Publications by authors named "Ronald G Collman"

133 Publications

Single- and Two-Stage, Closed-Tube, Point-of-Care, Molecular Detection of SARS-CoV-2.

Anal Chem 2021 Sep 20. Epub 2021 Sep 20.

Department of Mechanical Engineering and Applied Mechanics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.

Short of a vaccine, frequent and rapid testing, preferably at home, is the most effective strategy to contain the COVID-19 pandemic. Herein, we report on single-stage and two-stage molecular diagnostic tests that can be carried out with simple or no instrumentation. Our single-stage amplification is reverse transcription-loop mediated isothermal amplification (RT-LAMP) with custom-designed primers targeting the ORF1ab and the N gene regions of the virus genome. Our new two-stage amplification, dubbed Penn-RAMP, comprises recombinase isothermal amplification (RT-RPA) as its first stage and LAMP as its second stage. We compared various sample preparation strategies aimed at deactivating the virus while preserving its RNA and tested contrived and patient samples, consisting of nasopharyngeal swabs, oropharyngeal swabs, and saliva. Amplicons were detected either in real time with fluorescent intercalating dye or after amplification with the intercalating colorimetric dye LCV, which is insensitive to sample's PH. Our single RT-LAMP tests can be carried out instrumentation-free. To enable concurrent testing of multiple samples, we developed an inexpensive heat block that supports both the single-stage and two-stage amplification. Our RT-LAMP and Penn-RAMP assays have, respectively, analytical sensitivities of 50 and 5 virions/reaction. Both our single- and two-stage assays have successfully detected SARS-CoV-2 in patients with viral loads corresponding to the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) threshold cycle smaller than 32 while operating with minimally processed samples, without nucleic acid isolation. Penn-RAMP provides a 10-fold better sensitivity than RT-LAMP and does not need thermal cycling like PCR assays. All reagents are amenable to dry, refrigeration-free storage. The SARS-CoV-2 test described herein is suitable for screening at home, at the point of need, and in resource-poor settings.
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http://dx.doi.org/10.1021/acs.analchem.1c03016DOI Listing
September 2021

Lack of Atorvastatin Effect on Monocyte Gene Expression and Inflammatory Markers in HIV-1-infected ART-suppressed Individuals at Risk of non-AIDS Comorbidities.

Pathog Immun 2021 13;6(2):1-26. Epub 2021 Aug 13.

Department of Medicine; University of Pennsylvania Perelman School of Medicine, Philadelphia, PA.

Background: Many people living with HIV have persistent monocyte activation despite viral suppression by antiretroviral therapy (ART), which contributes to non-AIDS complications including neurocognitive and other disorders. Statins have immunomodulatory properties that might be beneficial by reducing monocyte activation.

Methods: We previously characterized monocyte gene expression and inflammatory markers in 11 HIV-positive individuals on long-term ART (HIV/ART) at risk for non-AIDS complications because of low nadir CD4+ counts (median 129 cells/uL) and elevated hsCRP. Here, these individuals participated in a double-blind, randomized, placebo-controlled crossover study of 12 weeks of atorvastatin treatment. Monocyte surface markers were assessed by flow cytometry, plasma mediators by ELISA and Luminex, and monocyte gene expression by microarray analysis.

Results: Among primary outcome measures, 12 weeks of atorvastatin treatment led to an unexpected increase in CCR2+ monocytes (=0.04), but did not affect CD16+ or CD163+ monocytes, nor levels in plasma of CCL2/MCP-1 or sCD14. Among secondary outcomes, atorvastatin treatment was associated with decreased plasma hsCRP (=0.035) and IL-2R (=0.012). Treatment was also associated with increased total CD14+ monocytes (=0.015), and increased plasma CXCL9 (=0.003) and IL-12 (<0.001). Comparable results were seen in a subgroup that had inflammatory marker elevations at baseline. Atorvastatin treatment did not significantly alter monocyte gene expression or normalize aberrant baseline transcriptional patterns.

