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19 pages, 2249 KiB

Open AccessArticle

Evaluation of Droplet Digital Polymerase Chain Reaction (ddPCR) for the Absolute Quantification of Aspergillus species in the Human Airway

by Tuang Yeow Poh, Nur A’tikah Binte Mohamed Ali, Louisa L.Y. Chan, Pei Yee Tiew and Sanjay H. Chotirmall

Int. J. Mol. Sci. 2020, 21(9), 3043; https://doi.org/10.3390/ijms21093043 - 26 Apr 2020

Cited by 18 | Viewed by 3982

Abstract

Background: Prior studies illustrate the presence and clinical importance of detecting Aspergillus species in the airways of patients with chronic respiratory disease. Despite this, a low fungal biomass and the presence of PCR inhibitors limits the usefulness of quantitative PCR (qPCR) for accurate [...] Read more.

Background: Prior studies illustrate the presence and clinical importance of detecting Aspergillus species in the airways of patients with chronic respiratory disease. Despite this, a low fungal biomass and the presence of PCR inhibitors limits the usefulness of quantitative PCR (qPCR) for accurate absolute quantification of Aspergillus in specimens from the human airway. Droplet digital PCR (ddPCR) however, presents an alternative methodology allowing higher sensitivity and accuracy of such quantification but remains to be evaluated in head-to-head fashion using specimens from the human airway. Here, we implement a standard duplex TaqMan PCR protocol, and assess if ddPCR is superior in quantifying airway Aspergillus when compared to standard qPCR. Methods: The molecular approaches of qPCR and ddPCR were applied to DNA fungal extracts in n = 20 sputum specimens obtained from non-diseased (n = 4), chronic obstructive pulmonary disease (COPD; n = 8) and non-cystic fibrosis bronchiectasis (n = 8) patients where Aspergillus status was known. DNA was extracted and qPCR and ddPCR performed on all specimens with appropriate controls and head-to-head comparisons performed. Results: Standard qPCR and ddPCR were both able to detect, even at low abundance, Aspergillus species (Aspergillus fumigatus - A. fumigatus and Aspergillus terreus - A. terreus) from specimens known to contain the respective fungi. Importantly, however, ddPCR was superior for the detection of A. terreus particularly when present at very low abundance and demonstrates greater resistance to PCR inhibition compared to qPCR. Conclusion: ddPCR has greater sensitivity for A. terreus detection from respiratory specimens, and is more resistant to PCR inhibition, important attributes considering the importance of A. terreus species in chronic respiratory disease states such as bronchiectasis. Full article

(This article belongs to the Special Issue Lung Infections: From Molecular Biology to Therapy)

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13 pages, 2858 KiB

Open AccessArticle

Optimisation and Benchmarking of Targeted Amplicon Sequencing for Mycobiome Analysis of Respiratory Specimens

by Nur A’tikah Binte Mohamed Ali, Micheál Mac Aogáin, Raika Francesca Morales, Pei Yee Tiew and Sanjay H. Chotirmall

Int. J. Mol. Sci. 2019, 20(20), 4991; https://doi.org/10.3390/ijms20204991 - 09 Oct 2019

Cited by 22 | Viewed by 4338

Abstract

(1) Background: Firm consensus has yet to be established in relation to taxonomic classification and primer choice in targeted amplicon sequencing of the mycobiome. While the nuclear ribosomal internal transcribed spacer (ITS) region are recognized as the formal fungal taxonomic barcode, appraisal of [...] Read more.

(1) Background: Firm consensus has yet to be established in relation to taxonomic classification and primer choice in targeted amplicon sequencing of the mycobiome. While the nuclear ribosomal internal transcribed spacer (ITS) region are recognized as the formal fungal taxonomic barcode, appraisal of different ITS sub-regions and the influence of DNA extraction methods have not been comprehensively undertaken using human respiratory specimens. (2) Methods: We performed ITS analysis of respiratory (sputum) samples by assessing (a) the effect of alternate DNA extraction techniques and (b) an evaluation of four different ITS primer pairs (ITS1F and ITS2; ITS1-30F and ITS1-217R; gITS7ngs and ITS4ng; and Fseq and Rseq) on the mycobiome profiles generated for mock fungal communities and their respective clinical (airway) specimens. (3) Results: Primer pairs varied in their resulting ITS mycobiome profiles, suggesting that particular pairs may be more relevant for analysis of respiratory samples compared to others. Assessment of DNA extraction methods highlighted lower final DNA concentrations achieved by mechanical disruption compared to enzymatic lysis. However, despite lower yields, DNA liberated by mechanical lysis more readily yielded ITS bands with highest success in combination with the Fseq and Rseq primers. (4) Conclusion: Choice of extraction method, primers used, and sequencing approach are all important considerations in sequencing the mycobiome and should be tailored to sample type. A standardization of approach to mycobiome studies using respiratory specimens will permit more reliable comparisons between studies and improve our understanding of the role of fungi in the human airway. Full article

