Lower Respiratory Tract Specimens Research Articles

Molecular epidemiological characterization of hypervirulent carbapenem-resistant Klebsiella pneumoniae in a hospital in Henan Province from 2020 to 2022

Objective: The study investigated the clinical distribution, antimicrobial resistance and epidemiologic characteristics of hypervirulent Carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) in a hospital in Henan Province to provide a scientific basis for antibiotic use and nosocomial infection prevention and control. Methods: A retrospective analysis of the clinical data from the cases was carried out in this study. Clinical data of patients infected with the CRKP strain isolated from the clinical microbiology laboratory of Henan Provincial Hospital of Traditional Chinese Medicine from January 2020 to December 2022 were retrospectively analyzed. A string test, virulence gene screening, serum killing, and a G. mellonella infection model were used to screen hv-CRKP isolates. The clinical characteristics of hv-CRKP and the drug resistance rate of hv-CRKP to twenty-five antibiotics were analyzed using WHONET 5.6. Carbapenemase phenotypic characterization of the hv-CRKP was performed by colloidal gold immunochromatographic assay, and Carbapenemase genotyping, multi-locus sequence typing (MLST) and capsular serotyping of hv-CRKP isolates were performed by PCR and Sanger sequencing. Results: A total of non-duplicate 264 CRKP clinical isolates were detected in the hospital from 2020 to 2022, and 23 hv-CRKP isolates were detected, so the corresponding detection rate of hv-CRKP was 8.71% (23/264). The hv-CRKP isolates in this study were mainly from the intensive care unit (10/23) and neurosurgery department (8/23), and the main sources of hv-CRKP isolates were sputum (10/23) and bronchoalveolar lavage fluid (6/23). The hv-CRKP isolates in this study were highly resistant to β-lactam antibiotics, fluoroquinolones and aminoglycosides, and were only susceptible to colistin, tigecycline and ceftazidime/avibactam. The detection rate of the blaKPC-2 among 23 hv-CRKP isolates was 91.30% (21/23) and none of the class B and class D carbapenemases were detected. Results of MLST and capsular serotypes showed that ST11 type hv-CRKP was the dominant strain in the hospital, accounting for 56.52% (13/23), and K64 (9/13) and KL47 (4/13) were the major capsular serotypes. Conclusion: The hv-CRKP isolates from the hospital are mainly from lower respiratory tract specimens from patients admitted to the intensive care department and the drug resistance is relatively severe. The predominant strains with certain polymorphisms are mainly composed of the KPC-2-producing ST11-K64 and ST11-KL47 hv-CRKP isolates in the hospital.

Diagnostic value of metagenomic next-generation sequencing of lower respiratory tract specimen for the diagnosis of suspected Pneumocystis jirovecii pneumonia

Aim To evaluate diagnostic performance of metagenomic next-generation sequencing (mNGS) for Pneumocystis jirovecii pneumonia (PCP), in comparison with polymerase chain reaction (PCR), Gomori methenamine silver (GMS) staining and serum 1,3-β-d-Glucan (BG) assay. Methods 52 PCP patients and 103 patients with non-pneumocystic jirovecii pneumonia (non-PCP) were enrolled, and comparative analysis was conducted of different diagnostic tests. Clinical features and co-pathogen characteristics were reviewed. Results The diagnostic sensitivity (92.3%) and specificity (87.4%) of mNGS did not show significant differences compared with that of PCR while mNGS had the advantage over PCR in the detection of co-pathogens. Despite its excellent specificity, the sensitivity of GMS staining (9.3%) was inferior to that of mNGS (p < .001). The combination of mNGS with serum BG statistically outperformed mNGS or serum BG alone in the areas under the receiver operating characteristic curves (AUCs, p = .0013 and p = .0015, respectively). Notably, all the blood samples showing positive mNGS for Pneumocystis jirovecii came from PCP patients. The leading co-pathogens among patients with PCP were cytomegalovirus, Epstein-Barr virus and Torque teno virus. Conclusions mNGS shows superiority over several common clinical methods in the diagnosis of suspected PCP. Serum BG in conjunction with mNGS further improved the diagnostic efficacy of mNGS.

Investigating Viral Involvement in Immunocompromised Patients Using Comprehensive Infectious Disease Testing Including FilmArray Respiratory Panel 2.1 on Bronchoscopy: A Retrospective Study.

