Respiratory Samples Of Patients Research Articles

P235 Microbiological parameters in respiratory samples of patients with cystic fibrosis before and after being treated with elexacaftor/tezacaftor/ivacaftor

P235 Microbiological parameters in respiratory samples of patients with cystic fibrosis before and after being treated with elexacaftor/tezacaftor/ivacaftor

Aspergillus Outbreak in an Intensive Care Unit: Source Analysis with Whole Genome Sequencing and Short Tandem Repeats.

Whole genome sequencing (WGS) is widely used for outbreak analysis of bacteriology and virology but is scarcely used in mycology. Here, we used WGS for genotyping Aspergillus fumigatus isolates from a potential Aspergillus outbreak in an intensive care unit (ICU) during construction work. After detecting the outbreak, fungal cultures were performed on all surveillance and/or patient respiratory samples. Environmental samples were obtained throughout the ICU. WGS was performed on 30 isolates, of which six patient samples and four environmental samples were related to the outbreak, and twenty samples were unrelated, using the Illumina NextSeq 550. A SNP-based phylogenetic tree was created from outbreak samples and unrelated samples. Comparative analysis (WGS and short tandem repeats (STRs), microsatellite loci analysis) showed that none of the strains were related to each other. The lack of genetic similarity suggests the accumulation of Aspergillus spores in the hospital environment, rather than a single source that supported growth and reproduction of Aspergillus fumigatus. This supports the hypothesis that the Aspergillus outbreak was likely caused by release of Aspergillus fumigatus spores during construction work. Indeed, no new Aspergillus cases were observed in the ICU after cessation of construction. This study demonstrates that WGS is a suitable technique for examining inter-strain relatedness of Aspergillus fumigatus in the setting of an outbreak investigation.

Association between Pneumonia Development and Virulence Gene Expression in Carbapenem-Resistant Acinetobacter baumannii Isolated from Clinical Specimens.

We investigated the virulence gene expression of carbapenem-resistant Acinetobacter baumanii (CRAB) isolated from the respiratory samples of patients with CRAB pneumonia and those with CRAB colonization to identify the virulence genes contributing to CRAB pneumonia's development and mortality. Patients with CRAB identified from respiratory specimens were screened at a tertiary university hospital between January 2018 and January 2019. Patients were classified into CRAB pneumonia or CRAB colonization groups according to predefined clinical criteria. A. baumannii isolated from respiratory specimens was examined for the expression levels of ompA, uspA, hfq, hisF, feoA, and bfnL by quantitative reverse-transcription polymerase chain reaction. Among 156 patients with CRAB from respiratory specimens, 17 and 24 met the criteria for inclusion in the pneumonia and colonization groups, respectively. The expression level of ompA was significantly higher in the pneumonia group than in the colonization group (1.45 vs. 0.63, P=0.03). The expression levels of ompA (1.97 vs. 0.86, P=0.02), hisF (1.06 vs. 0.10, P < 0.01), uspA (1.62 vs. 1.01, P < 0.01), and bfnL (3.14 vs. 2.14, P=0.03) were significantly higher in patients with 30-day mortality than in the surviving patients. Elevated expression of hisF (adjusted odds ratio = 5.93, P=0.03) and uspA (adjusted odds ratio = 7.36, P=0.02) were associated with 30-day mortality after adjusting for age and the Charlson score. uspA and hisF may serve as putative targets for novel therapeutic strategies.

