Ex-vivo mucolytic and anti-inflammatory activity of BromAc in tracheal aspirates from COVID-19

COVID-19 is a lethal disease caused by the pandemic SARS-CoV-2, which continues to be a public health threat. COVID-19 is principally a respiratory disease and is often associated with sputum retention, for which there are limited therapeutic options. In this regard, we evaluated the use of BromAc, a combination of Bromelain and Acetylcysteine (NAC). Both drugs present mucolytic effect and have been studied to treat COVID-19. Therefore, we sought to examine the mucolytic, antiviral, and anti-inflammatory effect of BromAc in tracheal aspirate samples from critically ill COVID-19 patients requiring mechanical ventilation. Method: Tracheal aspirate samples from COVID-19 patients were collected following next of kin consent and mucolysis, rheometry and cytokine storm analysis was performed. Results: BromAc displayed a robust mucolytic effect in a dose dependent manner. BromAc showed anti-inflammatory activity, reducing the action of cytokine storm, chemokines including MIP-1alpha, CXCL8, MIP-1b, MCP-1 and IP-10, and regulatory cytokines IL-5, IL-10, IL-13 IL-1RA and total reduction for IL-9 compared to NAC alone and control. BromAc acted on IL-6, demonstrating a reduction in G-CSF and VEGF-D at concentrations of 125 and 250ug. Conclusion: These results indicate robust mucolytic and anti-inflammatory effect of BromAc in tracheal aspirates from critically ill COVID-19 patients, indicating its potential as a therapeutic strategy to COVID-19.

Candida spp. in Lower Respiratory Tract Secretions – A Ten Years Retrospective Study

Introduction: Lower respiratory tract secretions (LRTS) like sputum and tracheal aspirates are frequently sent to the microbiology laboratory from patients with various respiratory pathologies. Improper collection techniques can lead to false-positive results, resulting in improper therapy. Aim of the study: To determine the percentage of contaminated samples sent to the microbiology laboratory, to establish the prevalence of Candida spp. in non-contaminated samples and therefore, the presence of Candida spp. originating in lower respiratory tract infections. Material and Methods: A 10-year data survey was conducted to assess the differences in Candida prevalence from contaminated versus non-contaminated samples, assessed and categorised by Bartlett grading system, and to emphasise the importance of quality control for potentially contaminated samples. The data were analysed according to gender, age, referring departments, and the species of Candida. For the statistical analysis, Kruskal-Wallis and Fisher tests were used, and the alpha value was set for 0.5. Results: The prevalence of Candida spp. in all analysed samples was 31.60%. After excluding the contaminated samples, the actual prevalence was 27.66%. Of all sputum samples, 31.6% were contaminated. Patients aged more than 40 years old were more prone to provide contaminated sputum samples. C. albicans is more prevalent in non-contaminated sputum samples. In both sputum and tracheal aspirates, the chances of identifying a single species are higher than the chances of identifying multiple species. Conclusions: The study emphasises the importance of assessing the quality of sputum samples because of the high number of improperly collected samples sent to the microbiology laboratory.

Human endogenous retrovirus K activation in the lower respiratory tract of severe COVID-19 patients associates with early mortality

Critically ill 2019 coronavirus disease patients (COVID-19) under invasive mechanical ventilation (IMV) are 10- to 40-times more likely to die than the general population. Although progression from mild to severe COVID-19 has been associated with hypoxia, uncontrolled inflammation and coagulopathy, the mechanisms involved in progression to severity are poorly understood. By analyzing the virome from tracheal aspirates (TA) of 25 COVID-19 patients under IMV, we found higher levels and differential expression of human endogenous retrovirus K (HERV-K) genes compared to nasopharyngeal swabs from mild cases and TA from non-COVID patients. Proteomic analysis and RT-PCR confirmed the presence of HERV-K in these patients. Moreover, increased HERV-K expression was triggered in human primary monocytes from healthy donors after experimental SARS-CoV-2 infection in vitro. In critically ill patients, higher HERV-K levels were associated with early mortality (within 14 days) in the intensive care unit. Increased HERV-K expression in deceased patients associated with IL-17-related inflammation, monocyte activation and higher consumption of clotting/fibrinolysis factors. Our data implicate the levels of HERV-K transcripts in the outcome of critical COVID-19 patients under invasive mechanical ventilation.