Introduction Reports are rare on the usefulness of the FilmArray Respiratory Panel 2.1 (FARP) using lower respiratory tract specimens. This retrospective study assessed its use, as part of a comprehensive infectious disease panel, to detect the viral causes of pneumonia using bronchoalveolar lavage samples from immunosuppressed patients. Methods This study included immunocompromised patients who underwent bronchoalveolar lavage or bronchial washing by bronchoscopy between April 1, 2021, and April 30, 2022. The collected samples were submitted for comprehensive testing, including FARP test; reverse transcription polymerase chain reaction (RT-PCR) for cytomegalovirus, varicella-zoster virus DNA, and herpes simplex virus; PCR for Pneumocystis jirovecii DNA; antigen testing for Aspergillus and Cryptococcus neoformans; and loop-mediated isothermal amplification method for Legionella. Results Out of 23 patients, 16 (70%) showed bilateral infiltrative shadows on computed tomography and three (13%) were intubated. The most common causes of immunosuppression were anticancer drug use (n=12, 52%) and hematologic tumors (n=11, 48%). Only two (9%) patients tested positive for severe acute respiratory syndrome coronavirus 2 and adenovirus by FARP. Four patients (17%) tested positive for cytomegalovirus by RT-PCR, but no inclusion bodies were identified cytologically. Nine (39%) patients tested positive for Pneumocystis jirovecii by PCR, but cytology confirmed the organism in only one case. Conclusions Comprehensive infectious disease testing, performed using bronchoalveolar lavage samples collected from lung lesions in immunosuppressed patients, showed low positive detection by FARP. The viruses currently detectable by FARP may be less involved in viral pneumonia diagnosed in immunocompromised patients.

Several issues in interpreting the results of metagenomic next generation sequencing of lower respiratory tract infection

Metagenome next generation sequencing (mNGS) is an important method for pathogen diagnosis of lower respiratory tract infection, but there are many difficulties in interpreting mNGS reports. The Expert Consensus on Clinical Interpretation Path of mNGS of Lower Respiratory Tract Infection, completed by Chinese Thoracic Society, provides a detailed path and guidance for report interpretation. The expert consensus covers clinical medicine, microbiology, molecular diagnosis and other aspects. On this basis, several important clinical issues need to be highlighted. First, the lower respiratory tract specimens used for mNGS must be obtained in a timely and qualified manner. Second, the correct interpretation of the mNGS report needs to be based on a full understanding of the patients and their conditions. Third, the quality of the report should be analyzed according to the main parameters in the mNGS report. Fourth, an understanding of basic microbiology knowledge is beneficial to identify valuable pathogens in the mNGS report. Fifth, other microbiological methods should be actively used during mNGS detection. Sixth, to seek the help of the team when necessary and organize multidisciplinary discussions are vital. Seventh, it is important to constantly adjust the diagnosis and treatment strategies according to the clinical treatment response to treatment and the evolution of the disease. In a word, the interpretation of mNGS results needs to refer to specimen types and sequencing parameters, to combine with the specific conditions of patients in detail, to collect a variety of microbiological test results, to carefully screen and make judgement according to the treatment effect and disease outcome, and finally to make the diagnosis. The interpretation of mNGS report also requires us to have more knowledge of microbiology, sequencing and bioinformatics analysis, and to pay more attention to the strength of the team to discriminate the truth in multidisciplinary collaboration.

Increase in invasive group A streptococcal infection notifications, England, 2022.

Increases in invasive group A streptococcal (iGAS) infection and associated deaths, particularly in children, above seasonally expected levels are being seen this season (772 notifications reported in weeks 37 to 48 in 2022) across England. Diagnoses of iGAS infection from lower respiratory tract specimens in children under 15 years increased to 28% in November 2022. Medical practitioners have been alerted to the exceptional increase in incidence, including unusual numbers of children presenting with pulmonary empyema.

Acinetobacter baumannii in suspected bacterial infections: Association between multidrug resistance, virulence genes, & biofilm production.

Acinetobacter baumannii has emerged as a nosocomial pathogen with a tendency of high antibiotic resistance and biofilm production. This study aimed to determine the occurrence of A. baumannii from different clinical specimens of suspected bacterial infections and furthermore to see the association of biofilm production with multidrug resistance and expression of virulence factor genes in A. baumannii. A. baumannii was confirmed in clinical specimens by the detection of the blaOXA-51-like gene. Biofilm production was tested by microtitre plate assay and virulence genes were detected by real-time PCR. A. baumannii was isolated from a total of 307 clinical specimens. The isolate which showed the highest number of A. baumannii was an endotracheal tube specimen (44.95%), then sputum (19.54%), followed by pus (17.26%), urine (7.49%) and blood (5.86%), and <2 per cent from body fluids, catheter-tips and urogenital specimens. A resistance rate of 70-81.43 per cent against all antibiotics tested, except colistin and tigecycline, was noted, and 242 (78.82%) isolates were multidrug-resistant (MDR). Biofilm was detected in 205 (66.78%) with a distribution of 54.1 per cent weak, 10.42 per cent medium and 2.28 per cent strong biofilms. 71.07 per cent of MDR isolates produce biofilm (P<0.05). Amongst virulence factor genes, 281 (91.53%) outer membrane protein A (OmpA) and 98 (31.92%) biofilm-associated protein (Bap) were detected. Amongst 100 carbapenem-resistant A. baumannii, the blaOXA-23-like gene was predominant (96%), the blaOXA-58-like gene (6%) and none harboured the blaOXA-24-like gene. The metallo-β-lactamase genes blaIMP-1 (4%) and blaVIM-1(8%) were detected, and 76 per cent showed the insertion sequence ISAba1. The majority of isolates studied were from lower respiratory tract specimens. The high MDR rate and its positive association with biofilm formation indicate the nosocomial distribution of A. baumannii. The biofilm formation and the presence of Bap were not interrelated, indicating that biofilm formation was not regulated by a single factor. The MDR rate and the presence of OmpA and Bap showed a positive association (P<0.05). The isolates co-harbouring different carbapenem resistance genes were the predominant biofilm producers, which will seriously limit the therapeutic options suggesting the need for strict antimicrobial stewardship and molecular surveillance in hospitals.