Incidence, Risk Factors and Clinical Impact of Sars-Cov-2 Prolonged Viral Shedding in Patients Diagnosed with Hematologic Malignancies

Patients with hematologic malignancies (HM) infected by SARS-CoV-2 present a higher risk of prolonged viral shedding, severe infection and mortality. However, data regarding clinical outcomes and viral evolution of prolonged COVID-19 in HM are still scarce. Our aim was to describe the incidence, risk factors, clinical outcomes and viral evolution of the protracted SARS-CoV-2 infection in patients with HM. We collected clinical data and respiratory samples of patients with HM who consulted at Vall d´Hebron Universitary Hospital and were diagnosed with a laboratory-confirmed SARS-CoV-2 infection between March 2020 and March 2023. Prolonged COVID-19 was defined as a positive real-time reverse transcription polymerase chain reaction (rRT-PCR) with a cycle threshold (Ct) &amp;lt;35 beyond 30 days since microbiological diagnosis with the same genotyped viral variant. Whole-genome sequencing of the virus was performed following the ARTIC, v4.1 protocol on a MiSeq platform. We considered an exacerbation of persistent COVID-19 the reappearance of symptoms and/or a positive SARS-CoV-2 rRT-PCR after initial clinical improvement or a previous negative rRT-PCR. Results: Among a total of 400 patients with HM developed COVID-19, 269 patients (67.25%) were eligible for prolonged COVID-19 assessment, whereas 52 patients (13%) died due to COVID-19 before day 30 and 79 patients (19.5%) did not have follow-up rRT-PCR. Prolonged COVID-19 was documented in 126 of 269 evaluable patients (46.8%) with a median time of rRT-PCR positivity of 60 days (IQR 43-95). Median age of patients with prolonged COVID-19 was 68 years and 46.8% were female. The main HM was lymphoma (58.7%), the majority of patients were under active treatment (78.6%), and the most frequent active therapy was anti-CD20 monoclonal antibodies+/- chemotherapy (25.4%). In the univariate analysis, active treatment with bispecific T-cell engagers antibodies (BiTEs) (OR 9.2; p=0.048) and anti-CD38 monoclonal antibodies (mAb) (OR 4.9; p=0.02), heavily pretreated patients (&amp;gt; 2 previous lines of therapy) (OR 3.1; p=0.02), and severe/critical acute COVID-19 (OR 7.4; p=&amp;lt;0.01) were significantly associated with prolonged viral shedding. Vaccination (OR 0.2; p=&amp;lt;0.01) and the development of an optimal humoral vaccine-induced immunogenicity (anti-spike IgG &amp;gt;260 BAU/mL) (OR 0.1; p=&amp;lt;0.01) were significantly associated with an inferior risk of developing prolonged COVID-19. In the multivariate analysis, active treatment with anti-CD20 mAb, BiTEs, and BTK inhibitors; lack of vaccination; an impaired humoral vaccine-induced immunogenicity and severe acute COVID-19 remained significantly associated with prolonged COVID-19 (Figure 1). CAR T-cell therapy also showed a trend to develop prolonged COVID-19 but did not reach statistical significance (OR 14.4; p=0.08). Prolonged COVID-19 clinical spectrum in HM ranged from a chronic asymptomatic infection in 29.4% of cases (37/126) to severe infection and COVID-19 related death in 16.7% of cases (21/126). During prolonged COVID-19, 51.6% of patients (65/126) experienced an increase in the severity of the infection, 54% (68/126) patients suffered a delay/interruption of their hematologic treatment, and 22.2% (28/126) had to suspend it indefinitely. A 55.6% of patients (70/126) experienced an exacerbation of persistent COVID-19 with a median of 1 episode (IQR 1-2) per patient. Through those exacerbations, 84.3% (59/70) required hospital readmission with a median of 17 days of length of stay (IQR 9-27). To treat prolonged COVID-19, 55.6% of patients (70/126) required a median of 1 course of antiviral treatment and 31% (39/126) received convalescent plasma. Of note, 37.3% of prolonged COVID-19 patients (47/126) developed a pneumopathy being organizing pneumonia the most frequent one in 28.6% (36/126) of cases. Finally, data of the kinetics of viral load and intra-host viral mutations in prolonged COVID-19 patients with a positive rRT-PCR beyond 90 days will be available for the congress. In conclusion, prolonged COVID-19 is frequent in patients with HM and clinical repercussion are severe. Patients with risk factors for prolonged viral replication, particularly those with a suboptimal humoral vaccine-induced immunogenicity and acute severe disease, require a personalized diagnostic, therapeutic and follow-up.

