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.

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The onset of clinical signs occurs within 5 weeks following the infectious challenge and immunosuppression can then be withdrawn.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.

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.

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.

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.

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.

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.

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.

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.

Human nasal microbiota shifts in healthy and chronic respiratory disease conditions.

BACKGROUND: An increasing number of studies investigate various human microbiotas and their roles in the development of diseases, maintenance of health states, and balanced signaling towards the brain. Current data demonstrate that the nasal microbiota contains a unique and highly variable array of commensal bacteria and opportunistic pathogens. However, we need to understand how to harness current knowledge, enrich nasal microbiota with beneficial microorganisms, and prevent pathogenic developments. RESULTS: In this study, we have obtained nasal, nasopharyngeal, and bronchoalveolar lavage fluid samples from healthy volunteers and patients suffering from chronic respiratory tract diseases for full-length 16 S rRNA sequencing analysis using Oxford Nanopore Technologies. Demographic and clinical data were collected simultaneously. The microbiome analysis of 97 people from Lithuania suffering from chronic inflammatory respiratory tract disease and healthy volunteers revealed that the human nasal microbiome represents the microbiome of the upper airways well. CONCLUSIONS: The nasal microbiota of patients was enriched with opportunistic pathogens, which could be used as indicators of respiratory tract conditions. In addition, we observed that a healthy human nasal microbiome contained several plant- and bee-associated species, suggesting the possibility of enriching human nasal microbiota via such exposures when needed. These candidate probiotics should be investigated for their modulating effects on airway and lung epithelia, immunogenic properties, neurotransmitter content, and roles in maintaining respiratory health and nose-brain interrelationships.

[Metagenomic next-generation sequencing-based retrospective investigation of the drug resistance sites of Mycoplasma pneumoniae in children].

Objective: To analyze the drug-resistant gene loci of Mycoplasma pneumoniae (MP) using metagenomic next-generation sequencing (mNGS). Methods: From November 2022 to October 2023, 697 clinical samples (including sputum, alveolar lavage fluid and blood) of 686 children with Mycoplasma pneumoniae positive detected by mNGS were retrospectively analyzed. Samples were divided into intensive care unit (ICU) group and non-ICU group, Chi-square test was used to compare groups, and Mann-Kendall trend test was used to analyze the change trend of the detection rate of drug resistance gene loci over time. Results: Of the 697 samples, 164 were from the ICU group and 533 were from the non-ICU group. The detection rate of Mycoplasma pneumoniae resistance gene was 44.3% (309/697), and all detected drug-resistant gene loci of MP were A2063G. The detection rate of Mycoplasma pneumoniae in ICU group was 50.0% (82/164), and the detection rates of Mycoplasma pneumoniae resistance gene loci in sputum, alveolus lavage fluid and blood samples were 75.0% (18/24) and 48.4% (62/128), respectively. The detection rate in sputum was higher than alveolus lavage fluid samples (χ2=5.72,P=0.017). The detection rate of Mycoplasma pneumoniae in non-ICU group was 42.6% (227/533), the detection rate of Mycoplasma pneumoniae resistance gene loci in sputum and alveolar lavage fluid was 40.0% (16/40), 44.3% (201/454), and no detection rate in blood samples (0/12). There was no significant difference in the detection rate of alveolar lavage fluid and sputum (χ2=0.27, P=0.602). From November 2022 to October 2023, the detection rate of submitted samples showed an increasing trend month by month (overall: Z=3.99, ICU inspection group: Z=2.93, non-ICU group: Z=3.01, all P<0.01). Among the bacteria commonly detected with Mycoplasma pneumoniae, Streptococcus pneumoniae accounted for the highest proportion, the detection rate was 15.5% (108/697), and Epstein-Barr virus accounted for the highest proportion of 17.6% (123/697). Conclusions: From November 2022 to October 2023, the detection rate of Mycoplasma pneumoniae drug resistance gene loci showed an increasing trend. The detection rate of drug resistance gene loci in sputum samples of ICU group was higher than alveolus lavage fluid. No new drug resistance site were detected.