Usefulness of GeneXpert MTB/RIF in the diagnosis of extra-pulmonary tuberculosis

Background: Diagnosis of extra-pulmonary tuberculosis (TB) is often delayed because of diverse clinical presentations and difficulties in establishing the bacteriological diagnosis. This study aimed to evaluate usefulness of GeneXpert MTB/RIF in the diagnosis of extra-pulmonary TB in Bangladeshi patients. Methods: This cross-sectional study was done in BIRDEM General Hospital, Dhaka, Bangladesh from 2013 to 2016 as a part of Bangladesh Diabetic Somiti (BADAS)-USAID-TB Care-II project. Representative samples from 590 clinically suspected extra-pulmonary TB cases were tested for GeneXpert MTB/RIF along with conventional methods. Results: Total patients were 590 [mean age 43.9 (range 1-95) years] with male predominance (326, 55.3%). Most (513, 86.9%) patients were diabetic and new (574, 97.3%) TB suspects; while 16 (2.7%) patients had past history of TB. Common samples were pleural fluid (125, 21.2%), urine (110, 18.6%), cerebrospinal fluid (CSF) (91, 15.4%), pus (82, 13.9%), tracheal aspirates (57, 9.7%), ascitic fluid (45, 7.6%), gastric lavage (31, 5.3%), broncho-alveolar lavage (BAL) (18, 3.1%), lymph node aspirates (11, 1.9%) and synovial fluid (8, 1.4%). Among 590 samples, 68 (11.5%) were positive for Mycobacterium tuberculosis. Diagnostic yield was common for lymph nodes (4/7, 57.1%), pus (25/82, 30.5%), BAL (4/18, 22.2%), tracheal aspirates (8/57, 14.0%), urine (7/110, 6.4%), CSF (6/91, 6.6%) and pleural fluid (7/125, 5.6%). Of the 68 GeneXpert MTB/RIF positive samples, 52 (76.1%) were rifampicin sensitive, 16 (23.9%) showed intermediate sensitivity and none of the samples was resistant to rifampicin. Conclusions: GeneXpert MTB/RIF appeared as useful tool for diagnosing extra-pulmonary TB. Birdem Med J 2021; 11(2): 121-124

SARS-CoV-2 RNA load in the lower respiratory tract, viral RNAemia and N-antigenemia in critically ill adult COVID-19 patients: relationship with biomarkers of disease severity

Background: Little is known about the comparative kinetics of SARS-CoV-RNA load in the lower respiratory tract and in blood compartment in patients admitted to the intensive care unit, and how these relate to biomarkers of COVID-19 severity. Methods: Seventy-three consecutive critically ill COVID-19 patients (median age, 65 years) were recruited. Serial lower respiratory tract (n=165) and plasma (n=340) specimens were collected. RT-PCR and lateral flow immunochromatography assay were used for SARS-CoV-2 RNA quantitation and N protein detection in plasma, respectively. Serum levels of inflammatory and tissue-damage biomarkers in paired specimens were analyzed. Results: SARS-CoV-RNA was detected in the lower respiratory tract of most patients (92%). Viral RNAemia and N-antigenemia were documented in 35.6% and 40.1% of patients, respectively. Viral RNAemia and N-antigenemia cleared at a faster rate than SARS-CoV-2 RNA in tracheal aspirates (TA). SARS-CoV-2 RNA load was higher (P<0.001) in TA than in plasma, and correlated significantly (Rho, 0.41; P<0.001). A modest correlation was found between SARS-CoV-2 RNA load in TA and plasma and levels of ferritin and lactose dehydrogenase (Rho≤0.3; P≤0.008) in paired serum specimens. Neither the dynamics of SARS-CoV-2 RNA load in TA and plasma, nor N-antigenemia detection rate differed between surviving and deceased patients. Yet, a trend towards a higher mortality was seen in patients with viral RNAemia (OR; 2.82; 95% CI, 0.94-8.47; P=0.06).

Emergence of multiple SARS-CoV-2 antibody escape variants in an immunocompromised host undergoing convalescent plasma treatment

SARS-CoV-2 Variants of Concerns (VOC), e.g., B.1.351 (20H/501Y.V2) and P1 (20J/501Y.V3), harboring N-terminal domain (NTD) or the receptor-binding domain (RBD) (e.g., E484K) mutations, exhibit reduced in vitro susceptibility to convalescent serum, commercial antibody cocktails, and vaccine neutralization, and have been associated with reinfection. The accumulation of these mutations could be the consequence of intra-host viral evolution due to prolonged infection in immunocompromised hosts. In this study, we document the microevolution of SARS-CoV-2 recovered from sequential tracheal aspirates from an immunosuppressed patient on tacrolimus, steroids and convalescent plasma therapy, and identify the emergence of multiple NTD and RBD mutations associated with reduced antibody neutralization as early as three weeks after infection. SARS-CoV-2 genomes from the first swab (Day 0) and three tracheal aspirates (Day 7, 21 and 27) were compared at the sequence level. We identified five different S protein mutations at the NTD or RBD regions from the second tracheal aspirate sample (21 Day). The S:Q493R substitution and S:243-244LA deletion had ~70% frequency, while ORF1a:A138T, S:141-144LGVY deletion, S:E484K and S:Q493K substitutions demonstrated ~30%, ~30%, ~20% and ~10% mutation frequency, respectively. However, the third tracheal aspirate sample collected one week later (Day 27) was predominated by the haplotype of ORF1a:A138T, S:141-144LGVY deletion and S:E484K (> 95% mutation frequency). Notably, S protein deletions (141-144LGVY and 243-244LA deletions in NTD region) and substitutions (Q493K/R and E484K in the RBD region) previously showed reduced susceptibly to monoclonal antibody or convalescent plasma. The observation supports the hypothesis that VOCs can independently arise and that immunocompromised patients on convalescent plasma therapy are potential breeding grounds for immune-escape mutants.