(This article belongs to the Special Issue Lung Infections: From Molecular Biology to Therapy)

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12 pages, 2357 KiB

Open AccessArticle

Molecular Characterization of Influenza Strains in Patients Admitted to Intensive Care Units during the 2017–2018 Season

by Antonio Piralla, Elena Pariani, Federica Giardina, Cristina Galli, Davide Sapia, Laura Pellegrinelli, Federica Novazzi, Giovanni Anselmi, Francesca Rovida, Francesco Mojoli, Danilo Cereda, Sabrina Senatore and Fausto Baldanti

Int. J. Mol. Sci. 2019, 20(11), 2664; https://doi.org/10.3390/ijms20112664 - 30 May 2019

Cited by 4 | Viewed by 2541

Abstract

This study aimed at assessing the frequency and the distribution of influenza virus types/subtypes in 172 laboratory-confirmed influenza-positive patients admitted to intensive care units (ICUs) during the 2017–2018 season in the Lombardy region (Northern Italy), and to investigate the presence of molecular pathogenicity [...] Read more.

This study aimed at assessing the frequency and the distribution of influenza virus types/subtypes in 172 laboratory-confirmed influenza-positive patients admitted to intensive care units (ICUs) during the 2017–2018 season in the Lombardy region (Northern Italy), and to investigate the presence of molecular pathogenicity markers. A total of 102/172 (59.3%) patients had influenza A infections (83 A/H1N1pdm09, 2 H3N2 and 17 were untyped), while the remaining 70/172 (40.7%) patients had influenza B infections. The 222G/N mutation in the hemagglutinin gene was identified in 33.3% (3/9) of A/H1N1pdm09 strains detected in the lower respiratory tract (LRT) samples and was also associated with more severe infections, whereas no peculiar mutations were observed for influenza B strains. A single-point evolution was observed in site 222 of A/H1N1pdm09 viruses, which might advantage viral evolution by favouring virus binding and replication in the lungs. Data from 17 paired upper respiratory tract (URT) and LRT samples showed that viral load in LRT samples was mostly higher than that detected in URT samples. Of note, influenza viruses were undetectable in 35% of paired URT samples. In conclusion, LRT samples appear to provide more accurate clinical information than URT samples, thus ensuring correct diagnosis and appropriate treatment of patients with severe respiratory infections requiring ICU admission. Full article

(This article belongs to the Special Issue Lung Infections: From Molecular Biology to Therapy)

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25 pages, 3206 KiB

Open AccessArticle

Transcriptomic Analysis Reveals Priming of The Host Antiviral Interferon Signaling Pathway by Bronchobini^® Resulting in Balanced Immune Response to Rhinovirus Infection in Mouse Lung Tissue Slices

by Stella Marie Reamon-Buettner, Monika Niehof, Natalie Hirth, Olga Danov, Helena Obernolte, Armin Braun, Jürgen Warnecke, Katherina Sewald and Sabine Wronski

Int. J. Mol. Sci. 2019, 20(9), 2242; https://doi.org/10.3390/ijms20092242 - 07 May 2019

Cited by 7 | Viewed by 3706

Abstract

Rhinovirus (RV) is the predominant virus causing respiratory tract infections. Bronchobini^® is a low dose multi component, multi target preparation used to treat inflammatory respiratory diseases such as the common cold, described to ease severity of symptoms such as cough and viscous [...] Read more.

Rhinovirus (RV) is the predominant virus causing respiratory tract infections. Bronchobini^® is a low dose multi component, multi target preparation used to treat inflammatory respiratory diseases such as the common cold, described to ease severity of symptoms such as cough and viscous mucus production. The aim of the study was to assess the efficacy of Bronchobini^® in RV infection and to elucidate its mode of action. Therefore, Bronchobini^®’s ingredients (BRO) were assessed in an ex vivo model of RV infection using mouse precision-cut lung slices, an organotypic tissue capable to reflect the host immune response to RV infection. Cytokine profiles were assessed using enzyme-linked immunosorbent assay (ELISA) and mesoscale discovery (MSD). Gene expression analysis was performed using Affymetrix microarrays and ingenuity pathway analysis. BRO treatment resulted in the significant suppression of RV-induced antiviral and pro-inflammatory cytokine release. Transcriptome analysis revealed a multifactorial mode of action of BRO, with a strong inhibition of the RV-induced pro-inflammatory and antiviral host response mediated by nuclear factor kappa B (NFkB) and interferon signaling pathways. Interestingly, this was due to priming of these pathways in the absence of virus. Overall, BRO exerted its beneficial anti-inflammatory effect by priming the antiviral host response resulting in a reduced inflammatory response to RV infection, thereby balancing an otherwise excessive inflammatory response. Full article

