Metagenomics for the microbiological diagnosis of hospital-acquired pneumonia and ventilator-associated pneumonia (HAP/VAP) in intensive care unit (ICU): a proof-of-concept study.

BACKGROUND: Hospital-acquired and ventilator-associated-pneumonia (HAP/VAP) are one of the most prevalent health-care associated infections in the intensive care unit (ICU). Culture-independent methods were therefore developed to provide faster route to diagnosis and treatment. Among these, metagenomic next-generation sequencing (mNGS) has shown considerable promise. METHODS: This proof-of-concept study describes the technical feasibility and evaluates the clinical validity of the mNGS for the detection and characterization of the etiologic agents causing hospital-acquired and ventilator-associated pneumonia. We performed a prospective study of all patients with HAP/VAP hospitalized in our intensive care unit for whom a bronchoalveolar lavage (BAL) was performed between July 2017 and November 2018. We compared BAL fluid culture and mNGS results of these patients. RESULTS: A total of 32 BAL fluids were fully analyzed. Of these, 22 (69%) were positive by culture and all pathogens identified were also reported by mNGS. Among the culture-positive BAL samples, additional bacterial species were revealed by mNGS for 12 patients, raising the issue of their pathogenic role (colonization versus coinfection). Among BALF with culture-negative test, 5 were positive in mNGS test. CONCLUSIONS: This study revealed concordant results for pneumonia panel pathogens between mNGS and culture-positive tests and identified additional pathogens potentially implicated in pneumonia without etiologic diagnosis by culture. mNGS has emerged as a promising methodology for infectious disease diagnoses to support conventional methods. Prospective studies with real-time mNGS are warranted to examine the impact on antimicrobial decision-making and clinical outcome.

Diagnostic utility of oropharyngeal swabs as an alternative to lower respiratory tract samples for PCR-based syndromic testing in patients with community-acquired pneumonia.

Syndromic PCR-based analysis of lower respiratory tract (LRT) samples in patients with community-acquired pneumonia (CAP) improves the bacterial yield and time-to-results compared to culture-based methods. However, obtaining adequate sputum samples can be challenging and is frequently not prioritized in the emergency department (ED). In this study, we assess the concordance of microbiological detections between oropharyngeal- (OP) and LRT samples from patients presenting to the ED with CAP using a syndromic PCR-based respiratory panel [Biofire FilmArray Pneumonia plus (FAP plus)]. Paired OP- and high-quality LRT samples were collected from 103 patients with confirmed CAP, who had been included in a randomized controlled trial (NCT04660084) or a subsequent observational study at Haukeland University Hospital, and analyzed using the FAP plus. The LRT samples were obtained mainly by sputum induction (88%). Using the LRT samples as a reference standard, the positive percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement for the most common bacterial pathogens in CAP, Streptococcus pneumoniae and Haemophilus influenzae, were 85%, 99% and 95%, and 86%, 98% and 93%, respectively. For Moraxella catarrhalis, the PPA was lower (74%), while the NPA was 100%. For bacteria that are less likely causes of uncomplicated CAP (e.g., Staphylococcus aureus and Enterobacterales) the results were more divergent. In conclusion, the FAP plus detects the most common CAP pathogens S. pneumoniae and H. influenzae from OP samples with high PPAs and excellent NPAs when compared with LRT samples. For these pathogens, the PPAs for OP samples were higher than previous reports for nasopharyngeal samples. This suggests that analysis of OP samples with syndromic PCR panels could represent an alternative approach for rapid microbiological testing in the ED, especially in patients where LRT samples are difficult to obtain. Divergent results for bacteria that are less likely to cause uncomplicated CAP do, however, emphasize the need for clinical evaluation of positive test results.

Retrospective Study of Lower Respiratory Tract Infections in the Intensive Care Unit Detected by the FilmArray Pneumonia Panel.

