Respiratory Pathogens
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2022 ◽  
Vol 2 (1) ◽  
Long T. Nguyen ◽  
Santosh R. Rananaware ◽  
Brianna L. M. Pizzano ◽  
Brandon T. Stone ◽  
Piyush K. Jain

Abstract Background The coronavirus disease (COVID-19) caused by SARS-CoV-2 has swept through the globe at an unprecedented rate. CRISPR-based detection technologies have emerged as a rapid and affordable platform that can shape the future of diagnostics. Methods We developed ENHANCEv2 that is composed of a chimeric guide RNA, a modified LbCas12a enzyme, and a dual reporter construct to improve the previously reported ENHANCE system. We validated both ENHANCE and ENHANCEv2 using 62 nasopharyngeal swabs and compared the results to RT-qPCR. We created a lyophilized version of ENHANCEv2 and characterized its detection capability and stability. Results Here we demonstrate that when coupled with an RT-LAMP step, ENHANCE detects COVID-19 samples down to a few copies with 95% accuracy while maintaining a high specificity towards various isolates of SARS-CoV-2 against 31 highly similar and common respiratory pathogens. ENHANCE works robustly in a wide range of magnesium concentrations (3 mM-13 mM), allowing for further assay optimization. Our clinical validation results for both ENHANCE and ENHANCEv2 show 60/62 (96.7%) sample agreement with RT-qPCR results while only using 5 µL of sample and 20 minutes of CRISPR reaction. We show that the lateral flow assay using paper-based strips displays 100% agreement with the fluorescence-based reporter assay during clinical validation. Finally, we demonstrate that a lyophilized version of ENHANCEv2 shows high sensitivity and specificity for SARS-CoV-2 detection while reducing the CRISPR reaction time to as low as 3 minutes while maintaining its detection capability for several weeks upon storage at room temperature. Conclusions CRISPR-based diagnostic platforms offer many advantages as compared to conventional qPCR-based detection methods. Our work here provides clinical validation of ENHANCE and its improved form ENHANCEv2 for the detection of COVID-19.

2022 ◽  
Vol 10 (1) ◽  
pp. 134
Chunli Li ◽  
Rahat Zaheer ◽  
Andrea Kinnear ◽  
Murray Jelinski ◽  
Tim A. McAllister

A comparative study of microbiota of the respiratory tract and joints of bovine respiratory disease (BRD) cattle mortalities was undertaken. Nasopharynx, trachea, lung and joint samples were collected from 32 cattle that died of BRD, “cases”, and 8 that died of other causes, “controls”. Bacterial diversity was lower (p < 0.05) in the nasopharynx, trachea and lungs of cases as compared to controls. In cases, alpha-diversity (p < 0.05) was lower in the lungs and joints than the nasopharynx. Proteobacteria, Tenericutes, Bacteroidetes, Firmicutes and Actinobacteria were the most abundant phyla in all samples. Relative abundances of Mycoplasma spp. in the lung, Pasteurella spp. in the trachea and lung, and Histophilus spp. in the lung, trachea and nasopharynx of cases were higher (p < 0.001) than controls. Mycoplasma spp. comprised 20.5% of bacterial flora in the joint, 36.0% in the lung, 22.4% in the trachea and 8.8% in the nasopharynx. Mannheimia spp. (21.8%) and Histophilus spp. (10.4%) were more abundant in lungs. Cattle that died of BRD possessed less diverse respiratory microbiomes with a higher abundance of respiratory pathogens. Mycoplasma spp. were prominent members of pneumonic lungs and joints displaying septic arthritis.

2022 ◽  
pp. 2101013
Alexander Y. Trick ◽  
Fan‐En Chen ◽  
Liben Chen ◽  
Pei‐Wei Lee ◽  
Alexander C. Hasnain ◽  

2022 ◽  
Vol 12 ◽  
Sylwia Jarzynka ◽  
Riccardo Spott ◽  
Tinatini Tchatchiashvili ◽  
Nico Ueberschaar ◽  
Mark Grevsen Martinet ◽  

Human milk oligosaccharides (HMOs) have been shown to exhibit plenty of benefits for infants, such as prebiotic activity shaping the gut microbiota and immunomodulatory and anti-inflammatory activity. For some pathogenic bacteria, antimicrobial activity has been proved, but most studies focus on group B streptococci. In the present study, we investigated the antimicrobial and antibiofilm activities of the total and fractionated HMOs from pooled human milk against four common human pathogenic Gram-negative species (Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Burkholderia cenocepacia) and three Gram-positive species (Staphylococcus aureus, Enterococcus faecium, and Enterococcus faecalis). The activity of HMOs against enterococci and B. cenocepacia are addressed here for the first time. We showed that HMOs exhibit a predominant activity against the Gram-positive species, with E. faecalis being the most sensitive to the HMOs, both in planktonic bacteria and in biofilms. In further tests, we could exclude fucosyllactose as the antibacterial component. The biological significance of these findings may lie in the prevention of skin infections of the mother’s breast as a consequence of breastfeeding-induced skin laceration and/or protection of the infants’ nasopharynx and lung from respiratory pathogens such as staphylococci.