MicroRNA Signatures Associated with Bronchopulmonary Dysplasia Severity in Tracheal Aspirates of Preterm Infants

Bronchopulmonary dysplasia (BPD) is a form of chronic lung disease that develops in neonates as a consequence of preterm birth, arrested fetal lung development, and inflammation. The incidence of BPD remains on the rise as a result of increasing survival of extremely preterm infants. Severe BPD contributes to significant health care costs and is associated with prolonged hospitalizations, respiratory infections, and neurodevelopmental deficits. In this study, we aimed to detect novel biomarkers of BPD severity. We collected tracheal aspirates (TAs) from preterm babies with mild/moderate (n = 8) and severe (n = 17) BPD, and we profiled the expression of 1048 miRNAs using a PCR array. Associations with biological pathways were determined with the Ingenuity Pathway Analysis (IPA) software. We found 31 miRNAs differentially expressed between the two disease groups (2-fold change, false discovery rate (FDR) < 0.05). Of these, 4 miRNAs displayed significantly higher expression levels, and 27 miRNAs had significantly lower expression levels in the severe BPD group when compared to the mild/moderate BPD group. IPA identified cell signaling and inflammation pathways associated with miRNA signatures. We conclude that TAs of extremely premature infants contain miRNA signatures associated with severe BPD. These may serve as potential biomarkers of disease severity in infants with BPD.

Validation of the loop-mediated isothermal amplification method for rapid and sensitive detection of Ureaplasma species in respiratory tracts of preterm infants

Introduction A simple and rapid diagnosis of Ureaplasma spp. is required for the choice of the appropriate antibiotic. However, an ideal detection method has not been available. This study examines the efficacy of the loop-mediated isothermal amplification (LAMP) assay, which provides rapid and sensitive results, to detect Ureaplasma spp. in respiratory tract samples of preterm infants. Methods The study included preterm infants born before 32 weeks of gestation admitted Kagoshima City Hospital from June 2018 to March 2020. Nasopharyngeal swabs and/or tracheal aspirates were obtained in the first seven postnatal days. One hundred sixty-seven nasopharyngeal swabs and 101 tracheal aspirates were analyzed by LAMP, culture, and quantitative real-time polymerase chain reaction. Results All 167 infants had a median (range) gestational age of 28.7 weeks (22.3–30.9) and birthweight 1030g (322–1828). One hundred sixty-seven nasopharyngeal swabs and 101 tracheal aspirates were obtained. In the results of nasopharyngeal swabs, the sensitivity and specificity of LAMP were 73.9% (17/23) and 97.2% (140/144), whereas those of quantitative real-time polymerase chain reaction were 73.9% (17/23) and 95.8% (138/144), compared to culture. In the results of tracheal aspirates, the sensitivity and specificity of LAMP were 89.5% (17/19) and 92.7% (76/82), whereas those of quantitative real-time polymerase chain reaction were 89.5% (17/19) and 93.9% (77/82), compared to culture. Conclusions The LAMP assay showed similar sensitivity and specificity with quantitative real-time polymerase chain reaction in the respiratory tracts of preterm infants including extremely preterm infants during the immediate postnatal period. Therefore, the LAMP is a practical alternative for the early detection so that appropriate antibiotics can be administered for preventing BPD.

Surveillance culture and Gram staining for detecting the causative bacteria of ventilator-associated pneumonia in neonates

Background To investigate the efficacy of surveillance culture for the rapid detection of pathogens associated with ventilator-associated pneumonia (VAP) in neonates. Methods A retrospective study was conducted on all patients with VAP in the neonatal intensive care unit. Whether causative bacteria on the culture of tracheal aspirates obtained at the onset of VAP matched with those on Gram staining and weekly surveillance culture was investigated. Result The concordance rate between surveillance culture using samples collected 4–10 days before VAP and causative bacteria was 53%. When Gram staining was performed at the onset of VAP, the concordance rate significantly increased to 86% (p = 0.022). Conclusions The concurrent use of surveillance culture and Gram staining for the detection of causative bacteria may be effective in determining initial treatment among neonates with VAP.