BACKGROUND: Lower respiratory tract infections (LRIs) are an important public health concern and a leading cause of death from infection worldwide. The current study aims to evaluate the distribution of viral and bacterial pathogens in lower respiratory tract specimens. METHODS: Between April 2022 and December 2022, specimens from lower respiratory tract from patients aged between 37 and 85 years in an intensive care unit (ICU) of Asia University Hospital were analysed by the FilmArrayTM pneumonia panel (PP) assay. RESULTS: There were 54 patients for whom the FilmArrayTM PP assay was analysed, and 25 (46.3%) of them showed positive results. Among the 54 specimens, 12 (22.2%, 12/54) had a single pathogen, 13 (24.1%, 13/54) had multiple pathogens, and 29 (53.7%, 29/54) had no pathogens. The overall positive rate of the specimens was 46.3% (25/54). CONCLUSIONS: The FilmArrayTM PP assay may act as a feasible diagnostic tool for LRIs in ICUs.

Evaluation of the usefulness of culture of induced sputum and the optimal timing for the collection of a good-quality sputum sample to identify causative pathogen of community-acquired pneumonia in young children: A prospective observational study.

BACKGROUND: The usefulness of an induced sputum in the identification of causative bacteria of community-acquired pneumonia (CAP) in young children is controversial. This study aimed to investigate the significance of the implementation of an induced sputum culture among children with CAP and the impact of prior use of antimicrobial agents on the quality of the sample and result of the culture. METHODS: This prospective study included 96 children hospitalized for acute bacterial CAP whose sputum samples were collected by suctioning from the hypopharynx through the nose. The samples were evaluated for their quality using Geckler classification, and the result of this conventional culture method was compared to that of a clone library analysis of the bacterial 16S rRNA gene sequence for each sample. RESULTS: The concordance between bacteria isolated by sputum culture and the most predominant bacteria identified by a clonal library analysis was significantly higher in the samples judged as a good quality (Geckler 5, 90%) than in others (70%). The rate of good-quality sputum sample was significantly higher in samples collected from patients without prior antimicrobial therapy (70%) than in those from patients with it (41%). The concordance between the two methods was significantly higher in the former (88%) than in the latter population (71%). CONCLUSION: Bacteria isolated by the culture using good-quality sputum samples collected from children with CAP were more likely to be causative pathogens. Sputum samples collected before starting antimicrobial therapy showed better quality and higher probability of the identification of causative pathogens.

Development of an anti-Pseudomonas aeruginosa therapeutic monoclonal antibody WVDC-5244.

The rise of antimicrobial-resistant bacterial infections is a crucial health concern in the 21st century. In particular, antibiotic-resistant Pseudomonas aeruginosa causes difficult-to-treat infections associated with high morbidity and mortality. Unfortunately, the number of effective therapeutic interventions against antimicrobial-resistant P. aeruginosa infections continues to decline. Therefore, discovery and development of alternative treatments are necessary. Here, we present pre-clinical efficacy studies on an anti-P. aeruginosa therapeutic monoclonal antibody. Using hybridoma technology, we generated a monoclonal antibody and characterized its binding to P. aeruginosa in vitro using ELISA and fluorescence correlation spectroscopy. We also characterized its function in vitro and in vivo against P. aeruginosa. The anti-P. aeruginosa antibody (WVDC-5244) bound P. aeruginosa clinical strains of various serotypes in vitro, even in the presence of alginate exopolysaccharide. In addition, WVDC-5244 induced opsonophagocytic killing of P. aeruginosa in vitro in J774.1 murine macrophage, and complement-mediated killing. In a mouse model of acute pneumonia, prophylactic administration of WVDC-5244 resulted in an improvement of clinical disease manifestations and reduction of P. aeruginosa burden in the respiratory tract compared to the control groups. This study provides promising pre-clinical efficacy data on a new monoclonal antibody with therapeutic potential for P. aeruginosa infections.

Natural Antibodies Mediate Protection Against Acinetobacter baumannii Respiratory Infections.