2022 ◽  
Vol 9 ◽  
Kun Li ◽  
Christine Wohlford-Lenane ◽  
Jennifer A. Bartlett ◽  
Paul B. McCray

Middle East respiratory syndrome coronavirus (MERS-CoV) causes respiratory infection in humans, with symptom severity that ranges from asymptomatic to severe pneumonia. Known risk factors for severe MERS include male sex, older age, and the presence of various comorbidities. MERS-CoV gains entry into cells by binding its receptor, dipeptidyl peptidase 4 (DPP4), on the surface of airway epithelia. We hypothesized that expression of this receptor might be an additional determinant of outcomes in different individuals during MERS-CoV infection. To learn more about the role of DPP4 in facilitating MERS-CoV infection and spread, we used ELISA and immunofluorescent staining to characterize DPP4 expression in well-differentiated primary human airway epithelia (HAE). We noted wide inter-individual variation in DPP4 abundance, varying by as much as 1000-fold between HAE donors. This variability appears to influence multiple aspects of MERS-CoV infection and pathogenesis, with greater DPP4 abundance correlating with early, robust virus replication and increased cell sloughing. We also observed increased induction of interferon and some interferon-stimulated genes in response to MERS-CoV infection in epithelia with the greatest DPP4 abundance. Overall, our results indicate that inter-individual differences in DPP4 abundance are one host factor contributing to MERS-CoV replication and host defense responses, and highlight how HAE may serve as a useful model for identifying risk factors associated with heightened susceptibility to serious respiratory pathogens.

2022 ◽  
Vol 8 ◽  
Jiajia Dong ◽  
Wei Li ◽  
Qi Wang ◽  
Jiahao Chen ◽  
Yue Zu ◽  

Oral microecosystem is a very complicated ecosystem that is located in the mouth and comprises oral microbiome, diverse anatomic structures of oral cavity, saliva and interactions between oral microbiota and between oral microbiota and the host. More and more evidence from studies of epidemiology, microbiology and molecular biology is establishing a significant link between oral microecosystem and respiratory diseases. Microbiota settling down in oral microecosystem is known as the main source of lung microbiome and has been associated with the occurrence and development of respiratory diseases like pneumonia, chronic obstructive pulmonary disease, lung cancer, cystic fibrosis lung disease and asthma. In fact, it is not only indigenous oral microbes promote or directly cause respiratory infection and inflammation when inhaled into the lower respiratory tract, but also internal environment of oral microecosystem serves as a reservoir for opportunistic respiratory pathogens. Moreover, poor oral health and oral diseases caused by oral microecological dysbiosis (especially periodontal disease) are related with risk of multiple respiratory diseases. Here, we review the research status on the respiratory diseases related with oral microecosystem. Potential mechanisms on how respiratory pathogens colonize oral microecosystem and the role of indigenous oral microbes in pathogenesis of respiratory diseases are also summarized and analyzed. Given the importance of oral plaque control and oral health interventions in controlling or preventing respiratory infection and diseases, we also summarize the oral health management measures and attentions, not only for populations susceptible to respiratory infection like the elderly and hospitalized patients, but also for dentist or oral hygienists who undertake oral health care. In conclusion, the relationship between respiratory diseases and oral microecosystem has been established and supported by growing body of literature. However, etiological evidence on the role of oral microecosystem in the development of respiratory diseases is still insufficient. Further detailed studies focusing on specific mechanisms on how oral microecosystem participate in the pathogenesis of respiratory diseases could be helpful to prevent and treat respiratory diseases.

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Asmae Lamrani Hanchi ◽  
Morad Guennouni ◽  
Meriem Rachidi ◽  
Toufik Benhoumich ◽  
Hind Bennani ◽  

Sever acute respiratory infections (SARIs) are a public health issue that are common in children and are associated with an important morbidity and mortality rate worldwide. Although SARI are mainly caused by viruses, they are still a cause of antibiotic overuse. The use of molecular methods especially real-time multiplex PCR allowed to detect a wide range of respiratory viruses and their subtype as well as some atypical bacteria. The aim of this study was to investigate the epidemiology of respiratory pathogens detected in children admitted with SARI and to highlight the role of real-time multiplex PCR in the rapid diagnosis of viral and bacterial SARI. This work is a descriptive observational study from January 2018 to December 2019 including nasopharyngeal secretions collected from 534 children hospitalised in paediatric department. The detection of respiratory viruses and bacteria was performed by the FilmArray® Respiratory Panel. A total of 387 (72.5%) children were tested positive for at least one respiratory pathogen, and 23.3% of them were coinfected with more than one pathogen. Viral aetiology was found in 91.2% (n = 340). The most common viruses detected were HRV (n = 201) and RSV (n = 124), followed by PIV (n = 35) influenza A (n = 29) and human metapneumovirus (n = 27). Bacteria was found in 8.8% (n = 47), and Bordetella pertussis was the most detected. Respiratory syncytial virus and Bordetella pertussis were significantly higher in infants less than 6 months old. The detection of RSV and influenza A presented a pic in winter, and HMPV was statistically significant in spring ( p < 0.01 ). This study described the epidemiology of respiratory pathogens involved in severe respiratory infections in children that were affected by several factors such as season and age group. It also highlighted the importance of multiplex PCR in confirming viral origin, thus avoiding irrational prescription of antibiotics in paediatric settings.