Conclusions: In this study of aviremic HIV+ individuals at high risk of non-AIDS events, 12 weeks of atorvastatin did not normalize monocyte gene expression patterns nor lead to significant changes in monocyte surface markers or plasma mediators linked to non-AIDS comorbidities.
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http://dx.doi.org/10.20411/pai.v6i2.461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8382234PMC
August 2021

Redondovirus diversity and evolution on global, individual, and molecular scales.

J Virol 2021 Aug 18:JVI0081721. Epub 2021 Aug 18.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

is a newly-established family of circular Rep-encoding single stranded (CRESS) DNA viruses found in the human oro-respiratory tract. Redondoviruses were previously found in ∼15% of respiratory specimens from US urban subjects; levels were elevated in individuals with periodontitis or critical illness. Here, we report higher redondovirus prevalence in saliva samples: four rural African populations showed 61-82% prevalence, and an urban US population showed 32% prevalence. Longitudinal, limiting-dilution single-genome sequencing revealed diverse strains of both redondovirus species ( and ) in single individuals, persistence over time, and evidence of inter-genomic recombination. Computational analysis of viral genomes identified a recombination hot spot associated with a conserved potential DNA stem loop structure. To assess the possible role of this site in recombination, we carried out studies which showed that this potential stem-loop was cleaved by the virus-encoded Rep protein. In addition, in reconstructed reactions, a Rep-DNA covalent intermediate was shown to mediate DNA strand transfer at this site. Thus, redondoviruses are highly prevalent in humans, found in individuals on multiple continents, heterogeneous even within individuals, and encode a Rep protein implicated in facilitating recombination. is a recently established family of DNA viruses predominantly found in the human respiratory tract and associated with multiple clinical conditions. In this study, we found high redondovirus prevalence in saliva from urban North American individuals and non-industrialized African populations from Botswana, Cameroon, Ethiopia, and Tanzania. Individuals on continents harbored both known redondovirus species. Global prevalence of both species suggests that redondoviruses have long been associated with humans but have remained undetected until recently due to their divergent genomes. By sequencing single redondovirus genomes in longitudinally-sampled humans, we found that redondoviruses persisted over time within subjects and likely evolve by recombination. The Rep protein encoded by redondoviruses catalyzes multiple reactions , consistent with a role in mediating DNA replication and recombination. In summary, we identify high redondovirus prevalence in humans across multiple continents, longitudinal heterogeneity and persistence, and potential mechanisms of redondovirus evolution by recombination.
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http://dx.doi.org/10.1128/JVI.00817-21DOI Listing
August 2021

Signatures of COVID-19 Severity and Immune Response in the Respiratory Tract Microbiome.

mBio 2021 08 17;12(4):e0177721. Epub 2021 Aug 17.

Pulmonary, Allergy and Critical Care Division, Department of Medicine, University of Pennsylvaniagrid.25879.31 Perelman School of Medicine, Philadelphia, Pennsylvania, USA.