BACKGROUND: Acinetobacter baumannii causes a wide range of dangerous infections due to the emergence of pandrug-resistant strains. Therefore, there is a need for alternative therapeutics to treat these infections, including those targeting the host immune responses. However, immune responses, especially the humoral response against this pathogen, are poorly understood. METHODS: This study investigated the lymphocyte-mediated innate immune resistance to A. baumannii AB5075 pulmonary infection using B- and T-cell-deficient (Rag2-/-) mice, the protective effect of natural antibodies (NAbs), and the expression of complement-mediated responses using a mouse pneumonia model. RESULTS: Our results showed that intranasally infected Rag2-/- mice are impaired in clearing bacteria from lung, liver, and spleen at 24 hours postinfection compared to wildtype mice. Animal pretreatment with normal mouse serum or purified antibodies from naive mice rescued Rag2-/- mice from infection. Analysis of C3 complement protein binding demonstrated that NAbs increased C3 protein deposition on A. baumannii cells, indicating the activation of the classical complement pathway by NAbs. CONCLUSIONS: Overall, our study shows that NAbs mediate innate immune resistance against A. baumannii, a finding that may lead to the development of effective therapies against human infections caused by this antibiotic-resistant A. baumannii.

"Hot Tub Lung" With M. avium complex in an Immunocompetent Adolescent.

We describe a case of pulmonary Mycobacterium avium complex (MAC) infection in an immunocompetent pediatric patient after a hot tub near drowning event with a literature review of pediatric MAC-associated disease after hot tub exposure.

Pretreatment with 3-methyladenine ameliorated Pseudomonas aeruginosa-induced acute pneumonia by inhibiting cell death of neutrophils in a mouse infection model.

Pseudomonas aeruginosa is one of the leading causes of nosocomial infections worldwide. Clinical isolates that are resistant to multiple antimicrobials make it intractable. The interactions between P. aeruginosa and host cell death have multiple effects on bacterial clearance and inflammation; however, the potential intervention effects remain to be defined. Herein, we demonstrated that intravenous administration of 3-methyladenine before, but not after, P. aeruginosa infection enhanced autophagy-independent survival, which was accompanied by a decrease in the bacterial load, alleviation of pathology and reduction in inflammatory cytokines, in an acute pneumonia mouse model. Interestingly, these beneficial effects were not dependent on neutrophil recruitment or phagocytosis, but on the enhanced killing capacity induced by inhibiting the cell death of 3-MA pretreated neutrophils. These findings demonstrate a novel protective role of 3-MA pretreatment in P. aeruginosa-induced acute pneumonia.

Anti-inflammatory effect of chlorogenic acid in Klebsiella pneumoniae-induced pneumonia by inactivating the p38MAPK pathway.

INTRODUCTION: Pneumonia is an inflammation-related respiratory infection and chlorogenic acid (CGA) possesses a wide variety of bioactive properties, such as anti-inflammation and anti-bacteria. AIM: This study explored the anti-inflammatory mechanism of CGA in Klebsiella pneumoniae (Kp)-induced rats with severe pneumonia. METHODS: The pneumonia rat models were established by infection with Kp and treated with CGA. Survival rates, bacterial load, lung water content, and cell numbers in the bronchoalveolar lavage fluid were recorded, lung pathological changes were scored, and levels of inflammatory cytokines were determined by enzyme-linked immunosorbent assay. RLE6TN cells were infected with Kp and treated with CGA. The expression levels of microRNA (miR)-124-3p, p38, and mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) in lung tissues and RLE6TN cells were quantified by real-time quantitative polymerase chain reaction or Western blotting. The binding of miR-124-3p to p38 was validated by the dual-luciferase and RNA pull-down assays. In vitro, the functional rescue experiments were performed using miR-124-3p inhibitor or p38 agonist. RESULTS: Kp-induced pneumonia rats presented high mortality, increased lung inflammatory infiltration and the release of inflammatory cytokines, and enhanced bacterial load, while CGA treatment improved rat survival rates and the above situations. CGA increased miR-124-3p expression, and miR-124-3p inhibited p38 expression and inactivated the p38MAPK pathway. Inhibition of miR-124-3p or activation of the p38MAPK pathway reversed the alleviative effect of CGA on pneumonia in vitro. CONCLUSION: CGA upregulated miR-124-3p expression and inactivated the p38MAPK pathway to downregulate inflammatory levels, facilitating the recovery of Kp-induced pneumonia rats.