(This article belongs to the Special Issue Lung Infections: From Molecular Biology to Therapy)

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12 pages, 4661 KiB

Open AccessArticle

How to Process Sputum Samples and Extract Bacterial DNA for Microbiota Analysis

by Leonardo Terranova, Martina Oriano, Antonio Teri, Luca Ruggiero, Camilla Tafuro, Paola Marchisio, Andrea Gramegna, Martina Contarini, Elisa Franceschi, Samantha Sottotetti, Lisa Cariani, Annamaria Bevivino, James D. Chalmers, Stefano Aliberti and Francesco Blasi

Int. J. Mol. Sci. 2018, 19(10), 3256; https://doi.org/10.3390/ijms19103256 - 20 Oct 2018

Cited by 24 | Viewed by 7449

Abstract

Different steps and conditions for DNA extraction for microbiota analysis in sputum have been reported in the literature. We aimed at testing both dithiothreitol (DTT) and enzymatic treatments of sputum samples and identifying the most suitable DNA extraction technique for the microbiota analysis [...] Read more.

Different steps and conditions for DNA extraction for microbiota analysis in sputum have been reported in the literature. We aimed at testing both dithiothreitol (DTT) and enzymatic treatments of sputum samples and identifying the most suitable DNA extraction technique for the microbiota analysis of sputum. Sputum treatments with and without DTT were compared in terms of their median levels and the coefficient of variation between replicates of both DNA extraction yield and real-time PCR for the 16S rRNA gene. Treatments with and without lysozyme and lysostaphin were compared in terms of their median levels of real-time PCR for S. aureus. Two enzyme-based and three beads-based techniques for DNA extraction were compared in terms of their DNA extraction yield, real-time PCR for the 16S rRNA gene and microbiota analysis. DTT treatment decreased the coefficient of variation between replicates of both DNA extraction yield and real-time PCR. Lysostaphin (either 0.18 or 0.36 mg/mL) and lysozyme treatments increased S. aureus detection. One enzyme-based kit offered the highest DNA yield and 16S rRNA gene real-time PCR with no significant differences in terms of alpha-diversity indexes. A condition using both DTT and lysostaphin/lysozyme treatments along with an enzymatic kit seems to be preferred for the microbiota analysis of sputum samples. Full article

(This article belongs to the Special Issue Lung Infections: From Molecular Biology to Therapy)

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Review

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16 pages, 307 KiB

Open AccessReview

Challenges in the Diagnosis and Management of Bacterial Lung Infections in Solid Organ Recipients: A Narrative Review

by Manuela Carugati, Letizia Corinna Morlacchi, Anna Maria Peri, Laura Alagna, Valeria Rossetti, Alessandra Bandera, Andrea Gori, Francesco Blasi and on behalf of the IFALT Working Group

Int. J. Mol. Sci. 2020, 21(4), 1221; https://doi.org/10.3390/ijms21041221 - 12 Feb 2020

Cited by 7 | Viewed by 2338

Abstract

Respiratory infections pose a significant threat to the success of solid organ transplantation, and the diagnosis and management of these infections are challenging. The current narrative review addressed some of these challenges, based on evidence from the literature published in the last 20 [...] Read more.

Respiratory infections pose a significant threat to the success of solid organ transplantation, and the diagnosis and management of these infections are challenging. The current narrative review addressed some of these challenges, based on evidence from the literature published in the last 20 years. Specifically, we focused our attention on (i) the obstacles to an etiologic diagnosis of respiratory infections among solid organ transplant recipients, (ii) the management of bacterial respiratory infections in an era characterized by increased antimicrobial resistance, and (iii) the development of antimicrobial stewardship programs dedicated to solid organ transplant recipients. Full article

(This article belongs to the Special Issue Lung Infections: From Molecular Biology to Therapy)

20 pages, 386 KiB

Open AccessReview

Animal Models of Pneumococcal pneumonia

by Noemi Borsa, Marta Di Pasquale and Marcos I. Restrepo

Int. J. Mol. Sci. 2019, 20(17), 4220; https://doi.org/10.3390/ijms20174220 - 28 Aug 2019

Cited by 15 | Viewed by 6549

Abstract

Streptococcus pneumoniae remains the most common bacterial pathogen causing lower respiratory tract infections and is a leading cause of morbidity and mortality worldwide, especially in children and the elderly. Another important aspect related to pneumococcal infections is the persistent rate of penicillin and [...] Read more.