Viral infection of the respiratory tract can be associated with propagating effects on the airway microbiome, and microbiome dysbiosis may influence viral disease. Here, we investigated the respiratory tract microbiome in coronavirus disease 2019 (COVID-19) and its relationship to disease severity, systemic immunologic features, and outcomes. We examined 507 oropharyngeal, nasopharyngeal, and endotracheal samples from 83 hospitalized COVID-19 patients as well as non-COVID patients and healthy controls. Bacterial communities were interrogated using 16S rRNA gene sequencing, and the commensal DNA viruses and were quantified by qPCR. We found that COVID-19 patients had upper respiratory microbiome dysbiosis and greater change over time than critically ill patients without COVID-19. Oropharyngeal microbiome diversity at the first time point correlated inversely with disease severity during hospitalization. Microbiome composition was also associated with systemic immune parameters in blood, as measured by lymphocyte/neutrophil ratios and immune profiling of peripheral blood mononuclear cells. Intubated patients showed patient-specific lung microbiome communities that were frequently highly dynamic, with prominence of Staphylococcus. and showed more frequent colonization and higher titers in severe disease. Machine learning analysis demonstrated that integrated features of the microbiome at early sampling points had high power to discriminate ultimate level of COVID-19 severity. Thus, the respiratory tract microbiome and commensal viruses are disturbed in COVID-19 and correlate with systemic immune parameters, and early microbiome features discriminate disease severity. Future studies should address clinical consequences of airway dysbiosis in COVID-19, its possible use as biomarkers, and the role of bacterial and viral taxa identified here in COVID-19 pathogenesis. COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of the respiratory tract, results in highly variable outcomes ranging from minimal illness to death, but the reasons for this are not well understood. We investigated the respiratory tract bacterial microbiome and small commensal DNA viruses in hospitalized COVID-19 patients and found that each was markedly abnormal compared to that in healthy people and differed from that in critically ill patients without COVID-19. Early airway samples tracked with the level of COVID-19 illness reached during hospitalization, and the airway microbiome also correlated with immune parameters in blood. These findings raise questions about the mechanisms linking SARS-CoV-2 infection and other microbial inhabitants of the airway, including whether the microbiome might regulate severity of COVID-19 disease and/or whether early microbiome features might serve as biomarkers to discriminate disease severity.
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http://dx.doi.org/10.1128/mBio.01777-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8406335PMC
August 2021

Femtomolar SARS-CoV-2 Antigen Detection Using the Microbubbling Digital Assay with Smartphone Readout Enables Antigen Burden Quantitation and Tracking.

Clin Chem 2021 Aug 12. Epub 2021 Aug 12.

Department of Pathology and Laboratory Medicine Penn Center for Research on Coronavirus and Other Emerging Pathogens.

Background: High sensitivity SARS-CoV-2 antigen assays are desirable to mitigate false negative results. Limited data are available to quantify and track SARS-CoV-2 antigen burden in respiratory samples from different populations.

Methods: We developed the Microbubbling SARS-CoV-2 Antigen Assay (MSAA) with smartphone readout, with a limit of detection (LOD) of 0.5 pg/mL (10.6 fmol/L) nucleocapsid (N) antigen or 4000 copies/mL inactivated SARS-CoV-2 virus in nasopharyngeal (NP) swabs. We developed a computer vision and machine learning-based automatic microbubble image classifier to accurately identify positives and negatives, and quantified and tracked antigen dynamics in ICU COVID inpatients and immunocompromised COVID patients.

Results: Compared to qualitative RT-PCR methods, the MSAA demonstrated a positive percent agreement (PPA) of 97% (95% confidence interval (CI), 92-99%) and a negative percent agreement (NPA) of 97% (95% CI, 94-100%) in a clinical validation study with 372 residual clinical NP swabs. In immunocompetent individuals, the antigen positivity rate in swabs decreased as days-after-symptom-onset increased, despite persistent nucleic acid positivity. Antigen was detected for longer and variable periods of time in immunocompromised patients with hematologic malignancies. Total microbubble volume, a quantitative marker of antigen burden, correlated inversely with Ct values and days-after-symptom-onset. Viral sequence variations were detected in patients with long duration of high antigen burden.

Conclusions: The MSAA enables sensitive and specific detection of acute infections, quantification and tracking of antigen burden, and may serve as a screening method in longitudinal studies to identify patients who are likely experiencing active rounds of ongoing replication and warrant close viral sequence monitoring.
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http://dx.doi.org/10.1093/clinchem/hvab158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8436368PMC
August 2021

The lung microbiome: progress and promise.