Feasibility of the modified mini-bronchoalveolar lavage techniques using a nasogastric suction catheter and polytetrafluoroethylene bronchoscopic suction catheter in diagnosing bilateral pneumonia: a pilot study.

The adequacy of actual lower respiratory tract samples collected using the current collection technique is debated. Endotracheal aspiration is commonly insufficient and can be contaminated with colonization from the proximal airway. Diagnostic bronchoscopy is the standard method for collecting specimens from the lower respiratory tract. However, it is usually unavailable in resource-limited settings. At present, noninvasive methods with the mini-bronchoalveolar lavage (BAL) catheter are used to collect specimens from the lower respiratory tract. Compared with the nasogastric (NG) tube, the polytetrafluoroethylene (PTFE) catheter, a modified mini-BAL catheter that suctions the more distal part of the tracheobronchial tree, can collect actual lower respiratory tract specimens. This prospective open-label pilot study included patients aged >18 years who were diagnosed with bilateral pneumonia and who required mechanical ventilation. Lower respiratory tract samples were collected via endotracheal aspiration, mini-BAL using an NG tube, and mini-BAL using a PTFE bronchoscopic catheter. Data on return fluid volume, white blood cell (WBC) count, microbiologic information obtained via quantitative culture, and each procedure-related complication were recorded. The return fluid volumes of the NG tube and PTFE groups were 50 and 40 mL, respectively. The median WBC counts were 245 cells/cumm3 in the NG tube group and 305 cells/cumm3 in the PTFE group. Culture from endotracheal aspiration detected polymicrobial organisms in 8 (20.0%) patients. Further, 19 (47.5%) patients in the NG tube group and 18 (45.0%) in the PTFE group presented with polymicrobial organisms. Approximately 10% of patients developed mini-BAL-related complications, including arrhythmia (2.5%), mild hypoxemia (2.5%), and mild bleeding (5.0%). The two modified mini-BAL techniques are feasible in diagnosing patients with pneumonia requiring mechanical ventilation. The mini-BAL technique is more likely to detect polymicrobial organisms compared with endotracheal aspiration, which can then identify the causative polymicrobial organism of ventilator associated pneumonia (VAP) and lead to antibiotic adjustment. Moreover, it is easy to perform, can yield adequate specimens, and has few complications.

Application of nested multiplex polymerase chain reaction respiratory and pneumonia panels in children with severe community-acquired pneumonia.

Community-acquired pneumonia (CAP) is a serious clinical concern. A lack of accurate diagnosis could hinder pathogen-directed therapeutic strategies. To solve this problem, we evaluated clinical application of nested multiplex polymerase chain reaction (PCR) in children with severe CAP. We prospectively enrolled 60 children with severe CAP requiring intensive care between December 2019 and November 2021 at a tertiary medical center. Nested multiplex PCR respiratory panel (RP) and pneumonia panel (PP) were performed on upper and lower respiratory tract specimens. We integrated standard-of-care tests and quantitative PCR for validation. The combination of RP, PP, and standard-of-care tests could detect at least one pathogen in 98% of cases and the mixed viral-bacterial detection rate was 65%. The positive percent agreement (PPA), and negative percent agreement (NPA) for RP were 94% and 99%; the PPA and NPA for PP were 89% and 98%. The distribution of pathogens was similar in the upper and lower respiratory tracts, and the DNA or RNA copies of pathogens in the lower respiratory tract were equal to or higher than those in the upper respiratory tract. PP detected bacterial pathogens in 40 (67%) cases, and clinicians tended to increase bacterial diagnosis and escalate antimicrobial therapy for them.RP and PP had satisfactory performance to help pediatricians make pathogenic diagnoses and establish therapy earlier. The pathogens in the upper respiratory tract had predictive diagnostic values for lower respiratory tract infections in children with severe CAP.

Detection of influenza and non-influenza respiratory viruses in lower respiratory tract specimens among hospitalized adult patients and analysis of the clinical outcome.

Lower respiratory tract infection (LRTI) is one of the most fatal diseases for adults. Influenza is a well-recognized cause of severe pneumonia; however, the outcomes of LRTI caused by non-influenza respiratory viruses (NIRVs) have not been sufficiently investigated. This study aimed to describe the characteristics and outcomes of LRTI associated with respiratory viruses (RVs) in adults. A retrospective review was performed using medical records of adult patients whose lower respiratory tract (LRT) specimens (endotracheal aspirate and bronchoalveolar lavage fluid) tested positive for RVs using multiplex PCR. Underlying comorbidities, laboratory data, and clinical outcomes were analyzed. Among the 808 LRT specimens collected from 666 adult patients, RV was identified in 115 specimens (14%) from 106 patients (16%). The underlying comorbidities and laboratory data did not differ between patients with influenza- and NIRV-related LRTI. The 14-day and 30-day mortality rates were higher in the influenza group than in the NIRV group (24% versus 7%, p=0.03 and 33% versus 13%, p=0.02, respectively), whereas the 90-day mortality rate did not. In a multivariate Cox model to predict 90-day mortality, shock and acute kidney injury independently predicted a higher mortality rate (hazard ratio (HR): 4.28, 95% CI: 1.46-12.58, p=0.01 and HR: 2.80, 95% CI: 1.28-6.15, p=0.01, respectively), whereas the detection of influenza did not. Influenza and NIRVs were associated with increased mortality due to LRTI in adults. Therefore, NIRVs are among key pathogens causing LRTI and should not be neglected by clinicians.

