Deciphering the microbial landscape of lower respiratory tract infections: insights from metagenomics and machine learning.

Background: Lower respiratory tract infections represent prevalent ailments. Nonetheless, current comprehension of the microbial ecosystems within the lower respiratory tract remains incomplete and necessitates further comprehensive assessment. Leveraging the advancements in metagenomic next-generation sequencing (mNGS) technology alongside the emergence of machine learning, it is now viable to compare the attributes of lower respiratory tract microbial communities among patients across diverse age groups, diseases, and infection types. Method: We collected bronchoalveolar lavage fluid samples from 138 patients diagnosed with lower respiratory tract infections and conducted mNGS to characterize the lung microbiota. Employing various machine learning algorithms, we investigated the correlation of key bacteria in patients with concurrent bronchiectasis and developed a predictive model for hospitalization duration based on these identified key bacteria. Result: We observed variations in microbial communities across different age groups, diseases, and infection types. In the elderly group, Pseudomonas aeruginosa exhibited the highest relative abundance, followed by Corynebacterium striatum and Acinetobacter baumannii. Methylobacterium and Prevotella emerged as the dominant genera at the genus level in the younger group, while Mycobacterium tuberculosis and Haemophilus influenzae were prevalent species. Within the bronchiectasis group, dominant bacteria included Pseudomonas aeruginosa, Haemophilus influenzae, and Klebsiella pneumoniae. Significant differences in the presence of Pseudomonas phage JBD93 were noted between the bronchiectasis group and the control group. In the group with concomitant fungal infections, the most abundant genera were Acinetobacter and Pseudomonas, with Acinetobacter baumannii and Pseudomonas aeruginosa as the predominant species. Notable differences were observed in the presence of Human gammaherpesvirus 4, Human betaherpesvirus 5, Candida albicans, Aspergillus oryzae, and Aspergillus fumigatus between the group with concomitant fungal infections and the bacterial group. Machine learning algorithms were utilized to select bacteria and clinical indicators associated with hospitalization duration, confirming the excellent performance of bacteria in predicting hospitalization time. Conclusion: Our study provided a comprehensive description of the microbial characteristics among patients with lower respiratory tract infections, offering insights from various perspectives. Additionally, we investigated the advanced predictive capability of microbial community features in determining the hospitalization duration of these patients.

Exploring the microbiota difference of bronchoalveolar lavage fluid between community-acquired pneumonia with or without COPD based on metagenomic sequencing: a retrospective study.

BACKGROUND: Community-acquired pneumonia (CAP) patients with chronic obstructive pulmonary disease (COPD) have higher disease severity and mortality compared to those without COPD. However, deep investigation into microbiome distribution of lower respiratory tract of CAP with or without COPD was unknown. METHODS: So we used metagenomic next generation sequencing (mNGS) to explore the microbiome differences between the two groups. RESULTS: Thirty-six CAP without COPD and 11 CAP with COPD cases were retrieved. Bronchoalveolar lavage fluid (BALF) was collected and analyzed using untargeted mNGS and bioinformatic analysis. mNGS revealed that CAP with COPD group was abundant with Streptococcus, Prevotella, Bordetella at genus level and Cutibacterium acnes, Rothia mucilaginosa, Bordetella genomosp. 6 at species level. While CAP without COPD group was abundant with Ralstonia, Prevotella, Streptococcus at genus level and Ralstonia pickettii, Rothia mucilaginosa, Prevotella melaninogenica at species level. Meanwhile, both alpha and beta microbiome diversity was similar between groups. Linear discriminant analysis found that pa-raburkholderia, corynebacterium tuberculostearicum and staphylococcus hominis were more enriched in CAP without COPD group while the abundance of streptococcus intermedius, streptococcus constellatus, streptococcus milleri, fusarium was higher in CAP with COPD group. CONCLUSIONS: These findings revealed that concomitant COPD have an mild impact on lower airway microbiome of CAP patients.

A Case of Invasive Pulmonary Aspergillosis.