Enhanced anti-biofilm activity of the minocycline-and-gallium-nitrate using niosome wrapping against Acinetobacter baumannii in C57/BL6 mouse pneumonia model.

Acinetobacter baumannii is a worldwide health issue in terms of its high antibiotic resistance and ability to form biofilms. Nanoparticles (NPs) with high biocompatibility, high penetrating ability, and low medication dose can successfully treat the antibiotic-resistant infections. In this research, the anti-biofilm activity of niosomes containing minocycline and gallium nitrate (GaN) against A. baumannii biofilm was determined. In order to improve their anti-biofilm properties, minocycline and GaN were encapsulated in niosomes as biocompatible drug carriers. The niosomes' size, zeta potential, shape, stability, drug entrapment efficacy, drug release pattern and antibacterial activity were assessed. Several clinical samples were isolated from the lungs of patients hospitalized at Loghman hospital, Tehran, Iran. The biofilm formation of most lethal clinical isolates of A. baumannii was analyzed. The pneumonia model was generated by intranasally administering A. baumannii suspension to anesthetized mice whose immune systems was compromised twice by cyclophosphamide. Lung infection of the mouse with A. baumannii was confirmed using PCR. After treatment, the lungs were excised under sterile conditions and stained with hematoxylin and eosin (H&E) to determine histological symptoms, inflammation and intercellular secretions. The niosomes contained minocycline and GaN had an average size of 230 nm and a zeta potential of -40 mV, respectively. The percentage of drug entrapment and delayed drug release was both high in niosomal formulations. Niosomes containing minocycline and GaN dispersed 1, 3 and 5 day old biofilms. The mice given the combination of two compounds required less time to be treated than the animals given the single medication (minocycline). The minocycline& GaN-loaded niosomes could be considered as promising candidates to treat the infections caused by A. baumannii biofilm.

Epidemiological characteristics of community-acquired pneumonia and effects from the COVID-19 pandemic in Shenzhen of China.

OBJECTIVE: This study aimed to observe the impact of the coronavirus disease 2019 (COVID-19) pandemic on the incidence of non-COVID-19 community-acquired pneumonia (CAP) in Shenzhen of China, offering new ideas for evaluating the effects of non-pharmaceutical interventions. METHODS: A retrospective analysis was conducted of inpatients with pneumonia from 2017 to 2021. Epidemiological characteristics of CAP and effects from the COVID-19 pandemic were analyzed by the basic characteristics, time distribution, etiology and disease burden. RESULTS: There were a total of 5746 CAP inpatient cases included from 2017 to 2021. The number of CAP hospitalizations decreased during the pandemic from 2020 to 2021, with seasonal variations of being higher in spring and winter and lower in summer and autumn, whereas it was prevalent throughout the year prior to the pandemic. The children group decreased significantly during the pandemic, with a 15% decrease in the share of CAP inpatients. The detection rates of bacteria and mycoplasma decreased in CAP patients, while the detection rate of the virus increased, and the number of moderate and severe cases reduced more than that of the mild. CONCLUSION: Non-pharmaceutical interventions from COVID-19 have led to a decrease in the number of CAP inpatients, especially for children, with a specific seasonal prevalence in spring and winter, when the prevention interventions should be strengthened further for adults during the pandemic.

Sodium houttuyfonate effectively treats acute pulmonary infection of Pseudomonas aeruginosa by affecting immunity and intestinal flora in mice.