J Clin Invest 2021 Aug;131(15)

The healthy lung was long thought of as sterile, but recent advances using molecular sequencing approaches have detected bacteria at low levels. Healthy lung bacteria largely reflect communities present in the upper respiratory tract that enter the lung via microaspiration, which is balanced by mechanical and immune clearance and likely involves limited local replication. The nature and dynamics of the lung microbiome, therefore, differ from those of ecological niches with robust self-sustaining microbial communities. Aberrant populations (dysbiosis) have been demonstrated in many pulmonary diseases not traditionally considered microbial in origin, and potential pathways of microbe-host crosstalk are emerging. The question now is whether and how dysbiotic microbiota contribute to initiation or perpetuation of injury. The fungal microbiome and virome are less well studied. This Review highlights features of the lung microbiome, unique considerations in studying it, examples of dysbiosis in selected disease, emerging concepts in lung microbiome-host interactions, and critical areas for investigation.
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http://dx.doi.org/10.1172/JCI150473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8321564PMC
August 2021

The lung microbiome in lung transplantation.

J Heart Lung Transplant 2021 Aug 7;40(8):733-744. Epub 2021 May 7.

Division of Pulmonary, Allergy and Critical Care Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania; Department of Microbiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.

Culture-independent study of the lower respiratory tract after lung transplantation has enabled an understanding of the microbiome - that is, the collection of bacteria, fungi, and viruses, and their respective gene complement - in this niche. The lung has unique features as a microbial environment, with balanced entry from the upper respiratory tract, clearance, and local replication. There are many pressures impacting the microbiome after transplantation, including donor allograft factors, recipient host factors such as underlying disease and ongoing exposure to the microbe-rich upper respiratory tract, and transplantation-related immunosuppression, antimicrobials, and postsurgical changes. To date, we understand that the lung microbiome after transplant is dysbiotic; that is, it has higher biomass and altered composition compared to a healthy lung. Emerging data suggest that specific microbiome features may be linked to host responses, both immune and non-immune, and clinical outcomes such as chronic lung allograft dysfunction (CLAD), but many questions remain. The goal of this review is to put into context our burgeoning understanding of the lung microbiome in the postlung transplant patient, the interactions between microbiome and host, the role the microbiome may play in post-transplant complications, and critical outstanding research questions.
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http://dx.doi.org/10.1016/j.healun.2021.04.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8335643PMC
August 2021

Transcriptomics of bronchoalveolar lavage cells identifies new molecular endotypes of sarcoidosis.

Eur Respir J 2021 Jun 3. Epub 2021 Jun 3.

Section of Pulmonary, Critical Care and Sleep Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA.

Sarcoidosis is a multisystem granulomatous disease of unknown origin with a variable and often unpredictable course and pattern of organ involvement. In this study we sought to identify specific bronchoalveolar lavage (BAL) cell gene expression patterns indicative of distinct disease phenotypic traits.RNA sequencing by Ion Torrent Proton was performed on BAL cells obtained from 215 well characterised patients with pulmonary sarcoidosis enrolled in the multicenter Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) study. Weighted Gene Co-expression Network Analysis (WGCNA) and non-parametric statistics were used to analyse genome wide BAL transcriptome. Validation of results was performed using a microarray expression data set of an independent sarcoidosis cohort (Freiburg, Germany (n=50)).Our supervised analysis found associations between distinct transcriptional programs and major pulmonary phenotypic manifestations of sarcoidosis including TH1 and TH17 pathways associated with hilar lymphadenopathy, TGFB1 and MTOR signaling with parenchymal involvement, and IL7 and IL2 with airway involvement. Our unsupervised analysis revealed gene modules that uncovered four potential sarcoidosis endotypes including hilar lymphadenopathy with increased acute T cell immune response; extraocular organ involvement with PI3 K activation pathways; chronic and multiorgan disease with increased immune response pathways; and multiorgan with increased IL-1 and IL-18 immune and inflammatory responses. We validated the occurrence of these endotypes using gene expression, pulmonary function tests and cell differentials from Freiburg. Taken together our results identify BAL gene expression programs that characterise major pulmonary sarcoidosis phenotypes and suggest the presence of distinct disease molecular endotypes.
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http://dx.doi.org/10.1183/13993003.02950-2020DOI Listing
June 2021

Detection of SARS-CoV-2 RNA using RT-LAMP and molecular beacons.