BACKGROUND: Invasive pulmonary aspergillosis (IPA) is a deep fungal infection caused by invasion of Aspergillus mycelium into the lung parenchyma resulting in tissue destruction and necrosis, which occurs more often in im-munosuppressed populations. The severity of the disease and the rapid progression of the lung lesions puts pa¬tients at high risk of death and poor prognosis if the correct therapeutic intervention is not given as early as possible. METHODS: Here we report a case of IPA, which was initially diagnosed as community-acquired pneumonia in a local hospital. The symptoms did not improve after receiving anti-infective treatment. The patient was diagnosed with IPA after completing a chest CT examination and an electronic bronchoscopy, as well as pathogenetic examination of the bronchoalveolar lavage fluid and pathological examination of the left bronchial mass in the respiratory department of our hospital, which was finally diagnosed as IPA. After one week of administration of voriconazole for anti-fungal infection treatment, the patient's symptoms improved significantly, and a repeat chest CT suggested that the lung lesions were better than before. In order to raise clinicians' awareness of this disease, we also conducted a literature analysis. RESULTS: The final diagnosis of IPA was made by analyzing the patient's history, symptoms, signs, and relevant findings. CONCLUSIONS: When the patient's clinical symptoms and imaging manifestations are consistent with IPA, electronic bronchoscopy and pathogenetic and pathological examinations may be appropriately performed to clarify the na-ture of the lesion. More consideration should be given to the possibility of disease diagnosis to avoid misdiagnosis and underdiagnosis. Appropriate treatment should be given at an early stage.

Neglected Mycobacterium Avium Complex Infection in a Patient with Prolonged Pneumonia.

BACKGROUND: Non-tuberculous mycobacterial pulmonary infections (NTM-PD) are becoming increasingly common in clinical practice, and early detection and accurate determination of the infecting pathogen is crucial for subsequent treatment. We report a case of NTM-PD in a healthy middle-aged female with Mycobacterium tuberculosis complex group (MAC) infection confirmed by mNGS examination. METHODS: Appropriate laboratory tests, chest CT scan, bronchoscopic alveolar lavage fluid (BALF) examination, and macrogenomic next-generation sequencing (mNGS) were performed to establish the diagnosis. RESULTS: Chest CT showed multiple inflammatory lesions in the right middle lobe, and BALF sent for mNGS finally confirmed the diagnosis of MAC infection. After symptomatic treatment with azithromycin combined with ethambutol and rifampicin, the patient improved and was discharged from the hospital. CONCLUSIONS: In patients with pulmonary infections, pathogens should be clarified early to determine the diagnosis. mNGS of BALF samples have high specificity in detecting pathogens of infectious diseases, especially complex mixed infectious disease pathogens.

First report of solitary cavitation nodules in the lungs with Mycobacterium canariasense.

INTRODUCTION: Mycobacterium canariasense is a relatively rare and rapidly growing nontuberculous mycobacterium (NTM) infection. CASE REPORT: This case report describes a 36-year-old man with a Canariasense infection in the lung with solitary cavitation nodules located subpleural on CT scan, for which the final diagnosis was made by metagenomic next-generation sequencing (mNGS) of bronchoalveolar lavage fluid (BALF-mNGS). It was successfully treated with levofloxacin and amikacin. CONCLUSIONS: This experience is instructive because clinical diagnostic and CT imaging characteristics and treatment strategy guidelines for pulmonary infections caused by M. canariasense have not yet been established.

Acute Q fever pneumonia diagnosed by metagenomic next-generation sequencing.

INTRODUCTION: Q fever, a zoonotic disease caused by Coxiella burnetii (C. burnetii), presents diagnostic challenges due to its clinical and radiological nonspecificity, which often mimics community-acquired pneumonia, coupled with the limitations of traditional diagnostic methods. Metagenomic next-generation sequencing (mNGS) has become an indispensable tool in clinical diagnostics for its high-throughput pathogen identification capabilities. Herein, we detail a case of acute Q fever pneumonia diagnosed with mNGS. CASE PRESENTATION: The patient exhibited symptoms of fever, cough, expectoration, and diarrhea for three days, with the pathogen undetected in initial laboratory assessments. Bronchoscopy and bronchoalveolar lavage (BAL) were conducted, leading to the identification of C. burnetii in the lavage fluid via mNGS. Consequently, the patient was promptly initiated on a treatment regimen of 100 mg doxycycline, administered orally every 12 hours. RESULTS: Post-treatment, the patient's temperature normalized, and a full recovery was observed. The follow-up chest CT scan revealed complete resolution of the right lower lobe consolidation. CONCLUSIONS: The clinical presentation of Q fever pneumonia lacks specificity, making diagnosis based solely on symptoms and imaging challenging. mNGS offers a superior alternative for identifying elusive or rarely cultured pathogens.