Introduction: Pseudomonas aeruginosa is a major nosocomial pathogen that frequently causes ventilator-associated pneumonia in specific populations. Sodium houttuyfonate (SH) has shown mild antibacterial activity against P. aeruginosa in vitro, but the mechanism of potent antimicrobial activity of SH against P. aeruginosa infection in vivo remains unclear. Methods: Here, using the mouse pneumonia model induced by P. aeruginosa nasal drip to explore the therapeutic effects of SH. Results: We found that SH exhibits dose-dependent therapeutic effects of reducing P. aeruginosa burden and systemic inflammation in pneumonia mice. SH ameliorates inflammatory gene expression and production of inflammatory proteins, such as interleukin-6 (IL-6), nuclear factor kappa-B (NF-κB) and toll-like receptor 4 (TLR4), associated with the TLR4/NF-κB pathway in mice with P. aeruginosa pneumonia. Furthermore, we analyzed the intestinal flora of mice and found that compared with the model group, the abundance and diversity of beneficial bacterial flora of SH treatment groups increased significantly, suggesting that SH can improve the intestinal flora disorder caused by inflammation. In addition, SH improves alpha and beta diversity index and reduces species abundance differences of intestinal flora in pneumonia mice. Discussion: Taken together, our presented results indicate that SH may effectively alleviate the acute pulmonary infection induced by P. aeruginosa by reducing the disturbance of regulating immunity and intestinal flora in mice.

Teeth record pneumonia-and racism.

Research raises standards for working with anthropological collections.

Novel care pathway to optimise antimicrobial prescribing for uncomplicated community-acquired pneumonia: study protocol for a prospective before-after cohort study in the emergency department of a tertiary care Canadian children's hospital.

INTRODUCTION: Evidence-based recommendations for paediatric community-acquired pneumonia (CAP) diagnosis and management are needed. Uncomplicated CAP is often caused by respiratory viruses, especially in younger children; these episodes self-resolve without antibiotic treatment. Unfortunately, there are no clinical criteria that reliably discriminate between viral and bacterial disease, and so the majority of children diagnosed with CAP are given antibiotics-even though these will often not help and may cause harm. We have developed a novel care pathway that incorporates point-of-care biomarkers, radiographic patterns, microbiological testing and targeted follow-up. The primary study objective is to determine if the care pathway will be associated with less antimicrobial prescribing. METHODS AND ANALYSIS: A prospective, before-after, study. Previously well children aged≥6 months presenting to a paediatric emergency department (ED) that have at least one respiratory symptom/sign, receive chest radiography, and are diagnosed with CAP by the ED physician will be eligible. Those with medical comorbidities, recently diagnosed pulmonary infection, or ongoing fever after≥4 days of antimicrobial therapy will be excluded. In the control (before) phase, eligible participants will be managed as per the standard of care. In the intervention (after) phase, eligible participants will be managed as per the novel care pathway. The primary outcome will be the proportion of participants in each phase who receive antimicrobial treatment for CAP. The secondary outcomes include: clinical cure; re-presentation to the ED; hospitalisation; time to resolution of symptoms; drug adverse events; caregiver satisfaction; child absenteeism from daycare/school; and caregiver absenteeism from work. ETHICS AND DISSEMINATION: All study documentation has been approved by the Hamilton Integrated Research Ethics Board and informed consent will be obtained from all participants. Data from this study will be presented at major conferences and published in peer-reviewed publications to facilitate collaborations with networks of clinicians experienced in the dissemination of clinical guidelines. TRIAL REGISTRATION NUMBER: NCT05114161.

Comparing the application of mNGS after combined pneumonia in hematologic patients receiving hematopoietic stem cell transplantation and chemotherapy: A retrospective analysis.

Rapid and accurate pathogen identification is essential for timely and effective treatment of pneumonia. Here, we describe the use of metagenomic next-generation sequencing (mNGS) of bronchoalveolar lavage (BALF) fluid to identify pathogens in patients with hematologic comorbid respiratory symptoms in a retrospective study with 84 patients. In the transplantation group, 8 cases (19.5%) and 47 cases (97.9%) were positive for BALF by conventional method detection and mNGS detection, respectively, and 6 cases (14.0%) and 41 cases (91.1%) in chemotherapy group, respectively. The detection rate of mNGS in both groups was significantly higher than that of conventional detection methods (all P<0.05). Pseudomonas aeruginosa and Streptococcus pneumoniae were the most common bacterial infections in the transplantation and chemotherapy groups, respectively. Aspergillus was the most common fungal infection in both groups. Human betaherpesvirus 5 (HHV-5), torque teno virus and human betaherpesvirus 7 (HHV-7) were the most common pathogen species in both groups. The most common type of infection in patients in the transplantation and chemotherapy groups was the mixed infection of bacteria-virus. Most patients in the transplantation group had mixed infections based on multiple viruses, with 42 cases of viral infections in the transplantation group and 30 cases of viral infections in the chemotherapy group, which were significantly higher in the transplantation group than in the chemotherapy group (χ2 = 5.766, P=0.016). and the mixed infection of virus-virus in the transplantation group was significantly higher than that in the chemotherapy group (27.1% vs 4.4%, P=0.003). The proportion of death due to pulmonary infection was significantly higher in the transplantation group than in the chemotherapy group (76.9% vs 16.7%, χ2 = 9.077, P=0.003). This study demonstrated the value of mNGS of BALF in improving the diagnosis and prognosis of hematologic comorbid pneumonia, helping patients to obtain timely and effective treatment, and giving guidance on the overall treatment plan for patients, with particular benefit for patients with hematologic chemotherapy comorbid pneumonia.