Antibiotic Sensitivity Pattern of Bacterial Isolates from Respiratory Samples of Patients with Respiratory Tract Infection in a Tertiary Care Teaching Hospital of Pune: A Hospital Record-Based Study

Antibiotic Sensitivity Pattern of Bacterial Isolates from Respiratory Samples of Patients with Respiratory Tract Infection in a Tertiary Care Teaching Hospital of Pune: A Hospital Record-Based Study

Levels of immunoglobulin isotypes in serum and respiratory samples of patients with chronic obstructive pulmonary disease: protocol for a systematic review and meta-analysis

IntroductionChronic obstructive pulmonary disease (COPD) is an inflammatory respiratory disorder characterised by the progressive worsening of lung function. Acute exacerbation of COPD (AECOPD) is a leading contributor to patient morbidity,...

Clinical outcomes associated with Achromobacter species infection in people with cystic fibrosis

BackgroundAchromobacter species are emerging pathogens isolated from respiratory samples of Patients with cystic fibrosis (pwCF) causing growing concerns in the CF community. The epidemiology and the clinical impact of Achromobacter in CF is unclear since data are restricted to small case control studies or selected populations. AimTo characterize the effect of Achromobacter respiratory infection on CF lung disease. MethodsEuropean CF Society Patient Registry data was analysed for association between Achromobacter infection and demographic/clinical characteristics and outcomes of pwCF. ResultsOf eligible 38,795 patients, Achromobacter infection was reported in 2,093 (prevalence (95% CI) of 5.40% (5.17 - 5.62). The prevalence varied significantly between the countries and increased with age peaking at the age 20–30. Achromobacter infection was more prevalent in pwCF carrying class minimal function mutations, having worse nutrition or lower pulmonary function, and more patients inhaled antibiotics against P. aeruginosa. Patient infected with Achromobacter had similar pulmonary function and BMI to patients infected with P. aeruginosa at all age groups. Being infected with both bacteria was associated with significantly lower pulmonary function and BMI at all age groups. ConclusionsAchromobacter infection was associated with disease severity similar to infection with P. aeruginosa. Being infected with both bacteria is associated with even more severe disease. This suggests to study if eradication will improve the outcome of pwCF.

Activity of newest generation β-lactam/β-lactamase inhibitor combination therapies against multidrug resistant Pseudomonas aeruginosa

Multidrug resistant (MDR) P. aeruginosa accounts for 35% of all P. aeruginosa isolated from respiratory samples of patients with cystic fibrosis (CF). The usefulness of β-lactam antibiotics for treating CF, such as carbapenems and later generation cephalosporins, is limited by the development of antibacterial resistance. A proven treatment approach is the combination of a β-lactam antibiotic with a β-lactamase inhibitor. New β-lactam/β-lactamase inhibitor combinations are available, but data are lacking regarding the susceptibility of MDR CF-associated P. aeruginosa (CFPA) to these new combination therapies. In this study we determined MIC values for three new combinations; imipenem-relebactam (I-R), ceftazidime-avibactam (CZA), and ceftolozane-tazobactam (C/T) against MDR CFPA (n = 20). The MIC90 of I-R, CZA, and C/T was 64/4, 32/4, and 16/8 (all µg/mL), respectively. The susceptibility of isolates to imipenem was not significantly improved with the addition of relebactam (p = 0.68). However, susceptibility to ceftazidime was significantly improved with the addition of avibactam (p < 0.01), and the susceptibility to C/T was improved compared to piperacillin/tazobactam (p < 0.05) These data provide in vitro evidence that I-R may not be any more effective than imipenem monotherapy against MDR CFPA. The pattern of susceptibility observed for CZA and C/T in the current study was similar to data previously reported for non-CF-associated MDR P. aeruginosa.

Development and Evaluation of Enzyme-Linked Viral Immune Capture Assay for Detection of SARS-CoV-2.