Genome Biol 2021 06 3;22(1):169. Epub 2021 Jun 3.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.

Background: Rapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse transcription-polymerase chain reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but instrument costs are high and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse transcription and loop-mediated isothermal amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection.

Results: Here we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in "single pot" reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes.

Conclusions: LAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.
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http://dx.doi.org/10.1186/s13059-021-02387-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8173101PMC
June 2021

An integrated self-powered 3D printed sample concentrator for highly sensitive molecular detection of HIV in whole blood at the point of care.

Analyst 2021 May 8;146(10):3234-3241. Epub 2021 Apr 8.

Department of Mechanical Engineering and Applied Mechanics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

Rapid and efficient biological sample preparation and pretreatment are crucial for highly sensitive, reliable and reproducible molecular detection of infectious diseases. Herein, we report a self-powered, integrated sample concentrator (SPISC) for rapid plasma separation, pathogen lysis, nucleic acid trapping and enrichment at the point of care. The proposed sample concentrator uses a combination of gravitational sedimentation of blood cells and capillary force for rapid, self-powered plasma separation. The pathogens (e.g., HIV virus) in separated plasma were directly lysed and pathogen nucleic acid was enriched by an integrated, flow-through FTA® membrane in the concentrator, enabling highly efficient nucleic acid preparation. The FTA® membrane of the SPISC is easy to store and transport at room temperature without need for uninterrupted cold chain, which is crucial for point of care sampling in resource-limited settings. The platform has been successfully applied to detect HIV virus in blood samples. Our experiments show that the sample concentrator can achieve a plasma separation efficiency as high as 95% and a detection sensitivity as low as 10 copies per 200 μL blood (∼100 copies per mL plasma) with variability less than 7%. The sample concentrator described is fully compatible with downstream nucleic acid detection and has great potential for early diagnostics, monitoring and management of infectious diseases at the point of care.
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http://dx.doi.org/10.1039/d0an02482aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8261191PMC
May 2021

Rengasvirus, a Circular Replication-Associated Protein-Encoding Single-Stranded DNA Virus-Related Genome That Is a Common Contaminant in Metagenomic Data.

Microbiol Resour Announc 2021 May 6;10(18). Epub 2021 May 6.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

We report the genome of a circular replication-associated protein (Rep)-encoding segmented or satellite virus, which we have provisionally named rengasvirus. In metagenomic studies of virus-enriched fractions, rengasvirus was detected widely, including in reagent-negative controls. We thus report this genome to help others recognize a probable contaminating sequence.
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http://dx.doi.org/10.1128/MRA.00273-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8103869PMC
May 2021

Signatures of COVID-19 severity and immune response in the respiratory tract microbiome.

medRxiv 2021 Apr 5. Epub 2021 Apr 5.

Pulmonary, Allergy and Critical Care Division; Department of Medicine; University of Pennsylvania Perelman School of Medicine; Philadelphia, PA 19104.

Rationale: Viral infection of the respiratory tract can be associated with propagating effects on the airway microbiome, and microbiome dysbiosis may influence viral disease.

Objective: To define the respiratory tract microbiome in COVID-19 and relationship disease severity, systemic immunologic features, and outcomes.

Methods And Measurements: We examined 507 oropharyngeal, nasopharyngeal and endotracheal samples from 83 hospitalized COVID-19 patients, along with non-COVID patients and healthy controls. Bacterial communities were interrogated using 16S rRNA gene sequencing, commensal DNA viruses and were quantified by qPCR, and immune features were characterized by lymphocyte/neutrophil (L/N) ratios and deep immune profiling of peripheral blood mononuclear cells (PBMC).