Diagnostics ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 9
Harshad Lade ◽  
Jung-Min Kim ◽  
Yousun Chung ◽  
Minje Han ◽  
Eun-Kyung Mo ◽  

Multiplex nucleic acid amplification assays that simultaneously detect multiple respiratory pathogens in a single nasopharyngeal swab (NPS) specimen are widely used for rapid clinical diagnostics. We evaluated Allplex Respiratory Panel (RP) 1, 2, 3, and the BioFire FilmArray RP assay for detecting respiratory pathogens from NPS specimens. In all, 181 NPS specimens obtained from patients suspected of having respiratory infections during the non-influenza season (August–December 2019) were included. The Allplex RP 1, 2, and 3 detected 154 samples positive for respiratory viruses, whereas the BioFire FilmArray detected viruses in 98 samples. Co-infection with two or more viruses was detected in 41 and 17 NPS specimens by Allplex RP and the BioFire FilmArray RP, respectively. For adenoviruses, Allplex RP 1 detected 31 specimens, compared to 34 by the BioFire FilmArray. In all, 64 NPS specimens were positive for human enterovirus (HEV) and human rhinovirus (HRV) on the Allplex RP, in contrast to 39 HEV/HRV on the BioFire FilmArray. The parainfluenza virus (PIV-1–4) detection rate differed between the two systems. Most discrepant results were observed for NPS specimens with high cycle threshold values obtained by Allplex RP. This study showed concordant performance of the Allplex RP 1, 2, 3, and the BioFire FilmArray RP for the simultaneous detection of multiple respiratory viruses.

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2519
Christoforos Rozario ◽  
Luis Martínez-Sobrido ◽  
Henry J. McSorley ◽  
Caroline Chauché

Influenza A viruses (IAVs) are important respiratory pathogens of horses and humans. Infected individuals develop typical respiratory disorders associated with the death of airway epithelial cells (AECs) in infected areas. Virulence and risk of secondary bacterial infections vary among IAV strains. The IAV non-structural proteins, NS1, PB1-F2, and PA-X are important virulence factors controlling AEC death and host immune responses to viral and bacterial infection. Polymorphism in these proteins impacts their function. Evidence from human and mouse studies indicates that upon IAV infection, the manner of AEC death impacts disease severity. Indeed, while apoptosis is considered anti-inflammatory, necrosis is thought to cause pulmonary damage with the release of damage-associated molecular patterns (DAMPs), such as interleukin-33 (IL-33). IL-33 is a potent inflammatory mediator released by necrotic cells, playing a crucial role in anti-viral and anti-bacterial immunity. Here, we discuss studies in human and murine models which investigate how viral determinants and host immune responses control AEC death and subsequent lung IL-33 release, impacting IAV disease severity. Confirming such data in horses and improving our understanding of early immunologic responses initiated by AEC death during IAV infection will better inform the development of novel therapeutic or vaccine strategies designed to protect life-long lung health in horses and humans, following a One Health approach.

Hermógenes Fernández-Marín ◽  
Gaspar Bruner-Montero ◽  
Ana Portugal-Loayza ◽  
Virginia Miranda ◽  
Alcibíades Villareal ◽  

Early in the SARS-CoV-2 pandemic, many national public health authorities implemented non-pharmaceutical interventions to mitigate disease outbreaks. Panamá established mandatory mask use two months after its first documented case. Initial compliance was high, but diverse masks were used in public areas. We studied behavioral dynamics of mask use through the first two COVID-19 waves in Panama, to improve the implementation of effective, low-cost public health containment measures when populations are exposed to novel air-borne pathogens. Mask use behavior was recorded from pedestrians in four Panamanian populations (August to December 2020). We recorded facial coverings and if used, the type of mask, and gender and estimated age of the wearer. Our results showed that people were highly compliant (>95%) with mask mandates and demonstrated important population-level behaviors: (1) decreasing use of cloth masks over time, and increasing use of surgical masks; (2) mask use was 3-fold lower in suburban neighborhoods than other public areas and (3) young people were least likely to wear masks. Results help focus on highly effective, low-cost, public health interventions for managing and controlling a pandemic. Considerations of behavioral preferences for different masks, relative to pricing and availability, are essential for optimizing public health policies. Policies to increase the availability of effective masks, and behavioral nudges to increase acceptance, and to facilitate mask usage, during the ongoing SARS-CoV-2 pandemic, and for future pandemics of respiratory pathogens, are key tools, especially for nations lagging in access to expensive vaccines and pharmacological approaches.

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