Exploratory Research for New Diagnostic Markers of Mucormycosis.

Mucormycosis is a fungal infectious disease caused by Rhizopus oryzae and other members of the order Mucorales, and it is known as one of the most lethal fungal infections. Early diagnosis of mucormycosis improves prognosis because of limited effective treatments and the rapid progression of the disease. On the other hand, the lack of characteristic clinical findings in mucormycosis and the challenge of early definitive diagnosis make early treatment difficult. Our goal was to establish a serodiagnostic method to detect Rhizopus specific antigen (RSA), and we have developed a diagnostic kit by Enzyme-linked immuno-sorbent assay (ELISA) using a monoclonal antibody against this antigen. RSA increased over time in the serum and alveolar lavage fluid of R. oryzae-infected mice. RSA was also detected in serum and alveolar fluid, even at an early stage (Day 1), when the tissue invasion of R. oryzae mycelium was not histopathologically detectable in the lungs of R. oryzae-infected mice. Further evaluation is needed to determine the feasibility of using this assay in clinical practice.

Associations of cold-inducible RNA-binding protein with bacterial load, proinflammatory cytokines and mortality from pneumonia.

Cold-inducible RNA-binding protein (CIRP) is a damage-associated molecular pattern that plays a critical role in triggering inflammatory responses. It remains unknown whether CIRP is strongly associated with bacterial load, inflammatory response, and mortality in sepsis model. Pneumonia was induced in specific pathogen-free 8-9-week old male rats by injecting bacteria via puncture of the tracheal cartilage. The expressions of CIRP and proinflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1ß] in lung tissues, alveolar macrophages (AMs), plasma, and bronchoalveolar lavage fluid (BALF) were determined by reverse transcription-polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. The numbers of bacteria recovered from the lungs were correlated with the bacterial loads injected and mortality. The expressions of CIRP increased sharply as the bacterial loads increased in the lung tissues and AMs. The amounts of TNF-α, IL-6 and IL-1ß proteins synthesized were dependent on the bacterial load in the lung tissues. Releases of CIRP, TNF-α, IL-6, and IL-1ß increased with the bacterial load in the blood plasma. The proteins confirmed similar patterns in the BALF. CIRP was strongly associated with the releases of TNF-α, IL-6, and IL-1ß in the lung tissues, blood plasma, and BALF, and showed a close correlation with mortality. CIRP demonstrated a strong association with bacterial load, which is new evidence, and close correlations with proinflammatory cytokines and mortality of pneumonia in rats, suggesting that it might be an interesting pneumonic biomarker for monitoring host response and predicting mortality, and a promising target for immunotherapy.

Universal digital high-resolution melting for the detection of pulmonary mold infections.

Invasive mold infections (IMIs) are associated with high morbidity, particularly in immunocompromised patients, with mortality rates between 40% and 80%. Early initiation of appropriate antifungal therapy can substantially improve outcomes, yet early diagnosis remains difficult to establish and often requires multidisciplinary teams evaluating clinical and radiological findings plus supportive mycological findings. Universal digital high-resolution melting (U-dHRM) analysis may enable rapid and robust diagnoses of IMI. A universal fungal assay was developed for U-dHRM and used to generate a database of melt curve signatures for 19 clinically relevant fungal pathogens. A machine learning algorithm (ML) was trained to automatically classify these pathogen curves and detect novel melt curves. Performance was assessed on 73 clinical bronchoalveolar lavage samples from patients suspected of IMI. Novel curves were identified by micropipetting U-dHRM reactions and Sanger sequencing amplicons. U-dHRM achieved 97% overall fungal organism identification accuracy and a turnaround time of ~4 hrs. U-dHRM detected pathogenic molds (Aspergillus, Mucorales, Lomentospora, and Fusarium) in 73% of 30 samples classified as IMI, including mixed infections. Specificity was optimized by requiring the number of pathogenic mold curves detected in a sample to be >8 and a sample volume to be 1 mL, which resulted in 100% specificity in 21 at-risk patients without IMI. U-dHRM showed promise as a separate or combination diagnostic approach to standard mycological tests. U-dHRM's speed, ability to simultaneously identify and quantify clinically relevant mold pathogens in polymicrobial samples, and detect emerging opportunistic pathogens may aid treatment decisions, improving patient outcomes. IMPORTANCE: Improvements in diagnostics for invasive mold infections are urgently needed. This work presents a new molecular detection approach that addresses technical and workflow challenges to provide fast pathogen detection, identification, and quantification that could inform treatment to improve patient outcomes.