Main Results: COVID-19 patients had upper respiratory microbiome dysbiosis, and greater change over time than critically ill patients without COVID-19. Diversity at the first time point correlated inversely with disease severity during hospitalization, and microbiome composition was associated with L/N ratios and PBMC profiles in blood. Intubated patients showed patient-specific and dynamic lung microbiome communities, with prominence of . and showed more frequent colonization and higher titers in severe disease. Machine learning analysis demonstrated that integrated features of the microbiome at early sampling points had high power to discriminate ultimate level of COVID-19 severity.

Conclusions: The respiratory tract microbiome and commensal virome are disturbed in COVID-19, correlate with systemic immune parameters, and early microbiome features discriminate disease severity. Future studies should address clinical consequences of airway dysbiosis in COVID-19, possible use as biomarkers, and role of bacterial and viral taxa identified here in COVID-19 pathogenesis.
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http://dx.doi.org/10.1101/2021.04.02.21254514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8043476PMC
April 2021

Femtomolar SARS-CoV-2 Antigen Detection Using the Microbubbling Digital Assay with Smartphone Readout Enables Antigen Burden Quantitation and Dynamics Tracking.

medRxiv 2021 Mar 26. Epub 2021 Mar 26.

Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA.

Background: Little is known about the dynamics of SARS-CoV-2 antigen burden in respiratory samples in different patient populations at different stages of infection. Current rapid antigen tests cannot quantitate and track antigen dynamics with high sensitivity and specificity in respiratory samples.

Methods: We developed and validated an ultra-sensitive SARS-CoV-2 antigen assay with smartphone readout using the Microbubbling Digital Assay previously developed by our group, which is a platform that enables highly sensitive detection and quantitation of protein biomarkers. A computer vision-based algorithm was developed for microbubble smartphone image recognition and quantitation. A machine learning-based classifier was developed to classify the smartphone images based on detected microbubbles. Using this assay, we tracked antigen dynamics in serial swab samples from COVID patients hospitalized in ICU and immunocompromised COVID patients.

Results: The limit of detection (LOD) of the Microbubbling SARS-CoV-2 Antigen Assay was 0.5 pg/mL (10.6 fM) recombinant nucleocapsid (N) antigen or 4000 copies/mL inactivated SARS-CoV-2 virus in nasopharyngeal (NP) swabs, comparable to many rRT-PCR methods. The assay had high analytical specificity towards SARS-CoV-2. Compared to EUA-approved rRT-PCR methods, the Microbubbling Antigen Assay demonstrated a positive percent agreement (PPA) of 97% (95% confidence interval (CI), 92-99%) in symptomatic individuals within 7 days of symptom onset and positive SARS-CoV-2 nucleic acid results, and a negative percent agreement (NPA) of 97% (95% CI, 94-100%) in symptomatic and asymptomatic individuals with negative nucleic acid results. Antigen positivity rate in NP swabs gradually decreased as days-after-symptom-onset increased, despite persistent nucleic acid positivity of the same samples. The computer vision and machine learning-based automatic microbubble image classifier could accurately identify positives and negatives, based on microbubble counts and sizes. Total microbubble volume, a potential marker of antigen burden, correlated inversely with Ct values and days-after-symptom-onset. Antigen was detected for longer periods of time in immunocompromised patients with hematologic malignancies, compared to immunocompetent individuals. Simultaneous detectable antigens and nucleic acids may indicate the presence of replicating viruses in patients with persistent infections.

Conclusions: The Microbubbling SARS-CoV-2 Antigen Assay enables sensitive and specific detection of acute infections, and quantitation and tracking of antigen dynamics in different patient populations at various stages of infection. With smartphone compatibility and automated image processing, the assay is well-positioned to be adapted for point-of-care diagnosis and to explore the clinical implications of antigen dynamics in future studies.
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http://dx.doi.org/10.1101/2021.03.17.21253847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010739PMC
March 2021

Detection of Microbial Agents in Oropharyngeal and Nasopharyngeal Samples of SARS-CoV-2 Patients.