Metagenomic next-generation sequencing indicates more precise pathogens in patients with pulmonary infection: A retrospective study.

Background: Timely identification of causative pathogens is important for the diagnosis and treatment of pulmonary infections. Metagenomic next-generation sequencing (mNGS), a novel approach to pathogen detection, can directly sequence nucleic acids of specimens, providing a wide range of microbial profile. The purpose of this study was to evaluate the diagnostic performance of mNGS in the bronchoalveolar lavage fluid (BALF) of patients with suspected pulmonary infection. Methods: From April 2019 to September 2021, 502 patients with suspected pneumonia, who underwent both mNGS of BALF and conventional microbiological tests (CMTs), were classified into different groups based on comorbidities. The diagnostic performances of mNGS and CMTs were compared. Comprehensive clinical analysis was used as the reference standard. Results: The diagnostic accuracy and sensitivity of mNGS were 74.9% (95% confidence interval [CI], 71.7-78.7%) and 72.5% (95% CI, 68.2-76.8%) respectively, outperformed those of CMTs (36.9% diagnostic accuracy, 25.4% sensitivity). For most pathogens, the detection rate of mNGS was higher than that of CMTs. Polymicrobial infections most often occurred in immunocompromised patients (22.1%). Only 2.3% patients without underlying diseases developed polymicrobial infections. Additionally, the spectrums of pathogens also varied among the different groups. We found the positive predictive values (PPV) to be dependent upon both the pathogen of interest as well as the immunologic status of the patient (e.g., the PPV of Mycobacterium tuberculosis was 94.9% while the PPV of Pneumocystis jirovecii in immunocompetent individuals was 12.8%). This information can help physicians interpret mNGS results. Conclusion: mNGS of BALF can greatly enhance the accuracy and detection rate of pathogens in patients with pulmonary infections. Moreover, the comorbidities and types of pathogens should be taken consideration when interpreting the results of mNGS.

Clinical diagnosis and etiology of patients with Chlamydia psittaci pneumonia based on metagenomic next-generation sequencing.

The incidence of severe Chlamydia psittaci (C. psittaci) pneumonia and coinfections is increasing. Early detection of this condition is needed to prevent negative outcomes, along with detailed descriptions of its associated clinical characteristics. Our study contributes by undertaking etiological analysis of patients with C. psittaci pneumonia based on metagenomic next-generation sequencing (mNGS). A retrospective analysis of 30 patients with C. psittaci pneumonia was undertaken and confirmed by mNGS or polymerase chain reaction (PCR). Clinical manifestations of the severe and non-severe C. psittaci pneumonia groups were compared for clinical reference. Etiological analyses were also performed to comprehensively understand pathogeny and coinfection with other respiratory pathogens in C. psittaci patients. The absolute value of lymphocytes (LYM) in the severe group was lower than in the non-severe group. At the same time, neutrophil-to-lymphocyte ratio (NLR), procalcitonin (PCT), alanine aminotransferase (ALT), D-II polymer, brain natriuretic peptide (BNP), myoglobin (MYO), and cardiac troponin I (cTnI) were significantly higher (P < 0.05) in the severe group. mNGS has a broader pathogen spectrum and can more sensitively detect C. psittaci and other low-abundance pathogens with a higher positive detection rate (100%, 13/13 vs. 46%, 6/13, P <0.05) than conventional culture methods. mNGS detected the following dominant species associated with C. psittaci in patients: bacteria (53.2%, 39% gram-positive, 61% gram-negative), fungi (12.9%), and viruses (33.9%). A total of 73.3% (11/15) of patients had suspected coinfections, with a coinfection rate of 91.7% (11/12) in the severe group. No coinfection or death occurred in the non-severe group. Prognosis in the severe group was poor, with a mortality rate of 27.3% (3/11) for patients with coinfection. Eight of 11 patients with coinfections (72.7%) recovered. In conclusion, the clinical symptoms of severe C. psittaci pneumonia manifested as abnormal inflammatory indicators, impaired liver function, myocardial injury, coagulation, and relatively low immune responses. The higher proportion of patients with coinfections in our study supports the use of mNGS for comprehensive early detection of respiratory infections in patients with C. psittaci pneumonia. Simultaneous early identification of coinfections would further improve the clinical treatment of these patients.

