death-related proteins such as caspase-3, poly (ADP-ribose) polymerase 1 (PARP-1), phosphorylation of Mixed Lineage Kinase domain-like protein (pMLKL), and gasdermin E (GSDME). Thus, EV-D68 infection activates caspase-8, which triggers the necroptosis and apoptosis pathways. Overall, our data suggest that caspase-8 activation is associated with the inflammatory response and cell death in EV-D68-infected RD cells. This mechanism represents a novel target for the treatment of EV-D68 infection by inhibiting caspase-8 activation.

PURPOSE To investigate the clinical features of endogenous Klebsiella pneumoniae endophthalmitis (EKPE) and the molecular characteristics of pathogenic K. pneumoniae. METHODS A retrospective study was conducted between January 2014 and January 2021. Clinical data were extracted. K. pneumoniae isolates cultured from EKPE patients' specimens were characterized by antimicrobial susceptibility testing, multilocus sequence typing, capsular serotyping, and virulence gene profiles. Virulence phenotypes were confirmed by mouse lethality assay. RESULTS Thirty-nine patients (47 eyes) were diagnosed with EKPE. Administration of systemic antibiotics and eye treatment within 24 hours of ocular symptom onset was statistically associated with better visual outcomes (p = .007). Sixteen K. pneumoniae isolates were collected, three of which were carbapenem-resistant and avirulent. Only salmochelin encoding gene was harbored by all isolates. CONCLUSIONS Systemic and eye treatment administered within 24 hours from the onset improves visual prognosis. Besides hypervirulent strains, avirulent carbapenem-resistant K. pneumoniae can cause EKPE.

儿童不明原因肝炎是对儿科临床一直存在且病因和发病机制未明确的一类肝病的统称,常呈散发性.自 2022 年 3 月,英国、美国等 35 个国家在数月时间内发现 1000 多例不明原因儿童严重急性肝炎(acute severe hepatitis of unknown aetiology,ASHep-UA)病例,引起全球关注;虽近半年来发病率有恢复常态趋势,但对其病因仍有争议,其中感染性病因假说为多 [1-3].本文对ASHep-UA感染相关理论进行归纳,进而对儿童不明原因肝炎的病原学检测技术的临床运用进行梳理.

Proton pump inhibitors (PPIs) are commonly used in the clinical treatment of abnormal gastric acid secretion and gastric acid related diseases. There are disputes about blood purification and PPIs reuse in patients with PPIs-induced rhabdomyolysis. Herein we reported an 84-year-old woman with a 10-year history of coronary heart disease and gastric acid. After 18 days of omeprazole therapy, the blood myoglobin of the patient rose progressively. Laboratory examination confirmed rhabdomyolysis, and PPIs-induced rhabdomyolysis was considered. Atorvastatin was initially discontinued. Additionally, omeprazole was altered to iprazole. Since blood myoglobin continued to exceed the highest value identified, continuous renal replacement therapy (CRRT) and hemoperfusion (HP) were administrated. When PPIs-induced rhabdomyolysis was considered, iprazole was discontinued. Two days after discontinuation of iprazole, blood myoglobin continuously decreased. After rhabdomyolysis was resolved, omeprazole was reused, and rhabdomyolysis did not reoccur. PPIs in combination with statins increase the risk of rhabdomyolysis. In the present case, switching to another PPIs or CRRT and HP therapy did not alleviate rhabdomyolysis. Rhabdomyolysis caused by statins is countless, but other reasons cannot be overlooked. In any case, the removal of etiology is the primary component of the treatment of rhabdomyolysis. When rhabdomyolysis is alleviated, PPIs can be reused safely under close monitoring.

Gut Microbiota (GM) are microorganisms that live in the host gastrointestinal tract, and their abundance varies throughout the host's life. With the development of sequencing technology, the role of GM in various diseases has been increasingly elucidated. Unlike earlier studies on orthopedic diseases, this review elucidates the correlation between GM health and bone health and discusses the potential mechanism of GM effects on host metabolism, inflammation, and ability to induce or aggravate some common orthopedic diseases, such as osteoarthritis, osteoporosis, rheumatoid arthritis, etc. Finally, the prospective methods of GM manipulation and evaluation of potential GM-targeting strategies in the diagnosis and treatment of orthopedic diseases are reviewed.

Pathogens that cause pediatric severe community-acquired pneumonia (SCAP) requiring bronchoscopy intervention are understudied. Through this study, we explore the etiology of SCAP form alveolar lavage fluid (ALF) samples by the RespiFinder 2SMART multi-PCR assay.