The changing spectrum of microbial aetiology of respiratory tract infections in hospitalized patients before and during the COVID-19 pandemic

BackgroundThe COVID-19 pandemic was met with strict containment measures. We hypothesized that societal infection control measures would impact the number of hospital admissions for respiratory tract infections, as well as, the spectrum of pathogens detected in patients with suspected community acquired pneumonia (CAP).MethodsThis study is based on aggregated surveillance data from electronic health records of patients admitted to the hospitals in Bergen Hospital Trust from January 2017 through June 2021, as well as, two prospective studies of patients with suspected CAP conducted prior to and during the COVID-19 pandemic (pre-COVID cohort versus COVID cohort, respectively). In the prospective cohorts, microbiological detections were ascertained by comprehensive PCR-testing in lower respiratory tract specimens. Mann–Whitney’s U test was used to analyse continuous variables. Fisher’s exact test was used for analysing categorical data. The number of admissions before and during the outbreak of SARS-CoV-2 was compared using two-sample t-tests on logarithmic transformed values.ResultsAdmissions for respiratory tract infections declined after the outbreak of SARS-CoV-2 (p < 0.001). The pre-COVID and the COVID cohorts comprised 96 and 80 patients, respectively. The proportion of viruses detected in the COVID cohort was significantly lower compared with the pre-COVID cohort [21% vs 36%, difference of 14%, 95% CI 4% to 26%; p = 0.012], and the proportion of bacterial- and viral co-detections was less than half in the COVID cohort compared with the pre-COVID cohort (19% vs 45%, difference of 26%, 95% CI 13% to 41%; p < 0.001). The proportion of bacteria detected was similar (p = 0.162), however, a difference in the bacterial spectrum was observed in the two cohorts. Haemophilus influenzae was the most frequent bacterial detection in both cohorts, followed by Streptococcus pneumoniae in the pre-COVID and Staphylococcus aureus in the COVID cohort.ConclusionDuring the first year of the COVID-19 pandemic, the number of admissions with pneumonia and the microbiological detections in patients with suspected CAP, differed from the preceding year. This suggests that infection control measures related to COVID-19 restrictions have an overall and specific impact on respiratory tract infections, beyond reducing the spread of SARS-CoV-2.

P257 Amplified fragment length polymorphism fingerprinting supports the absence of correlation of genotype with clinical phenotype or source of the isolate in Aspergillus flavus infections

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjectives Aspergillus flavus accounts for ∼ 10% of bronchopulmonary aspergillosis and is the second leading etiological agent of invasive aspergillosis worldwide. It is the commonest cause of fungal rhinosinusitis and ocular mycoses in tropical countries including India. We report amplified fragment length polymorphism genotyping of a set of clinical and environmental isolates to unravel the genetic diversity of A. flavus in India and to further determine correlation between isolate genotype and the source/clinical phenotype of the Aspergillus infection.MethodsTwo sets of morphologically identified isolates of A. flavus from clinical (n = 71) and environmental sources (n = 22) were included in the study using a stratified random sampling method. Clinical strains were isolated from lower respiratory tract specimens (n = 22), sinus and sino-nasal biopsies (n = 25), corneal samples (n = 12), and others (n = 12). Environmental strains were isolated from different niches like air, soil, and infected crop samples. Aspergillus fumigatus F145 was used as an out-group. DNA was extracted from the fungal broth culture following the method of Lee et al. AFLP was done as per an earlier described method using HpyCH41V and Mse1 (New England BioLabs) for restriction digestion followed by selective amplification of restriction-digested products with 1 mm HpyCH4 IV primer with one selective residue (5-Flu-GTA-GACTGCGTACCCGTC-3’), 1 mm MseI primer with four selective residues (5-GATGAGTCCTGACTAATGAA). Fingerprint data was analyzed in BioNumerics software version 7.6 (Applied Maths, Belgium). The phylogenetic tree was constructed using Pearson's Correlation coefficient. An AFLP genotype was assigned to a cluster using an arbitrary cut-off value of ≥90% fingerprint similarity. The distribution of genotypes among different categories was analyzed by Fisher's exact test or Pearson's χ 2 goodness of fit test with Yates correction factor as appropriate and two-tailed P-values of <.05 were considered statistically significant.ResultsThe analyses revealed a total of 16 AFLP genotypes with 5 major clusters (≥ 5 isolates) reflecting the extent of genetic diversity in A. flavus. Genotype VIII encompassed predominantly clinical isolates (P <.01) and genotype XI with majority of isolates from environmental sources (P <.0001). The strains which were isolated from invasive and non-invasive forms or from different sites (pulmonary, sinus, and ocular) did not diverge into separate or unique clusters. Although the genotypes had an asymmetric distribution in different clinical presentations as revealed by the χ2 goodness of fit test, none of the genotypes was exclusively responsible for causing a particular infection. Isolates from the north zone of India shared genotypes with those from the southern region of the country. Three isolates formed a separate genotype XVI and diverged from the A. flavus cluster by 42% fingerprint similarity. Partial β-tubulin and calmodulin gene sequencing-based phylogeny reconstruction placed those three isolates in A. tamarii clade of the A. flavus species complex.Conclusions: This study suggests that every genotype of A. flavus has the potential of causing an allergic, non-invasive or invasive infection. Further, A. flavus sensu stricto was predominantly (97%) isolated from clinical specimens revealing the majority of infections/colonization are caused by this species compared to other members in the Flavi complex.