Streptococcus pneumoniae remains the most common bacterial pathogen causing lower respiratory tract infections and is a leading cause of morbidity and mortality worldwide, especially in children and the elderly. Another important aspect related to pneumococcal infections is the persistent rate of penicillin and macrolide resistance. Therefore, animal models have been developed to better understand the pathogenesis of pneumococcal disease and test new therapeutic agents and vaccines. This narrative review will focus on the characteristics of the different animal pneumococcal pneumonia models. The assessment of the different animal models will include considerations regarding pneumococcal strains, microbiology properties, procedures used for bacterial inoculation, pathogenesis, clinical characteristics, diagnosis, treatment, and preventive approaches. Full article

(This article belongs to the Special Issue Lung Infections: From Molecular Biology to Therapy)

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15 pages, 1795 KiB

Open AccessReview

Cytomegalovirus (CMV) Pneumonitis: Cell Tropism, Inflammation, and Immunity

by Luís Fonseca Brito, Wolfram Brune and Felix R. Stahl

Int. J. Mol. Sci. 2019, 20(16), 3865; https://doi.org/10.3390/ijms20163865 - 08 Aug 2019

Cited by 23 | Viewed by 6593

Abstract

Human cytomegalovirus (HCMV) is an opportunistic pathogen causing disease mainly in immunocompromised patients or after congenital infection. HCMV infection of the respiratory tract leads to pneumonitis in the immunocompromised host, which is often associated with a bad clinical course. The related mouse cytomegalovirus [...] Read more.

Human cytomegalovirus (HCMV) is an opportunistic pathogen causing disease mainly in immunocompromised patients or after congenital infection. HCMV infection of the respiratory tract leads to pneumonitis in the immunocompromised host, which is often associated with a bad clinical course. The related mouse cytomegalovirus (MCMV) likewise exhibits a distinct tropism for the lung and thus provides an elegant model to study host-pathogen interaction. Accordingly, fundamental features of cytomegalovirus (CMV) pneumonitis have been discovered in mice that correlate with clinical data obtained from humans. Recent studies have provided insight into MCMV cell tropism and localized inflammation after infection of the respiratory tract. Accordingly, the nodular inflammatory focus (NIF) has been identified as the anatomical correlate of immune control in lungs. Several hematopoietic cells involved in antiviral immunity reside in NIFs and their key effector molecules have been deciphered. Here, we review what has been learned from the mouse model with focus on the microanatomy of infection sites and antiviral immunity in MCMV pneumonitis. Full article

(This article belongs to the Special Issue Lung Infections: From Molecular Biology to Therapy)

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18 pages, 251 KiB

Open AccessReview

Biomarkers in Pneumonia—Beyond Procalcitonin

by Meropi Karakioulaki and Daiana Stolz

Int. J. Mol. Sci. 2019, 20(8), 2004; https://doi.org/10.3390/ijms20082004 - 24 Apr 2019

Cited by 75 | Viewed by 9135

Abstract

Pneumonia is the leading infectious cause of mortality worldwide and one of the most common lower respiratory tract infections that is contributing significantly to the burden of antibiotic consumption. Due to the complexity of its pathophysiology, it is widely accepted that clinical diagnosis [...] Read more.

Pneumonia is the leading infectious cause of mortality worldwide and one of the most common lower respiratory tract infections that is contributing significantly to the burden of antibiotic consumption. Due to the complexity of its pathophysiology, it is widely accepted that clinical diagnosis and prognosis are inadequate for the accurate assessment of the severity of the disease. The most challenging task for a physician is the risk stratification of patients with community-acquired pneumonia. Herein, early diagnosis is essential in order to reduce hospitalization and mortality. Procalcitonin and C-reactive protein remain the most widely used biomarkers, while interleukin 6 has been of particular interest in the literature. However, none of them appear to be ideal, and the search for novel biomarkers that will most sufficiently predict the severity and treatment response in pneumonia has lately intensified. Although our insight has significantly increased over the last years, a translational approach with the application of genomics, metabolomics, microbiomics, and proteomics is required to better understand the disease. In this review, we discuss this rapidly evolving area and summarize the application of novel biomarkers that appear to be promising for the accurate diagnosis and risk stratification of pneumonia. Full article

(This article belongs to the Special Issue Lung Infections: From Molecular Biology to Therapy)

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