Front Microbiol 2021 9;12:637202. Epub 2021 Mar 9.

Department of Otorhinolaryngology-Head and Neck Surgery, and Microbiology, The Tumor Virology Program, Abramson Comprehensive Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States.

The novel coronavirus outbreak started in December 2019 and rapidly spread around the globe, leading to a global pandemic. Here we reported the association of microbial agents identified in oropharyngeal and nasopharyngeal samples from patients with SARS-CoV-2 infection, using a Pan-microarray based technology referred to as PathoChIP. To validate the efficiency of PathoChIP, reference viral genomes obtained from BEI resource and 25 SARS-CoV-2 positive clinical samples were tested. This technology successfully detected femtogram levels of SARS-CoV-2 viral RNA, which demonstrated greater sensitivity and specificity than conventional diagnostic techniques. Simultaneously, a broad range of other microorganisms, including other viruses, bacteria, fungi and parasites can be detected in those samples. We identified 7 viral, 12 bacterial and 6 fungal agents common across all clinical samples suggesting an associated microbial signature in individuals who are infected with SARS-CoV-2. This technology is robust and has a flexible detection methodology that can be employed to detect the presence of all human respiratory pathogens in different sample preparations with precision. It will be important for differentiating the causative agents of respiratory illnesses, including SARS-CoV-2.
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http://dx.doi.org/10.3389/fmicb.2021.637202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8006406PMC
March 2021

Decreased Intestinal Microbiome Diversity in Pediatric Sepsis: A Conceptual Framework for Intestinal Dysbiosis to Influence Immunometabolic Function.

Crit Care Explor 2021 Mar 17;3(3):e0360. Epub 2021 Mar 17.

Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA.

Objectives: The intestinal microbiome can modulate immune function through production of microbial-derived short-chain fatty acids. We explored whether intestinal dysbiosis in children with sepsis leads to changes in microbial-derived short-chain fatty acids in plasma and stool that are associated with immunometabolic dysfunction in peripheral blood mononuclear cells.

Design: Prospective observational pilot study.

Setting: Single academic PICU.

Patients: Forty-three children with sepsis/septic shock and 44 healthy controls.

Measurements And Main Results: Stool and plasma samples were serially collected for sepsis patients; stool was collected once for controls. The intestinal microbiome was assessed using 16S ribosomal RNA sequencing and alpha- and beta-diversity were determined. We measured short-chain fatty acids using liquid chromatography, peripheral blood mononuclear cell mitochondrial respiration using high-resolution respirometry, and immune function using ex vivo lipopolysaccharide-stimulated whole blood tumor necrosis factor-α. Sepsis patients exhibited reduced microbial diversity compared with healthy controls, with lower alpha- and beta-diversity. Reduced microbial diversity among sepsis patients (mainly from lower abundance of commensal obligate anaerobes) was associated with increased acetic and propionic acid and decreased butyric, isobutyric, and caproic acid. Decreased levels of plasma butyric acid were further associated with lower peripheral blood mononuclear cell mitochondrial respiration, which in turn, was associated with lower lipopolysaccharide-stimulated tumor necrosis factor-α. However, neither intestinal dysbiosis nor specific patterns of short-chain fatty acids were associated with lipopolysaccharide-stimulated tumor necrosis factor-α.

Conclusions: Intestinal dysbiosis was associated with altered short-chain fatty acid metabolites in children with sepsis, but these findings were not linked directly to mitochondrial or immunologic changes. More detailed mechanistic studies are needed to test the role of microbial-derived short-chain fatty acids in the progression of sepsis.
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http://dx.doi.org/10.1097/CCE.0000000000000360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7994045PMC
March 2021

CD4 receptor diversity represents an ancient protection mechanism against primate lentiviruses.