Successful management of Mycobacterium abscessus pneumonia in a 53-day-old immunocompetent infant.

Pulmonary infection due to Mycobacterium abscessus complex (MABC) usually occurs in children with underlying risk factors including cystic fibrosis (CF), chronic lung disease, and immunocompromised status, but rarely in immunocompetent children without underlying lung disease, especially in infants. We present a case of MABC pulmonary disease (MABC-PD) in an otherwise healthy 53-day-old male infant with one week of cough and respiratory distress. Computed tomography showed multiple masses across both lungs. Isolated mycobacteria from his bronchoalveolar lavage fluid were identified as MABC. We describe our complete evaluation, including immunodeficiency evaluation incorporating whole exome sequencing and our therapeutic process given complicated susceptibility pattern of the M. abscessus isolate, and review literature for MABC-PD in immunocompetent children. The infant was successfully treated through prolonged treatment with parenteral Amikacin, Cefoxitin, Linezolid, and Clarithromycin, combined with inhaled Amikacin.

The clinical value of mNGS of bronchoalveolar lavage fluid versus traditional microbiological tests for pathogen identification and prognosis of severe pneumonia (NT-BALF):study protocol for a prospective multi-center randomized clinical trial.

BACKGROUND: Early, rapid, and accurate pathogen diagnosis can help clinicians select targeted treatment options, thus improving prognosis and reducing mortality rates of severe pneumonia. Metagenomic next-generation sequencing (mNGS) has a higher sensitivity and broader pathogen spectrum than traditional microbiological tests. However, the effects of mNGS-based antimicrobial treatment procedures on clinical outcomes and cost-effectiveness in patients with severe pneumonia have not been evaluated. METHODS: This is a regional, multi-center, open, prospective, randomized controlled trial to evaluate that whether the combination of mNGS and traditional testing methods could decrease 28-day call-cause mortality with moderate cost-effectiveness. A total of 192 patients with severe pneumonia will be recruited from four large tertiary hospitals in China. Bronchoalveolar lavage fluid will be obtained in all patients and randomly assigned to the study group (mNGS combined with traditional microbiological tests) or the control group (traditional microbiological tests only) in a 1:1 ratio. Individualized antimicrobial treatment and strategy will be selected according to the analysis results. The primary outcome is 28-day all-cause mortality. The secondary outcomes are ICU and hospital length of stay (LOS), ventilator-free days and ICU-free days, consistency between mNGS and traditional microbiological tests, detective rate of mNGS and traditional microbiological tests, turn-out time, time from group allocation to start of treatment, duration of vasopressor support, types and duration of anti-infective regimens, source of drug-resistant bacteria or fungi, and ICU cost. DISCUSSION: The clinical benefits of mNGS are potentially significant, but its limitations should also be considered. TRIAL REGISTRATION: ChineseClinicalTrialRegistry.org, ChiCTR2300076853. Registered on 22 October 2023.

Performance of the galactomannan test for the diagnosis of invasive pulmonary aspergillosis using non-invasive proximal airway samples.