The pandemic of COVID-19 was caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 and it has prompted unprecedented research activities for vaccines, therapeutics, and diagnostics. The real-time reverse transcriptase-polymerase chain reaction (RT-PCR) is the gold standard method of diagnosis; however, immune-based assays offer cost-effective, deployable, easy-to-read solutions for diagnosis and surveillance. Here, we present the development, optimization, and testing of an enzyme-linked viral immune capture assay (ELVICA). It utilizes the spike antigen as the detected target of the virus and antibody-coated beads to capture the virus and enrich the detection. This method can be readout by luminescent and colorimetric equipment. It can also be visualized by the imaging system, offering a variety of detection approaches. ELVICA showed specificity to SARS-CoV-2-pseudotyped viruses as compared to MERS-CoV-pseudotyped viruses. As compared to RT-PCR, ELVICA showed high compatibility in detecting the virus in patient respiratory samples, especially for samples that are below a Ct value of 32 in RT-PCR. This assay is readily adaptable for detecting other pathogens and serves as a quick and affordable diagnostic tool.

Culture-Free Phylogenetic Analysis of Legionella pneumophila Using Targeted CRISPR/Cas9 Next-Generation Sequencing.

ABSTRACTCurrently available methods for the laboratory investigation of Legionella pneumophila outbreaks require organism culture. The ability to sequence L. pneumophila directly from clinical samples would significantly reduce delays. Here, we develop a method for targeted next-generation sequencing (NGS) of selected L. pneumophila genes utilizing a CRISPR/Cas9-based target enrichment system. We determine the method’s utility by typing cultured L. pneumophila isolates and subsequently apply the method directly to patient samples. We sequenced 10 L. pneumophila isolates by 2 methods, (i) whole-genome sequencing (WGS) and (ii) targeted (CRISPR/Cas9-based) finding low-abundance sequences by hybridization (FLASH)-NGS, sequencing 57 selected genes. The targeted NGS of 57 genes was more efficient than WGS, and phylogenetic analysis of the 57 genes yielded the same classification of the L. pneumophila isolates as that based on analysis of whole-genome data. Furthermore, targeted NGS of L. pneumophila performed directly on patient respiratory samples correctly classified the patients according to their corresponding cultured isolates. This provides proof of concept that targeted NGS can be used to sequence L. pneumophila directly from patient samples. Studies on a larger number of patient samples will further validate this method. Nonetheless, CRISPR/Cas9 targeted NGS methods have the potential to be widely applicable to microbial-outbreak investigations in the future, particularly in the context of difficult and slow-growing organisms.IMPORTANCE The bacterium Legionella pneumophila is responsible for outbreaks of serious and life-threatening pneumonia called Legionnaires’ disease. There is a need for new molecular methods that allow investigation of Legionella outbreaks directly from patient samples, without the need for prior microbiological culture, which causes delays. Our study aims to address this problem. We have utilized a CRISPR/Cas9-based targeted next-generation sequencing (NGS) method that can be applied directly on human specimens. Furthermore, we show that analysis of the sequences of a small number of targeted genes offers the same classification of L. pneumophila as that based on data derived from the whole genome. Given the rising interest globally in sequencing pathogens directly from human samples, CRISPR/Cas9 targeted NGS methods have the potential to be widely applicable to microbial-outbreak investigations in the future, particularly in the context of difficult and slow-growing organisms.

Microbiological monitoring of COVID-19 patients in the ICU: a prospective observational study