Chinese expert consensus on the management of lower respiratory tract infections of Pseudomonas aeruginosa in adults(2022)

Pseudomonas aeruginosa (PA) is the second common Gram-negative bacterium for hospital acquired pneumonia (HAP) in China (16.9%-22.0%). The proportion of PA in community acquired pneumonia (CAP) was about 1.0%, while increased to 1.8%-8.3% in severe CAP. PA accounted for 67.0% of CAP in patients with a history of PA infection, bronchiectasis, very severe chronic obstructive pulmonary disease (COPD) or tracheotomy. Considering the high disease burden of lower respiratory tract infections (LRTIs) caused by PA, together with the progress in this field in recent years, the Pulmonary Infection Assembly of Chinese Thoracic Society updated the "Chinese expert consensus on the management of lower respiratory tract infections of Pseudomonas aeruginosa in adults (2014 version)", focusing on pathogen detection, diagnosis, antimicrobial therapy, comprehensive management, infection prevention and control.PA causes both acute and chronic LTRIs. Acute LRTIs mainly include pneumonia (CAP, HAP and ventilator-associated pneumonia), tracheobronchitis, lung abscess and empyema. The diagnosis of chronic LTRIs should be based on a comprehensive assessment of (1) underlying chronic structural lung diseases, such as bronchiectasis, cystic fibrosis, COPD, or immunocompromised conditions; (2) the presence of clinical manifestations of LRTIs; and (3) ≥ two times (at least 3 months apart) of PA detected from eligible lower respiratory tract specimens within 1 year. It is important to distinguish infection from colonization when PA is isolated from lower respiratory tract specimens. Drug susceptibility test is a conventional method for PA resistance detection and serves as a basis for target therapy. When drug susceptibility test shows limited activity of available agents, combined susceptibility test is suggested to select antimicrobial drugs with additive or synergistic effect in vitro for combination therapy. Rapid test of resistance mechanisms of PA isolates, such as carbapenemase phenotype confirmation tests, is recommended if available. It is recommended not to routinely detect resistance genes for choosing therapeutic agents.For patients with acute LRTIs in critical condition or with high risk factors for PA infection, empirical antimicrobial therapy covering PA should be initiated after collecting specimens for microbiological tests. In patients with suspected PA pneumonia who are not critically ill, single antimicrobial drug of anti-PA activity with high lung concentration should be selected for empirical treatment. However, for patients with a serious condition such as sepsis or with risk factors for multidrug-resistant (MDR) PA, a combination of two different classes of antimicrobial drugs that are both potentially susceptible should be used. The antimicrobial regimen should follow pharmacokinetics/pharmacodynamics principles to ensure adequate dosage and administration frequency. For confirmed PA LRTIs, antibiotics should be selected based on drug sensitivity. In patients without significant underlying diseases, single therapy of an active antimicrobial with adequate pulmonary concentration is recommended rather than combination therapy; when all the available active agents have poor intrapulmonary concentrations, combination therapy is obligatory. For LRTIs caused by carbapenem-resistant PA (CRPA) or difficult-to-treat resistance PA (DTR-PA), if an agent of new enzyme inhibitor, such as ceftolozane/tazobactam, ceftazidime/avibactam, and imipenem/cilastatin/relebactam shows in vitro sensitivity, it is recommended as the first-line choice; cefiderocol may serve as the second-line treatment. Combination therapy based on polymyxins may also be considered. Other potentially successful approaches for drug-resistant PA LRTIs include extended infusion time of β-lactams, combination therapy and inhaled antimicrobial therapy.In patients with underlying chronic structural lung diseases, the antimicrobial regimen (drug, dosage, route of administration, and duration of therapy) should be decided according to clinical features, drug sensitivity, and treatment goals (control of exacerbated symptoms, eradication of new-emerging PA, or prevention of flare-ups in patients with frequent exacerbation).Along with antimicrobial therapy, comprehensive care including airway clearance therapy (ACT), oxygen therapy, nutritional support and organ function protection should be provided. From the perspective of nosocomial infection prevention and control, isolation and prophylaxis of contact transmission are recommended to block PA transmission in addition to standard prevention measures. Targeted active screening, timely monitoring and feedback can help the prevention and control of MDR-PA. The systemic and topical use of prophylactic antimicrobials is not recommended.