GM-CSF+ Tc17 cells are required to bolster vaccine immunity against lethal fungal pneumonia without causing overt pathology.

GM-CSF co-expressing T17 cells instigate pathologic inflammation during autoimmune disorders, but their function in immunity to infections is unclear. Here, we demonstrate the role of GM-CSF+Tc17 cells for vaccine immunity against lethal fungal pneumonia and the cytokine requirements for their induction and memory homeostasis. Vaccine-induced GM-CSF+ Tc17 cells are necessary to bolster pulmonary fungal immunity without inflating pathology. Although GM-CSF expressing Tc17 cells preferentially elevate during the memory phase, their phenotypic attributes strongly suggest they are more like Tc17 cells than IFNγ-producing Tc1 cells. IL-1 and IL-23, but not GM-CSF, are necessary to elicit GM-CSF+ Tc17 cells following vaccination. IL-23 is dispensable for memory Tc17 and GM-CSF+ Tc17 cell maintenance, but recall responses of effector or memory Tc17 cells in the lung require it. Our study reveals the beneficial, nonpathological role of GM-CSF+ Tc17 cells during fungal vaccine immunity.

Application of metagenomic next-generation sequencing in the diagnosis of pulmonary invasive fungal disease.

Background: Metagenomic next-generation sequencing (mNGS) is increasingly being used to detect pathogens directly from clinical specimens. However, the optimal application of mNGS and subsequent result interpretation can be challenging. In addition, studies reporting the use of mNGS for the diagnosis of invasive fungal infections (IFIs) are rare. Objective: We critically evaluated the performance of mNGS in the diagnosis of pulmonary IFIs, by conducting a multicenter retrospective analysis. The methodological strengths of mNGS were recognized, and diagnostic cutoffs were determined. Methods: A total of 310 patients with suspected pulmonary IFIs were included in this study. Conventional microbiological tests (CMTs) and mNGS were performed in parallel on the same set of samples. Receiver operating characteristic (ROC) curves were used to evaluate the performance of the logarithm of reads per kilobase per million mapped reads [lg(RPKM)], and read counts were used to predict true-positive pathogens. Result: The majority of the selected patients (86.5%) were immunocompromised. Twenty species of fungi were detected by mNGS, which was more than was achieved with standard culture methods. Peripheral blood lymphocyte and monocyte counts, as well as serum albumin levels, were significantly negatively correlated with fungal infection. In contrast, C-reactive protein and procalcitonin levels showed a significant positive correlation with fungal infection. ROC curves showed that mNGS [and especially lg(RPKM)] was superior to CMTs in its diagnostic performance. The area under the ROC curve value obtained for lg(RPKM) in the bronchoalveolar lavage fluid of patients with suspected pulmonary IFIs, used to predict true-positive pathogens, was 0.967, and the cutoff value calculated from the Youden index was -5.44. Conclusions: In this study, we have evaluated the performance of mNGS-specific indicators that can identify pathogens in patients with IFIs more accurately and rapidly than CMTs, which will have important clinical implications.