Objective. To study spectrum of pathogens and the time to colonization of respiratory samples in patients with severe and critical COVID-19 as well as to analyze incidence of nosocomial infections and structure of prescribed antibacterial drugs. Materials and Methods. The prospective observational study included patients aged 18 years and older with confirmed severe and critical COVID-19 from December 2021 to February 2022. During the first 48 hours and then every 2–3 days of hospitalization, a respiratory sample was collected: sputum, tracheal aspirate (if intubated), bronchoalveolar lavage (if bronchoscopy was performed) for microscopy and microbiological examination. Some patients were screened for invasive aspergillosis. Clinical and demographic data, comorbidities, pathogenetic therapy for COVID-19, antibiotic therapy, cases of probable/documented bacterial nosocomial infections, antibiotic-associated diarrhea, and hospital treatment outcomes were recorded. Results. A total of 82 patients were included in this study. Patients with lung parenchyma involvement of more than 50% by computer tomography predominated; most of them (77%) required intubation and mechanical ventilation due to progression of respiratory failure, and 76% of patients had a lethal outcome. During the first 48 hours, a respiratory sample was obtained from 47 patients; the rest of the patients presented with non-productive cough. No growth of microorganisms was detected in 31 (36.8%) cases; clinically significant pathogens were detected in 16 (19.5%) patients. A subsequent analysis included data from 63 patients with a sufficient number of samples for dynamic observation were used. During the first 3 days of ICU stay, the most common bacterial pathogens were Klebsiella pneumoniae without acquired antibiotic resistance and methicillin-susceptible Staphylococcus aureus. From 3rd day and afterwards, an increase in the proportion of Acinetobacter baumannii, other non-fermenting bacteria, and carbapenemresistant Enterobacterales was noted. Among the pathogens causing lower respiratory tract infections, A. baumannii and carbapenem-resistant K. pneumoniae were predominant pathogens and accounted for 76% of cases. Positive galactomannan test results were obtained in 4 cases. Conclusions. The study confirmed importance of bacterial nosocomial infections in patients with severe and critical COVID-19. In the case of the development of nosocomial lower respiratory tract infections, empirical antimicrobial therapy should take into account the predominance of carbapenem-resistant Enterobacteria and A. baumannii, as well as the possibility of invasive aspergillosis.

Evaluation of Three Culture Media for Isolation of Burkholderia cepacia Complex from Respiratory Samples of Patients with Cystic Fibrosis.

Burkholderia cepacia complex (BCC) is a significant pathogen causing respiratory disease in individuals with cystic fibrosis (CF). Diagnosis is typically achieved by isolation of BCC on selective culture media following culture of sputum or other respiratory samples. The aim of this study was to compare the efficacy of three commercially available selective media for the isolation of BCC. The three media comprised Burkholderia cepacia selective agar (BCSA; bioMérieux), BD Cepacia medium (BD: Becton–Dickinson) and MAST Cepacia medium (MAST laboratories). Each medium was challenged with 270 respiratory samples from individuals with CF as well as an international collection of BCC (n = 26) and 14 other isolates of Burkholderia species at a range of inocula. The international collection was also used to artificially “spike” 26 respiratory samples. From a total of 34 respiratory samples containing BCC, 97% were recovered on BD and 94% were detected on MAST and BCSA. All three media were effective for isolation of BCC. BCSA was much more selective than the other two media (p < 0.0001) meaning that fewer isolates required processing to exclude the presence of BCC.

Occurrence of the Legionella species in the respiratory samples of patients with pneumonia symptoms from Ahvaz, Iran; first detection of Legionella cherrii.

This study aimed to investigate the occurrence of Legionella species in the respiratory samples of patients with pneumonia symptoms from Ahvaz, Iran by culture and the real-time PCR of 23S-5S rRNA gene spacer region. A total of 123 clinical respiratory samples including 63 pleural aspirates, 57 bronchoalveolar lavage (BAL), and 3 sputum were collected from 65 males and 58 females with pneumonia symptoms. All samples were cultured on the Modified Wadowsky-Yee (MWY) agar. The Legionella species was identified by routine bacteriological tests. The presence of the 16S-23S rRNA spacer region gene was investigated by real-time PCR. The Legionella species were differentiated by sequencing of 16S-23S rRNA gene. A total of 2 (1.6%) BAL specimens were positive for Legionella species by culture method. No Legionella spp. were identified in pleural aspirates and sputum samples by the culture method. Using real-time PCR, 9 (7.3%) samples including 6 BAL, 1 sputum, and 2 pleural aspirates were positive for legionella species. These species were detected in 3 (5.2%) females and 6 males (9.2%). The results of sequencing showed that eight species were L. pneumophila while one was L. cherrii. Also, the 2 isolates that were identified by culture method, were confirmed as L. pneumophila by sequencing. The results showed that using the real-time PCR has a more efficacy for detecting of Legionella species in respiratory samples. Also, L. pneumophila was the most prevalent species circulating in the southwest region of Iran. So, periodic monitoring programs is recommended to prevent epidemics due to this bacterium.