Metagenomic next-generation sequencing-guided antimicrobial treatment versus conventional antimicrobial treatment in early severe community-acquired pneumonia among immunocompromised patients (MATESHIP): A study protocol.

BackgroundSevere community-acquired pneumonia (SCAP) is the main cause of mortality in immunocompromised patients. Compared with conventional microbiological tests (CMT), metagenomic next-generation sequencing (mNGS) can quickly and simultaneously detect a wide array of bacteria, viruses, and fungi in an unbiased manner. It is increasingly used for severe respiratory infectious diseases, especially for immunocompromised patients. However, the effects of mNGS-based antimicrobial treatment procedures on clinical outcomes in immunocompromised patients with SCAP have not been evaluated.Methods/DesignThe MATESHIP study is a prospective, multicenter, parallel-group, open-label, randomized controlled trial from 20 ICUs in university hospitals and academic teaching hospitals across Shandong Province, China. We will enroll 342 immunocompromised patients with early onset SCAP who are admitted to an intensive care unit (ICU). Participants will be randomly allocated to an mNGS-guided treatment group or a conventional treatment group (guided by CMT), according to centrally computer-based block randomization stratified by participating centers. Participants will undergo CMT tests using appropriate lower respiratory tract (LRT) and other necessary specimens, with or without mNGS tests using LRT specimens. The primary outcomes will be: (1) The relative change in Sequential Organ Failure Assessment (SOFA) score from randomization to day 5, day 7, day 10, or the day of ICU discharge/death; and (2) the consumption of antimicrobial agents during ICU stay (expressed as defined daily doses). The secondary outcome measures will be: days from randomization to initiation of definitive antimicrobial treatment; overall antimicrobial agent use and cost; total cost of hospitalization; length of ICU stay; 28- and 90-day mortality; and clinical cure rate. This study hypothesizes that mNGS-guided treatment will decrease the degree of organ dysfunction/failure, the consumption of antimicrobial agents, and mortality, while the cure rate will be increased, and the time to initiation of appropriate therapy will be advanced.DiscussionThe MATESHIP study will evaluate for the first time whether mNGS-guided antimicrobial therapy improves the outcomes of SCAP in an immunocompromised population, and provide high-level evidence on the application of mNGS in the management of this population.Clinical Trial Registration[ClinicalTrials.gov], identifier [NCT05290454].

Metagenomic prediction of antimicrobial resistance in critically ill patients with lower respiratory tract infections

BackgroundAntimicrobial resistance (AMR) is rising at an alarming rate and complicating the management of infectious diseases including lower respiratory tract infections (LRTI). Metagenomic next-generation sequencing (mNGS) is a recently established method for culture-independent LRTI diagnosis, but its utility for predicting AMR has remained unclear. We aimed to assess the performance of mNGS for AMR prediction in bacterial LRTI and demonstrate proof of concept for epidemiological AMR surveillance and rapid AMR gene detection using Cas9 enrichment and nanopore sequencing.MethodsWe studied 88 patients with acute respiratory failure between 07/2013 and 9/2018, enrolled through a previous observational study of LRTI. Inclusion criteria were age ≥ 18, need for mechanical ventilation, and respiratory specimen collection within 72 h of intubation. Exclusion criteria were decline of study participation, unclear LRTI status, or no matched RNA and DNA mNGS data from a respiratory specimen. Patients with LRTI were identified by clinical adjudication. mNGS was performed on lower respiratory tract specimens. The primary outcome was mNGS performance for predicting phenotypic antimicrobial susceptibility and was assessed in patients with LRTI from culture-confirmed bacterial pathogens with clinical antimicrobial susceptibility testing (n = 27 patients, n = 32 pathogens). Secondary outcomes included the association between hospital exposure and AMR gene burden in the respiratory microbiome (n = 88 patients), and AMR gene detection using Cas9 targeted enrichment and nanopore sequencing (n = 10 patients).ResultsCompared to clinical antimicrobial susceptibility testing, the performance of respiratory mNGS for predicting AMR varied by pathogen, antimicrobial, and nucleic acid type sequenced. For gram-positive bacteria, a combination of RNA + DNA mNGS achieved a sensitivity of 70% (95% confidence interval (CI) 47–87%) and specificity of 95% (CI 85–99%). For gram-negative bacteria, sensitivity was 100% (CI 87–100%) and specificity 64% (CI 48–78%). Patients with hospital-onset LRTI had a greater AMR gene burden in their respiratory microbiome versus those with community-onset LRTI (p = 0.00030), or those without LRTI (p = 0.0024). We found that Cas9 targeted sequencing could enrich for low abundance AMR genes by > 2500-fold and enabled their rapid detection using a nanopore platform.ConclusionsmNGS has utility for the detection and surveillance of resistant bacterial LRTI pathogens.

Transplanting thoracic COVID-19 positive donors: An institutional protocol and report of the first 14 cases.