Comparison of different lateral flow assays on bronchoalveolar lavage fluid for invasive aspergillosis screening in non-hematological patients.

Several lateral flow assays (LFA) capable of detecting Aspergillus fumigatus in serum and broncho-alveolar lavage fluid (BALF) within the hour, thereby potentially accelerating the screening process, are now commercially available. We prospectively compared three LFA targeting A. fumigatus on BALF collected from non-surgical intensive care patients between June 2022 and February 2023. The three LFA tested were Sõna Aspergillus galactomannan LFA (Immy), Fungadia Aspergillus antigen (Gadia), and AspLFD (OLM Diagnostics). We compared the results of these LFA with those of the galactomannan (GM) Platelia Aspergillus enzyme immunoassay (Bio-Rad), culture on Sabouraud medium and Aspergillus qPCR. We tested 97 BALF samples from 92 patients. In total 84 BALF samples tested negative with all three LFA, and four BALF samples tested positive with the AspLFD assay only (OLM). Only one BALF sample tested positive with the three LFA. In addition, three BALF samples tested positive only with the GM Platelia immunoassay. Four diagnosis of probable invasive aspergillosis were retained for the 92 patients tested. This prospective series included very few positive samples. From a practical point of view, the LFA from OLM presented the simplest protocol for use.

Tropheryma whipplei detected by metagenomic next-generation sequencing in bronchoalveolar lavage fluid.

Whipple's disease is a chronic systemic infectious disease that mainly affects the gastrointestinal tract. In some cases, Tropheryma whipplei can cause infection at the implant site or even throughout the body. In this study, we collected alveolar lavage fluid samples from patients with Tropheryma whipplei from 2020 to 2022, and retrospectively analyzed the clinical data of Tropheryma whipplei positive patients. Patient's past history, clinical manifestations, laboratory examinations, chest CT findings, treatment, and prognosis were recorded. 16 BALFs (70/1725, 4.0 %) from 16 patients were positive for Tropheryma whipplei. 8 patients were male with an average age of 50 years. The main clinical symptoms of patients included fever (9/16), cough (7/16), dyspnea (7/16), and expectoration (5/16), but neurological symptoms and arthralgia were rare. Cardiovascular and cerebrovascular diseases were the most common comorbidity (n=8). The main laboratory characteristics of the patient are red blood cell count, hemoglobin, total protein and albumin below normal levels (11/16), and/or creatinine above normal levels(14/16). Most chest computed tomography mainly show focal or patchy heterogeneous infection (n=5) and pleural effusion (n=8). Among the 6 samples, Tropheryma whipplei was the sole agent, and Klebsiella pneumoniae was the most common detected other pathogens. Metagenomic next-generation sequencing technology has improved the detection rate and attention of Tropheryma whipplei. Further research is needed to distinguish whether Tropheryma whipplei present in respiratory samples is a pathogen or an innocent bystander.

Comparison of the upper and lower airway microbiome in early postoperative lung transplant recipients.