Lower Respiratory Tract Pathogens and Their Antimicrobial Susceptibility Pattern: A 5-Year Study.

Lower respiratory tract infections (LRTIs) are the most common infections in humans. It is estimated that 2.74 million deaths worldwide occur each year due to LRTIs. The aim of the study was to determine the frequency and antibiotic susceptibility pattern of microorganisms isolated from respiratory samples of patients with LRTIs. Between January 2015 and December 2019, a total of 7038 sputum and bronchoaspirate samples from suspected LRTI patients were collected. Among them, 2753 samples (39.1%) showed significant microbial growth on culture media. The LRTI rate was higher in patients with male gender (67.1%) and with age between 40–59 years (48.6%). The microorganism identification and antibiotic susceptibility testing were performed with Vitek 2. Out of 4278 isolates species, 3102 (72.5%) were Gram-negative bacteria, 1048 (24.5%) were Gram-positive bacteria, and 128 (3.0%) were Candida spp. Major microorganisms isolated were Acinetobacter baumannii (18.6%), Staphylococcus aureus (15.2%), Pseudomonas aeruginosa (14.2%), and Klebsiella pneumoniae (10.9%). In antimicrobial susceptibility testing, Staphylococcus aureus isolates were mostly resistant to Penicillin G (84.1%) and Oxacillin (48.1%), whereas they demonstrated maximum sensitivity to Tigecycline (100%) and Linezolid (99.5%). Among Gram-negative isolates, Acinetobacter baumannii showed maximum sensitivity to Colistin but was resistant to other antibiotics (95–99%). Klebsiella pneumoniae isolates were mostly resistant to Cefotaxime (72.7%) and sensitive to Gentamicin (54.3%), and Pseudomonas aeruginosa was resistant to Ciprofloxacin (40.3%) and sensitive to Amikacin (85.9%). Gram-negative bacteria represented the species most commonly isolated. A high rate of antimicrobial resistance was observed in this study. In conclusion, the correct identification of causative microorganisms and their susceptibility patterns to antibiotics is crucial for choosing targeted and effective antibiotic therapy in LRTIs, and to prevent the emergence of multidrug-resistant bacteria.

Frequency of occurrence and antimicrobial susceptibility of bacteria isolated from respiratory samples of patients hospitalized with pneumonia in Western Europe, Eastern Europe and the USA: results from the SENTRY Antimicrobial Surveillance Program (2016-19).

BackgroundThe SENTRY Antimicrobial Surveillance Program monitors the frequency of occurrence and antimicrobial susceptibility of organisms from various infection types worldwide.ObjectivesTo evaluate the SENTRY programme results for organisms isolated from respiratory samples of patients hospitalized with probable pneumonia.MethodsA total of 28 918 bacterial isolates were consecutively collected (one per patient) in 2016–19 from 121 medical centres located in western Europe (W-EU; n = 7966), eastern Europe (E-EU; n = 3182) and the USA (n = 17 770) and then susceptibility tested by reference broth microdilution methods in a central laboratory.ResultsGram-negative bacilli (GNB) represented 76.3%, 88.6% and 69.1% of organisms; non-fermentative (NF) GNB accounted for 26.9%, 51.8% and 34.6% of organisms in W-EU, E-EU and USA, respectively. Pseudomonas aeruginosa susceptibility to piperacillin/tazobactam and meropenem was 75.4% and 76.9% in W-EU, 57.4% and 48.3% in E-EU, and 76.1% and 74.8% in the USA, respectively. Only 10.4% of Acinetobacter baumannii isolates from E-EU were meropenem susceptible compared with 45.8% in W-EU and 58.8% in the USA. Overall MRSA rates were 21.4% in W-EU and 28.7% in E-EU. In the USA, MRSA rates decreased from 44.8% in 2016 to 40.1% in 2019. Carbapenem resistance among Enterobacterales decreased continuously in the USA from 3.0% in 2016 to 1.7% in 2019 (2.4% overall) and was higher in E-EU (16.6%) than W-EU (2.2%). Klebsiella pneumoniae susceptibility to meropenem was 91.3%, 72.5% and 95.3% in W-EU, E-EU and the USA, respectively.ConclusionsRank order and antimicrobial susceptibility of bacteria isolated from patients with pneumonia widely varied by geography. MDR NF-GNB represented an important cause of pneumonia.