We present our institution's protocol for evaluating and transplanting thoracic organs from COVID-19 positive donors and report the outcomes to date. Hearts from donors testing positive for COVID-19 on any test were eligible for transplantation at our institution provided the donor exhibited no evidence of hypercoagulability or COVID-19 induced hyperinflammatory state during terminal hospitalization. Lungs were eligible if the donor first tested PCR positive on nasopharyngeal swab (NPS) for COVID-19 > 20 days prior to procurement and had a negative lower respiratory tract specimen. We performed 14 thoracic transplants in 13 recipients using organs from COVID-19 positive donors. None of the recipients or healthcare members acquired COVID-19. No recipients suffered unexpected acute rejection. Patient survival is 92% to date, with graft survival 93%. The use of hearts from COVID-19 positive donors may be safe and effective. Transplantation of lungs is unresolved but may be cautiously pursued under the restricted circumstances.

BioFire FilmArray Pneumonia Panel enhances detection of pathogens and antimicrobial resistance in lower respiratory tract specimens

BackgroundThis study investigated the diagnostic utility of the BioFire FilmArray Pneumonia Panel (PN panel), an automated and multiplexed nucleic acid detection system that rapidly detects 26 pathogens (18 bacteria and eight viruses) and seven antimicrobial resistance markers in a single assay.MethodsWe analyzed the targets in lower respiratory tract specimens using the PN panel and compared the detection results with those of bacterial culture methods and antimicrobial susceptibility testing.ResultsOf the 57 samples analyzed, the PN panel detected 97 targets (84 bacteria, four viruses, and nine antimicrobial resistance markers). Detection of bacteria and antimicrobial resistance was three times greater than that of the bacterial culture (25 bacteria and two resistant isolates) against the targets available in the panel. The overall positive and negative percent agreements between the PN panel and culture methods for bacterial detection were 100.0% and 92.9%, respectively. Multiple pathogens were detected by the PN panel in 24 samples (42.1%), ranging from two pathogens in 11 samples (19.3%) to six pathogens in one sample (1.8%). The PN panel semiquantitatively detected higher copies (≥ 106 copies/mL) of bacterial targets if the bacteria were positive by the culture method. In contrast, the semiquantitative values obtained by the panel varied (104 to 107 ≤ copies/mL) among bacteria that were negative by the culture method.ConclusionsThe PN panel enhanced the detection of pathogens and antimicrobial resistance markers in lower respiratory tract specimens.

Developing national benchmarks for antimicrobial resistance–NHSN, 2019

Background: The emergence and spread of drug-resistant pathogens continues to significantly impact patient safety and healthcare systems. Although antimicrobial susceptibility test (AST) results of clinical specimens are used by individual facilities for antimicrobial resistance surveillance, accurate tracking and benchmark comparison of a facility’s antimicrobial resistance using national data requires risk-adjusted methods to be more meaningful. The CDC NHSN Antimicrobial Resistance (AR) Option collects patient-level, deduplicated, isolate information, including AST results, for &gt;20 organisms from cerebrospinal fluid, lower respiratory tract (LRT), blood, and urinary specimens. To provide risk-adjusted national benchmarks, we developed prediction models for incidence of hospital-onset isolates with antimicrobial resistance. Methods: We analyzed AST results of isolates reported through the NHSN AR Option for January through December 2019. Isolates from facilities that had &gt;10% missing AST results for the organism-drug combinations or from hospitals that used outdated breakpoints were excluded. We assessed associations between facility-level factors and incidence rates of hospital-onset (specimen collected 3 days or more after hospital admission) isolates of specific drug-resistant phenotypes from blood, LRT, and urinary specimens. Factors included number of beds, length of stay, and prevalence of community onset isolates of the same phenotype. Drug-resistant phenotypes assessed included methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant (MDR) Pseudomonas aeruginosa, carbapenem-resistant Enterobacterales (CRE), fluoroquinolone-resistant Pseudomonas aeruginosa, fluoroquinolone-resistant Enterobacterales, and extended-spectrum cephalosporin-resistant Enterobacterales. Isolates of different phenotypes and from different specimen sources were modeled separately. Negative binomial regression was used to evaluate the factors associated with antimicrobial resistance incidence. Variable entry into the models is based on significance level P Among the models, 1 for each drug-resistant phenotype-specimen type combination, the number of isolates with AST results ranged from 718 (Pseudomonas aeruginosa–fluoroquinolones, blood) to 16,412 (Enterobacterales–fluoroquinolones, urine). The pooled incidence rate was highest for fluoroquinolone-resistant Enterobacterales in urinary specimens (0.2179 isolates per 1,000 patient days) among all phenotype-specimen combinations evaluated (Table 1). The incidence of drug-resistant isolates was consistently associated with community-onset prevalence across models evaluated. Other associated factors varied across phenotype-specimen combinations (Table 2). Conclusions: We developed statistical models to predict facility-level incidence rates of hospital-onset antimicrobial resistant isolates based on community-onset drug-resistant prevalence and facility characteristics. These models will enable facilities to compare antimicrobial resistance rates to the national benchmarks and therefore to inform their antimicrobial stewardship and infection prevention efforts.Funding: NoneDisclosures: None