The upper and lower respiratory tract may share microbiome because they are directly continuous, and the nasal microbiome contributes partially to the composition of the lung microbiome. But little is known about the upper and lower airway microbiome of early postoperative lung transplant recipients (LTRs). Using 16S rRNA gene sequencing, we compared paired nasal swab (NS) and bronchoalveolar lavage fluid (BALF) microbiome from 17 early postoperative LTRs. The microbiome between the two compartments were significantly different in Shannon diversity and beta diversity. Four and eight core NS-associated and BALF-associated microbiome were identified, respectively. NS samples harbored more Corynebacterium, Acinetobacter, and Pseudomonas, while BALF contained more Ralstonia, Stenotrophomonas, Enterococcus, and Pedobacter. The within-subject dissimilarity was higher than the between-subject dissimilarity, indicating a greater impact of sampling sites than sampling individuals on microbial difference. There were both difference and homogeneity between NS and BALF microbiome in early postoperative LTRs. High levels of pathogens were detected in both samples, suggesting that both of them can reflect the diseases characteristics of transplanted lung. The differences between upper and lower airway microbiome mainly come from sampling sites instead of sampling individuals. IMPORTANCE: Lung transplantation is the only therapeutic option for patients with end-stage lung disease, but its outcome is much worse than other solid organ transplants. Little is known about the NS and BALF microbiome of early postoperative LTRs. Here, we compared paired samples of the nasal and lung microbiome from 17 early postoperative LTRs and showed both difference and homogeneity between the two samples. Most of the "core" microbiome in both NS and BALF samples were recognized respiratory pathogens, suggesting that both samples can reflect the diseases characteristics of transplanted lung. We also found that the differences between upper and lower airway microbiome in early postoperative LTRs mainly come from sampling sites instead of sampling individuals.

Lipid A modification of colistin-resistant Klebsiella pneumoniae does not alter innate immune response in a mouse model of pneumonia.

Polymyxin resistance in carbapenem-resistant Klebsiella pneumoniae bacteria is associated with high morbidity and mortality in vulnerable populations throughout the world. Ineffective antimicrobial activity by these last resort therapeutics can occur by transfer of mcr-1, a plasmid-mediated resistance gene, causing modification of the lipid A portion of lipopolysaccharide (LPS) and disruption of the interactions between polymyxins and lipid A. Whether this modification alters the innate host immune response or carries a high fitness cost in the bacteria is not well established. To investigate this, we studied infection with K. pneumoniae (KP) ATCC 13883 harboring either the mcr-1 plasmid (pmcr-1) or the vector control (pBCSK) ATCC 13883. Bacterial fitness characteristics of mcr-1 acquisition were evaluated. Differentiated human monocytes (THP-1s) were stimulated with KP bacterial strains or purified LPS from both parent isolates and isolates harboring mcr-1. Cell culture supernatants were analyzed for cytokine production. A bacterial pneumonia model in WT C57/BL6J mice was used to monitor immune cell recruitment, cytokine induction, and bacterial clearance in the bronchoalveolar lavage fluid (BALF). Isolates harboring mcr-1 had increased colistin MIC compared to the parent isolates but did not alter bacterial fitness. Few differences in cytokines were observed with purified LPS from mcr-1 expressing bacteria in vitro. However, in a mouse pneumonia model, no bacterial clearance defect was observed between pmcr-1-harboring KP and parent isolates. Consistently, no differences in cytokine production or immune cell recruitment in the BALF were observed, suggesting that other mechanisms outweigh the effect of these lipid A mutations in LPS.

The impact of Sangju Qingjie Decoction on the pulmonary microbiota in the prevention and treatment of chronic obstructive pulmonary disease.

Objective: Exploring the effect of SJQJD on the pulmonary microbiota of chronic obstructive pulmonary disease (COPD) rats through 16S ribosomal RNA (rRNA) sequencing. Methods: A COPD rat model was constructed through smoking and lipopolysaccharide (LPS) stimulation, and the efficacy of SJQJD was evaluated by hematoxylin and eosin (H&E) staining and Enzyme-Linked Immunosorbnent Assay (ELISA). The alveolar lavage fluid of rats was subjected to 16S rRNA sequencing. The diversity of lung microbiota composition and community structure was analyzed and differential microbiota were screened. Additionally, machine learning algorithms were used for screening biomarkers of each group of the microbiota. Results: SJQJD could improve lung structure and inflammatory response in COPD rats. 16s rRNA sequencing analysis showed that SJQJD could significantly improve the abundance and diversity of bacterial communities in COPD rats. Through differential analysis and machine learning methods, potential microbial biomarkers were identified as Mycoplasmataceae, Bacillaceae, and Lachnospiraceae. Conclusion: SJQJD could improve tissue morphology and local inflammatory response in COPD rats, and its effect may be related to improve pulmonary microbiota.