Bacterial co-infections in COVID-19 pneumonia in a tertiary care hospital: Surfing the first wave

The aim of this study is to review bacterial isolates from respiratory samples of patients with severe COVID-19 disease during the first 2 months of the first wave in our hospital. A single-center retrospective observational study in critically ill adult patients was performed. A total of 1251 respiratory samples from 1195 patients were processed. Samples from 66 patients (5.52%) were determined to be microbiologically significant by a semi-quantitative culture. All patients received broad spectrum antibiotherapy as an empirical treatment. The isolated bacteria were mainly Enterobacterales followed by Staphylococcus aureus and Pseudomonas aeruginosa. Bacterial co-infections in ICU stay could seem not dependent on the virus that has produced the viral pneumonia similarly as with other respiratory viruses such as Influenza virus.

Respiratory and intestinal epithelial cells exhibit differential susceptibility and innate immune responses to contemporary EV-D68 isolates.

Enterovirus D68 (EV-D68) has been implicated in outbreaks of severe respiratory illness and is associated with acute flaccid myelitis (AFM). EV-D68 is often detected in patient respiratory samples but has also been detected in stool and wastewater, suggesting the potential for both respiratory and enteric routes of transmission. Here, we used a panel of EV-D68 isolates, including a historical pre-2014 isolate and multiple contemporary isolates from AFM outbreak years, to define the dynamics of viral replication and the host response to infection in primary human airway cells and stem cell-derived enteroids. We show that some recent EV-D68 isolates have decreased sensitivity to acid and temperature compared with earlier isolates and that the respiratory, but not intestinal, epithelium induces a robust type III interferon response that restricts infection. Our findings define the differential responses of the respiratory and intestinal epithelium to contemporary EV-D68 isolates and suggest that a subset of isolates have the potential to target both the human airway and gastrointestinal tracts.

Viral load dynamics in intubated patients with COVID-19 admitted to the intensive care unit.

BackgroundProlonged viral RNA detection in respiratory samples from patients with COVID-19 has been described, but the clinical relevance remains unclear. We studied the dynamics of SARS-CoV-2 on a group and individual level in intubated ICU patients.MethodsIn a cohort of 86 patients, we analysed SARS-CoV-2 RT-PCR results on nasopharyngeal and sputum samples (obtained as part of clinical care twice a week) according to time after intubation. Subsequently, we performed survival analyses.Results870 samples were tested by RT-PCR. Overall viral load was highest in the first week (median nasopharynx 3.5, IQR 1.5–4.3; median sputum 4.3, IQR 3.3–5.6) and decreased over time. In 20% of patients a relapsing pattern was observed. Nasopharyngeal and sputum PCR status on day 14 was not significantly associated with survival up to day 60 in this small cohort.ConclusionIn general SARS-CoV-2 RNA levels in respiratory samples in patients with severe COVID-19 decrease after the first week after intubation, but individual SARS-CoV-2 RNA levels can show a relapsing pattern. Larger studies are needed to address the association of clearance of SARS-CoV-2 RNA from respiratory samples with survival, because we observed a trend towards better survival in patients with early clearance from sputum.