OBJECTIVE: To diagnose invasive pulmonary aspergillosis (IPA), galactomannan (GM) detection in serum or bronchoalveolar lavage fluid (BALF) is widely used. However, the utility of proximal airway GM test (from induced sputum or tracheal aspirate) has not been well elucidated. METHODS: In this retrospective cohort study, we evaluated the diagnostic performance of proximal airway GM in diagnosis of IPA including COVID-19 associated pulmonary aspergillosis (CAPA). Between January 2022 and January 2023, patients who had been tested for GM with clinical suspicion or for surveillance from any specimen (serum, induced sputum, tracheal aspirate, and BALF) were screened. IPA was diagnosed using EORTC/MSGERC criteria, and CAPA was diagnosed following the 2020 ECMM/ISHAM consensus criteria. RESULTS: Of 624 patients with GM results, 70 met the criteria for proven/probable IPA and 427 had no IPA. The others included possible IPA and chronic form of aspergillosis. The sensitivities and specificities of serum, proximal airway, and BALF GM for proven/probable IPA versus no IPA were 78.9% and 70.6%, 93.1% and 78.7%, and 78.6% and 91.0%, respectively. Areas under the receiver operating characteristic curve (AUCs) were 0.742 for serum GM, 0.935 for proximal airway GM, and 0.849 for BALF GM (serum GM vs proximal airway GM, p = 0.014; proximal airway GM vs BALF GM, p = 0.334; serum GM vs BALF GM, p = 0.286). CONCLUSION: This study demonstrates that the performance of GM test from non-invasive proximal airway samples is comparable or even better than those from serum and distal airway sample (BALF).

The longitudinal microbial and metabolic landscape of infant cystic fibrosis: the gut-lung axis.

BACKGROUND AND AIM: In cystic fibrosis, gastrointestinal dysfunction and lower airway infection occur early and are independently associated with poorer outcomes in childhood. This study aimed to define the relationship between the microbiota at each niche during the first 2 years of life, its association with growth and airway inflammation, and explanatory features in the metabolome. MATERIALS AND METHODS: 67 bronchoalveolar lavage fluid (BALF), 62 plasma and 105 stool samples were collected from 39 infants with cystic fibrosis between 0 and 24 months who were treated with prophylactic antibiotics. 16S rRNA amplicon and shotgun metagenomic sequencing were performed on BALF and stool samples, respectively; metabolomic analyses were performed on all sample types. Sequencing data from healthy age-matched infants were used as controls. RESULTS: Bacterial diversity increased over the first 2 years in both BALF and stool, and microbial maturation was delayed in comparison to healthy controls from the RESONANCE cohort. Correlations between their respective abundance in both sites suggest stool may serve as a noninvasive alternative for detecting BALF Pseudomonas and Veillonella. Multisite metabolomic analyses revealed age- and growth-related changes, associations with neutrophilic airway inflammation, and a set of core systemic metabolites. BALF Pseudomonas abundance was correlated with altered stool microbiome composition and systemic metabolite alterations, highlighting a complex gut-plasma-lung interplay and new targets with therapeutic potential. CONCLUSION: Exploration of the gut-lung microbiome and metabolome reveals diverse multisite interactions in cystic fibrosis that emerge in early life. Gut-lung metabolomic links with airway inflammation and Pseudomonas abundance warrant further investigation for clinical utility, particularly in non-expectorating patients.

Evaluation of analyte-specific reagents for the direct detection of Pneumocystis jirovecii.

Pneumocystis jirovecii pneumonia (PJP) is a serious and sometimes fatal infection occurring in immunocompromised individuals. High-risk patients include those with low CD4 counts due to human immunodeficiency virus infection and transplant recipients. The incidence of PJP is increasing, and rapid detection of PJP is needed to effectively target treatment and improve patient outcomes. A common method used is an immunofluorescent assay (IFA), which has limitations, including labor costs, low sensitivity, and requirement for expert interpretation. This study evaluates the performance of the DiaSorin Molecular Pneumocystis jirovecii analyte-specific reagent (ASR) in a laboratory-developed test (LDT) for the direct detection of P. jirovecii DNA without prior nucleic acid extraction. Respiratory samples (n = 135) previously tested by IFA from 111 patients were included. Using a composite standard of in-house IFA and reference lab PJP PCR, the percent positive agreement for the LDT using the DiaSorin ASR was 97.8% (90/92). The negative percent agreement was 97.7% (42/43). The lower limit of detection of the assay was determined to be 1,200 copies/mL in bronchoalveolar lavage fluid. Analytical specificity was assessed using cultures of oropharyngeal flora and common respiratory bacterial and fungal pathogens. No cross-reactivity was observed. Our study suggests that the DiaSorin Pneumocystis ASR accurately detects P. jirovecii DNA and demonstrates improved sensitivity compared to the IFA method. IMPORTANCE: Our study is unique compared to other previously published studies on the DiaSorin analyte-specific reagent (ASR) because we focused on microbiological diagnostic methods commonly used (immunofluorescent assay) as opposed to pathology findings or reference PCR. In addition, in our materials and methods, we describe the protocol for the use of the DiaSorin ASR as a singleplex assay, which will allow other users to evaluate the ASR for clinical use in their lab.