Abstract Background There is little evidence about consistency between nasopharyngeal and pulmonary pathogens in children with severe pneumonia. This study aims to compare the difference of pathogens between nasopharyngeal aspirates (NPAs) collected before bronchoscopy and bronchoalveolar lavage fluids (BALFs) in children with severe community-acquired pneumonia (SCAP). Methods NPAs and BALFs were collected form pediatric SCAP cases hospitalized from January 2018 to March 2019. NPAs were colleced within 3 days before bronchoscopy. Samples were detected by direct immunofluorescence assay (DFA) for seven respiratory viruses and by routine bacterial culture in the clinical microbiology laboratory. Respiratory syncytial virus (RSV), Adenovirus (ADV), Influenza virus types A, B (IV-A and IV-B), Parainfluenza virus 1–3 (PIV1-3) were detected with a commercial assay. The virological and bacteriological detention results of NPAs were compared with the results of BALFs. Results In total 204 cases with mean age of 3.4 ± 2.8 years (IQR, 1 month-14 years) were included in the study. Both NPA and BALF were collected from those cases. The positive rates of pathogen in NPAs and BALFs were 25.0% (51/204) and 36.7% (75/204), respectively (x2 = 6.614, P = 0.010). Respiratory viruses were found in 16.1% (33/204) from NPAs and 32.3% (66/204) from BALFs (x2 = 14.524, P < 0.001). RSV and ADV were the two most frequent detected viruses in NPAs and BALFs. High consistentcy of pathogens between NPAs and BALFs was observed, and 96.9% (32/33) viruses detected in NPAs were also found in BALFs. While bacteria were isolated from 12.7% (26/204) and 10.7% (22/204) of the two kinds of samples, respectively (x2 = 0.378, P = 0.539). In addition, Haemophilus influenzae (HI) was the dominant germ in both samples. Conclusion The DFA method used to detect seven respiratory viruses from NPAs collected within 3 days before bronchoscopy can partially reflect the pathogens in the lungs in children with SCAP.

Background: We isolated the carbapenemase-producing Enterobacteriaceae (CPE) strains from children during 2016-2021 in Shanghai, China and investigated the antimicrobial resistance, molecular and epidemiological features of these isolates.Methods: Antimicrobial susceptibility tests were performed to confirm the carbapenem resis-tance. Carbapenemase production was assessed by the rapid phenotypic identification of five major carbapenemases (KPC, NDM, VIM, IMP, and OXA-48), which were further confirmed by PCR amplification and sequencing. Multilocus sequence typing (MLST) was conducted for phylo-genetic analyses. Results: A total of 320 CPE strains were collected from 2016 to 2021, consisting of carbapenemase-producing Klebsiella pneumoniae (CP-Kpn, 55.0%), Escherichia coli (CP -Eco, 24.5%) and Enterobacter cloacae (CP-Ecl, 20.4%) and others (2, 0.1%). NDM was the primary carbapenemase (67.6%) in children, followed by KPC(26.4%), IMP(5.3%) and OXA-48 (0.6%). The minimum inhibitory concentration (MIC) for imipenem has been increasing from 2016 to 2021. NDM and KPC isolates are high resistant while IMP strains show the lower resistant to imipe-nem. Invasive infection accounted for 10.7% of CPE-related infections and was mainly caused by CP-Kpn (70.6%). NDM-Kpn was detected in 51.8% of infants (70.8% of neonates), while KPC-Kpn was mainly isolated from non-infants (56.3%w64.3%). ST11 was the primary clone (64.6%) of KPC-Kpn and presented an increasing trend from 2016 to 2021.Conclusion: NDM is widely prevalent and transfers among CPE strains in children. NDM-Kpn shows the most serious threat to infants, especially to neonates. High-risk clone of ST11 KPC-Kpn should be paid more attention and monitored continuously in children. Copyright 2022, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Objective: To evaluate the diagnostic value of a high-throughput gene targeted amplicon sequencing (TAS) assay for detecting pathogenic microorganisms in alveolar lavage fluid (ALF) from children with severe community-acquired pneumonia (SCAP). Methods: A retrospective study was performed on 48 frozen ALF samples from 47 severe pneumonia cases admitted to Children's Hospital of Fudan University from January 1, 2019, to March 31, 2019. All samples were tested by a multiplex PCR (Multi-PCR) assay and a TAS assay. The results of the TAS panels were parallel compared with Multi-PCR and Conventional Tests (CT) including culture, direct fluorescent antibody method (DFA), and singleplex polymerase chain reaction (PCR). Results: The proportion of pathogens detection by CT was 81.2% (39/48). The 8 common respiratory viruses including respiratory syncytial virus (RSV), adenovirus (ADV), influenza A virus (FLUA), influenza B virus (FLUB), parainfluenza virus 1–3 (PIV1-3), and human Metapneumovirus (hMPV) were found in 31.2% (15/48) of the 48 samples by DFA. With the criteria of CT results used as “Golden Standard” for determing of TAS results, the proportion of pathogens detection by TAS was 70.8% (34/48). The difference of proportion of pathogens detection between TAS and CT was not statistically significant ( p = 0.232). The sensitivity and specificity of TAS for pathogens detection based on CT were 87.1% (95% CI, 71.77–95.18%) and 100.0% (95% CI, 62.88–100%), the positive predictive value (PPV) and negative predictive value (NPV) were 100.0% (95% CI, 87.35–100%) and 64.2% (95% CI, 35.62–86.02%), respectively. While Multi-PCR results were used as “Golden Standard,” the total pathogens detection rate of TAS was 83.3% (40/48), which had a significant difference with that of Multi-PCR ( p = 0.003). The sensitivity and PPV of TAS compared with Multi-PCR were 83.3% (95% CI, 69.23–92.03%) and 100.0% (95% CI, 89.08–100%), respectively. High rates of co-infection were proved by CT, Multi-PCR, and TAS. Mycoplasma pneumoniae (MP) and ADV were the two most frequently detected pathogens in all three assays. Conclusion: Compared with the CT and Multi-PCR methods, this TAS assay had a good performance in detecting bacteriological and viral pathogens from ALF. More research is needed to establish interpretation criteria based on TAS reads or analysis platforms.