Antibiotic resistance of bacteria responsible for lower respiratory tract infections seen in the University Hospital of Befelatanana Antananarivo Madagascar

The majority of bacteria responsible for Lower Respiratory Tract Infections (LRTIs) are multiresistant to antibiotics. The objectives of this study are to describe the antibiotic resistance of these bacteria in LRTIs and to describe the factors associated with these infections. It is a prospective study of 54 respiratory samples of patients with LRTIs for a period of six months from July 2019 to December 2019 in the laboratory of the University Hospital of Befelatanana Antananarivo. The microbiological results showed 12 (22%) isolates of nonfermenting gram-negative bacilli, 10 (19%) isolates of streptococci and 6 (11%) isolates of enterobacteria. Regarding the isolates of nonfermenting gram-negative bacilli, the antibiotic resistance, varies from 42% (amikacin) to 100% (cotrimoxazole). Concerning the isolates of streptococci, it varies from 0% (vancomycin) to 80% (penicillin G). And the antibiotic resistance of the isolates of enterobacteria varies from 0% (imipenem and amikacin) to 100% (amoxicillin). Concerning the associated factors, subjects aged 40 and over (58.6%) (p = 0.84; NS), men (71.4%) (p = 0.01) and subjects hospitalized in intensive care units (70.3%) (p = 0.004) are the most affected by LRTIs. In brief, the knowledge of antibiotic resistance of bacteria responsible for LRTIs allows better patient management.

744. Clinical Performance of Real-time PCR in the Diagnosis of Pneumocystis jirovecii Pneumonia compared with Immunofluorescence Assay

BackgroundThe laboratory diagnosis of Pneumocystis jirovecii pneumonia (PJP) has been traditionally based on microscopy techniques, which have suboptimal sensitivity and depends on the experience and skills of the microbiologist. Molecular detection assays based in PCR (Polymerase chain reaction) could improve sensitivity.Our aim was to evaluate the utility of real-time PCR in the diagnosis of PJP compared with IFA (Immunofluorescence assay) performed in different respiratory samples of patients with PJP suspicion for routine use in a clinical laboratory setting.MethodsFrom September 2015 to April 2018, we studied by a real-time PCR targeting the large subunit of rRNA gene of P. jirovecii (PJ-PCR RealCycler PJIR kit Progenie Molecular) and Immunofluorescence assay (MONOFLUO P. carinii IFA BioRad) in all respiratory samples received for microbiological diagnosis of PJP. The definite clinical diagnosis of PJP was established by infectious disease physicians considering symptoms, radiological and laboratory findings.ResultsOverall, 302 samples were included (182 bronchoalveolar lavage, 67 sputum, 53 tracheal aspirates). PJ-PCR was positive in 51 (16.9%) and IFA in 11 (3.6%) of the patients with PJP. There were not IFA positive/PCR negative samples. Sensitivity, specificity, PPV and NPV for IFA were 26% (95%CI 15.9-39.6%), 100% (95%CI 98.5-100%), 100% (95% CI 77.2-100%) and 87.2% (95% CI 82.6-90.6%). Whereas, sensitivity, specificity, PPV and NPV for PCR was 92% (95%CI 81.2-96.8%), 98% (95% CI 95.4-99.2%), 90.2% (95% CI 79.0-95.7%) and 98.4% (95% CI 96.0-99.4%).PJ-PCR had sensitivity > 80% and specificity > 90% in all type of samples included.A definitive diagnosis of PJP was considered in 50 (16.6%) patients, including 4 (1.3%) cases with negative PJ-PCR. Five cases (9.8%) with positive PJ-PCR were considered as colonization.Conclusion P. jirovecii PCR improves the sensitivity and NPV of PJP diagnosis respecting to IFA, regardless of respiratory sample type. Our results suggest that Microbiology laboratories should use PCR techniques to diagnose PJP better than IFA.Disclosures All Authors: No reported disclosures