Prospective and systematic screening for invasive aspergillosis in the ICU during the COVID-19 pandemic, a proof of principle for future pandemics.

The diagnostic performance of a prospective, systematic screening strategy for COVID-19 associated pulmonary aspergillosis (CAPA) during the COVID-19 pandemic was investigated. Patients with COVID-19 admitted to the ICU were screened for CAPA twice weekly by collection of tracheal aspirate (TA) for Aspergillus culture and PCR. Subsequently, bronchoalveolar lavage (BAL) sampling was performed in patients with positive screening results and clinical suspicion of infection. Patient data were collected from April 2020-February 2022. Patients were classified according to 2020 ECMM/ISHAM consensus criteria. In total, 126/370 (34%) patients were positive in screening and CAPA frequency was 52/370 (14%) (including 13 patients negative in screening). CAPA was confirmed in 32/43 (74%) screening positive patients who underwent BAL sampling. ICU mortality was 62% in patients with positive screening and confirmed CAPA, and 31% in CAPA cases who were screening negative. The sensitivity, specificity, positive and negative predictive value (PPV & NPV) of screening for CAPA were 0.71, 0.73, 0.27, and 0.95, respectively. The PPV was higher if screening was culture positive compared to PCR positive only, 0.42 and 0.12 respectively. CAPA was confirmed in 74% of screening positive patients, and culture of TA had a better diagnostic performance than PCR. Positive screening along with clinical manifestations appeared to be a good indication for BAL sampling since diagnosis of CAPA was confirmed in most of these patients. Prospective, systematic screening allowed to quickly gain insight into the epidemiology of fungal superinfections during the pandemic and could be applicable for future pandemics.

Intact lung tissue and bronchoalveolar lavage fluid are both suitable for the evaluation of murine lung microbiome in acute lung injury.

BACKGROUND: Accumulating clinical evidence suggests that lung microbiome is closely linked to the progression of pulmonary diseases; however, it is still controversial which specimen type is preferred for the evaluation of lung microbiome. METHODS AND RESULTS: To address this issue, we established a classical acute lung injury (ALI) mice model by intratracheal instillation of lipopolysaccharides (LPS). We found that the bacterial DNA obtained from the bronchoalveolar lavage fluid (BALF), intact lung tissue [Lung(i)], lung tissue after perfused [Lung(p)], and feces of one mouse were enough for 16S rRNA sequencing, except the BALF of mice treated with phosphate buffer saline (PBS), which might be due to the biomass of lung microbiome in the BALF were upregulated in the mice treated with LPS. Although the alpha diversity among the three specimens from lungs had minimal differences, Lung(p) had higher sample-to-sample variation compared with BALF and Lung(i). Consistently, PCoA analysis at phylum level indicated that BALF was similar to Lung(i), but not Lung(p), in the lungs of mice treated with LPS, suggesting that BALF and Lung(i) were suitable for the evaluation of lung microbiome in ALI. Importantly, Actinobacteria and Firmicutes were identified as the mostly changed phyla in the lungs and might be important factors involved in the gut-lung axis in ALI mice. Moreover, Actinobacteria and Proteobacteria might play indicative roles in the severity of lung injury. CONCLUSION: This study shows both Lung(i) and BALF are suitable for the evaluation of murine lung microbiome in ALI, and several bacterial phyla, such as Actinobacteria, may serve as potential biomarkers for the severity of ALI. Video Abstract.