Implications of deduplication on the detection rates of multidrug-resistant organism (MDRO) in various specimens: insights from the hospital infection surveillance program.

BACKGROUND: Currently, different guidelines recommend using different methods to determine whether deduplication is necessary when determining the detection rates of multidrug-resistant organisms (MDROs). However, few studies have investigated the effect of deduplication on MDRO monitoring data. In this study, we aimed to investigate the influence of deduplication on the detection rates of MDROs in different specimens to assess its impact on infection surveillance outcomes. METHODS: Samples were collected from hospitalized patients admitted between January 2022 and December 2022; four types of specimens were collected from key monitored MDROs, including sputum samples, urine samples, blood samples, and bronchoalveolar lavage fluid (BALF) samples. In this study, we compared and analysed the detection rates of carbapenem-resistant Klebsiella pneumoniae (CRKP), carbapenem-resistant Escherichia coli (CRECO), carbapenem-resistant Acinetobacter baumannii (CRAB), carbapenem-resistant Pseudomonas aeruginosa (CRPA), and methicillin-resistant Staphylococcus aureus (MRSA) under two conditions: with and without deduplication. RESULTS: When all specimens were included, the detection rates of CRKP, CRAB, CRPA, and MRSA without deduplication (33.52%, 77.24%, 44.56%, and 56.58%, respectively) were significantly greater than those with deduplication (24.78%, 66.25%, 36.24%, and 50.83%, respectively) (all P < 0.05). The detection rates in sputum samples were significantly different between samples without duplication (28.39%, 76.19%, 46.95%, and 70.43%) and those with deduplication (19.99%, 63.00%, 38.05%, and 64.50%) (all P < 0.05). When deduplication was not performed, the rate of detection of CRKP in urine samples reached 30.05%, surpassing the rate observed with deduplication (21.56%) (P < 0.05). In BALF specimens, the detection rates of CRKP and CRPA without deduplication (39.78% and 53.23%, respectively) were greater than those with deduplication (31.62% and 42.20%, respectively) (P < 0.05). In blood samples, deduplication did not have a significant impact on the detection rates of MDROs. CONCLUSION: Deduplication had a significant effect on the detection rates of MDROs in sputum, urine, and BALF samples. Based on these data, we call for the Infection Prevention and Control Organization to align its analysis rules with those of the Bacterial Resistance Surveillance Organization when monitoring MDRO detection rates.

The diagnostic value of metagenomics next-generation sequencing in HIV-infected patients with suspected pulmonary infections.

Background: Traditional microbiological detection methods used to detect pulmonary infections in people living with HIV (PLHIV) are usually time-consuming and have low sensitivity, leading to delayed treatment. We aimed to evaluate the diagnostic value of metagenomics next-generation sequencing (mNGS) for microbial diagnosis of suspected pulmonary infections in PLHIV. Methods: We retrospectively analyzed PLHIV who were hospitalized due to suspected pulmonary infections at the sixth people hospital of Zhengzhou from November 1, 2021 to June 30, 2022. Bronchoalveolar lavage fluid (BALF) samples of PLHIV were collected and subjected to routine microbiological examination and mNGS detection. The diagnostic performance of the two methods was compared to evaluate the diagnostic value of mNGS for unknown pathogens. Results: This study included a total of 36 PLHIV with suspected pulmonary infections, of which 31 were male. The reporting period of mNGS is significantly shorter than that of CMTs. The mNGS positive rate of BALF samples in PLHIV was 83.33%, which was significantly higher than that of smear and culture (44.4%, P<0.001). In addition, 11 patients showed consistent results between the two methods. Futhermore, mNGS showed excellent performance in identifying multi-infections in PLHIV, and 27 pathogens were detected in the BALF of 30 PLHIV by mNGS, among which 15 PLHIV were found to have multiple microbial infections (at least 3 pathogens). Pneumocystis jirovecii, human herpesvirus type 5, and human herpesvirus type 4 were the most common pathogen types. Conclusions: For PLHIV with suspected pulmonary infections, mNGS is capable of rapidly and accurately identifying the pathogen causing the pulmonary infection, which contributes to implement timely and accurate anti-infective treatment.