Risk of Severe Acute Respiratory Syndrome Coronavirus 2 Transmission Through Solid Organ Transplantation and Outcomes of Coronavirus Disease 2019 Among Recent Transplant Recipients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is transmissible through lung transplantation, and outcomes among infected organ recipients may be severe. Transmission risk to extrapulmonary organ recipients and recent (within 30 days of transplantation) SARS-CoV-2-infected recipient outcomes are unclear. During March 2020-March 2021, potential SARS-CoV-2 transmissions through solid organ transplantation were investigated. Assessments included SARS-CoV-2 testing, medical record review, determination of likely transmission route, and recent recipient outcomes. During March 2020-March 2021, approximately 42 740 organs were transplanted in the United States. Forty donors, who donated 140 organs to 125 recipients, were investigated. Nine (23%) donors and 25 (20%) recipients were SARS-CoV-2 positive by nucleic acid amplification test (NAAT). Most (22/25 [88%]) SARS-CoV-2-infected recipients had healthcare or community exposures. Nine SARS-CoV-2-infected donors donated 21 organs to 19 recipients. Of these, 3 lung recipients acquired SARS-CoV-2 infections from donors with negative SARS-CoV-2 testing of pretransplant upper respiratory tract specimens but from whom posttransplant lower respiratory tract (LRT) specimens were SARS-CoV-2 positive. Sixteen recipients of extrapulmonary organs from SARS-CoV-2-infected donors had no evidence of posttransplant COVID-19. All-cause mortality within 45 days after transplantation was 6-fold higher among SARS-CoV-2-infected recipients (9/25 [36%]) than those without (6/100 [6%]). Transplant-transmission of SARS-CoV-2 is uncommon. Pretransplant NAAT of lung donor LRT specimens may prevent transmission of SARS-CoV-2 through transplantation. Extrapulmonary organs from SARS-CoV-2-infected donors may be safely usable, although further study is needed. Reducing recent recipient exposures to SARS-CoV-2 should remain a focus of prevention.

Comparative Diagnostic Accuracy of the STANDARD M10 Assay for the Molecular Diagnosis of SARS-CoV-2 in the Point-of-Care and Critical Care Settings.

Accurate and rapid molecular diagnosis of COVID-19 is a crucial step to tackle the ongoing pandemic. The primary objective of this study was to estimate the real-world performance of the novel RT-PCR STANDARD M10 SARS-CoV-2 assay in a large number of nasopharyngeal (NP) specimens eluted in universal transport medium. The secondary objective was to evaluate the compatibility of this kit in testing NP samples eluted in an inactivated transport medium (essential for point-of-care testing) and lower respiratory tract (LRT) specimens, which are commonly collected in critical care. A total of 591 samples were analyzed. Compared with the standard extraction-based RT-PCR Allplex 2019-nCoV (time-to-result of 270 min), the sensitivities of the STANDARD M10 were 100% (95% CI: 98.1–100%), 95.5% (95% CI: 91.7–97.6%), and 99.5% (95% CI: 97.2–99.9%) for ≥1 gene, the ORF1ab gene, and the E gene, respectively, while the specificity was 100% (95% CI: 98.7–100%). The diagnostic accuracy was 100% in testing both NP samples eluted in an inactivated transport medium and LRT specimens. STANDARD M10 reliably detects SARS-CoV-2 in 60 min, may be used as a POC tool, and is suitable for testing LRT specimens in the critical care setting.

Transplanting Thoracic COVID-19 Positive Donors: Overcoming the Pandemic

PurposeThe COVID19 pandemic has exacerbated the thoracic organ shortage. Safe donation from COVID19 infected donors would increase the donor pool.MethodsWe present our institutional protocol and early results for thoracic organ transplantation using COVID19 positive donors.EndpointsTo date, we performed 10 thoracic organ transplants in 9 recipients using organs from COVID19 positive donors (9 hearts;1 pair of lungs). Patient and graft survival to date is 100%, 91%.Hearts were procured from donors testing positive for COVID19 on upper and/or lower respiratory tract specimens, provided severe COVID pneumonia or myocarditis was not the cause of death, and hypercoagulable complications were absent. Lungs were procured only if donors were first positive >20 days prior and were PCR-negative on bronchoalveolar lavage. Cycle threshold, duration of infectivity and urgency of recipient need were considered in addition to routine evaluations.8/10 donors were first detected positive during terminal illness, yet no heart recipients acquired COVID19 infection through transplant and no unexpected rejection occurred. 1 heart-liver recipient required a redo heart transplant due to massive hemorrhage followed by hypercoagulability and coronary thrombus. An RV biopsy from the donor heart demonstrated ischemic changes and SARS-CoV-2 immunohistochemical findings suspicious for myocyte infiltration. Urgent re-transplantation was successfully completed utilizing a heart from another COVID19+ donor with no recurrence of hypercoagulability. One patient received lungs from a donor with mild COVID19, first detected 38 days earlier, yet still COVID19 positive on nasopharyngeal swab but not on BAL. No procurement or care team members became infected with covid as a result of this protocol.While limited, our experience to date supports that use of hearts from COVID19 positive donors is safe and effective. Lung transplantation from COVID19 positive donors is unresolved but may be cautiously pursued under the restricted circumstances.