Description of a Murine Model of Pneumocystis Pneumonia.

Pneumocystis pneumonia is a serious lung infection caused by an original ubiquitous fungus with opportunistic behavior, referred to as Pneumocystis jirovecii. P. jirovecii is the second most common fungal agent among invasive fungal infections after Candida spp. Unfortunately, there is still an inability to culture P. jirovecii in vitro, and so a great impairment to improve knowledge on the pathogenesis of Pneumocystis pneumonia. In this context, animal models have a high value to address complex interplay between Pneumocystis and the components of the host immune system. Here, we propose a protocol for a murine model of Pneumocystis pneumonia. Animals become susceptible to Pneumocystis by acquiring an immunocompromised status induced by iterative administration of steroids within drinking water. Thereafter, the experimental infection is completed by an intranasal challenge with homogenates of mouse lungs containing Pneumocystis murina. The onset of clinical signs occurs within 5 weeks following the infectious challenge and immunosuppression can then be withdrawn. At termination, lungs and bronchoalveolar lavage (BAL) fluids from infected mice are analyzed for fungal load (qPCR) and immune response (flow cytometry and biochemical assays). The model is a useful tool in studies focusing on immune responses initiated after the establishment of Pneumocystis pneumonia.

Throat microbiota drives alterations in pulmonary alveolar microbiota in patients with septic ARDS.

OBJECTIVES: The translocation of intestinal flora has been linked to the colonization of diverse and heavy lower respiratory flora in patients with septic ARDS, and is considered a critical prognostic factor for patients. METHODS: On the first and third days of ICU admission, BALF, throat swab, and anal swab were collected, resulting in a total of 288 samples. These samples were analyzed using 16S rRNA analysis and the traceability analysis of new generation technology. RESULTS: On the first day, among the top five microbiota species in abundance, four species were found to be identical in BALF and throat samples. Similarly, on the third day, three microbiota species were found to be identical in abundance in both BALF and throat samples. On the first day, 85.16% of microorganisms originated from the throat, 5.79% from the intestines, and 9.05% were unknown. On the third day, 83.52% of microorganisms came from the throat, 4.67% from the intestines, and 11.81% were unknown. Additionally, when regrouping the 46 patients, the results revealed a significant predominance of throat microorganisms in BALF on both the first and third day. Furthermore, as the disease progressed, the proportion of intestinal flora in BALF increased in patients with enterogenic ARDS. CONCLUSIONS: In patients with septic ARDS, the main source of lung microbiota is primarily from the throat. Furthermore, the dynamic trend of the microbiota on the first and third day is essentially consistent.It is important to note that the origin of the intestinal flora does not exclude the possibility of its origin from the throat.

Aspergillus hubkae, a Novel Species Isolated from a Patient with Probable Invasive Pulmonary Aspergillosis.

A 50-year-old man, previously diagnosed with pulmonary tuberculosis and lung cavities, presented with symptoms including fever, shortness of breath, and cough. A pulmonary CT scan revealed multiple cavities, consolidation and tree-in-bud in the upper lungs. Further investigation through direct examination of bronchoalveolar lavage fluid showed septate hyphae with dichotomous acute branching. Subsequent isolation and morphological analysis identified the fungus as belonging to Aspergillus section Nigri. The patient was diagnosed with probable invasive pulmonary aspergillosis and successfully treated with a three-month oral voriconazole therapy. Phylogenetic analysis based on partial ß-tubulin, calmodulin and RNA polymerase second largest subunit sequences revealed that the isolate represents a putative new species related to Aspergillus brasiliensis, and is named Aspergillus hubkae here. Antifungal susceptibility testing demonstrated that the isolate is resistant to itraconazole but susceptible to voriconazole. This phenotypic and genetic characterization of A. hubkae, along with the associated case report, will serve as a valuable resource for future diagnoses of infections caused by this species. It will also contribute to more precise and effective patient management strategies in similar clinical scenarios.