Proc Natl Acad Sci U S A 2021 Mar;118(13)

Lukuru Wildlife Research Foundation, Tshuapa-Lomami-Lualaba Project, BP 2012, Kinshasa, Democratic Republic of the Congo.

Infection with human and simian immunodeficiency viruses (HIV/SIV) requires binding of the viral envelope glycoprotein (Env) to the host protein CD4 on the surface of immune cells. Although invariant in humans, the Env binding domain of the chimpanzee CD4 is highly polymorphic, with nine coding variants circulating in wild populations. Here, we show that within-species CD4 diversity is not unique to chimpanzees but found in many African primate species. Characterizing the outermost (D1) domain of the CD4 protein in over 500 monkeys and apes, we found polymorphic residues in 24 of 29 primate species, with as many as 11 different coding variants identified within a single species. D1 domain amino acid replacements affected SIV Env-mediated cell entry in a single-round infection assay, restricting infection in a strain- and allele-specific fashion. Several identical CD4 polymorphisms, including the addition of -linked glycosylation sites, were found in primate species from different genera, providing striking examples of parallel evolution. Moreover, seven different guenons ( spp.) shared multiple distinct D1 domain variants, pointing to long-term trans-specific polymorphism. These data indicate that the HIV/SIV Env binding region of the primate CD4 protein is highly variable, both within and between species, and suggest that this diversity has been maintained by balancing selection for millions of years, at least in part to confer protection against primate lentiviruses. Although long-term SIV-infected species have evolved specific mechanisms to avoid disease progression, primate lentiviruses are intrinsically pathogenic and have left their mark on the host genome.
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http://dx.doi.org/10.1073/pnas.2025914118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8020793PMC
March 2021

Dynamic Changes in the Nasal Microbiome Associated With Disease Activity in Patients With Granulomatosis With Polyangiitis.

Arthritis Rheumatol 2021 09 31;73(9):1703-1712. Epub 2021 Jul 31.

University of Pennsylvania, Philadelphia.

Objective: Little is known about temporal changes in nasal bacteria in granulomatosis with polyangiitis (GPA). This study was undertaken to examine longitudinal changes in the nasal microbiome in association with relapse in GPA patients.

Methods: Bacterial 16S ribosomal RNA gene sequencing was performed on nasal swabs from 19 patients with GPA who were followed up longitudinally for a total of 78 visits, including 9 patients who experienced a relapse and 10 patients who remained in remission. Relative abundance of bacteria and ratios between bacteria were examined. Generalized estimating equation models were used to evaluate the association between bacterial composition and 1) disease activity and 2) levels of antineutrophil cytoplasmic antibody (ANCA) with specificity for proteinase 3 (PR3), adjusted for medication.

Results: Corynebacterium and Staphylococcus were the most abundant bacterial genera across all nasal samples. Patients with quiescent disease maintained a stable ratio of Corynebacterium to Staphylococcus across visits. In contrast, in patients who experienced a relapse, a significantly lower ratio was observed at the visit prior to relapse, followed by a higher ratio at the time of relapse (adjusted P < 0.01). Species-level analysis identified an association between a higher abundance of nasal Corynebacterium tuberculostearicum and 1) relapse (adjusted P = 0.04) and 2) higher PR3-ANCA levels (adjusted P = 0.02).

Conclusion: In GPA, significant changes occur in the nasal microbiome over time and are associated with disease activity. The occurrence of these changes months prior to the onset of relapse supports a pathogenic role of nasal bacteria in GPA. Our results uphold existing hypotheses implicating Staphylococcus as an instigator of disease and have generated a novel finding involving Corynebacterium as a potential mediator of disease in GPA.
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http://dx.doi.org/10.1002/art.41723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8403103PMC
September 2021

SARS-CoV-2 Genomic Variation in Space and Time in Hospitalized Patients in Philadelphia.

mBio 2021 01 19;12(1). Epub 2021 Jan 19.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

The severe acute respiratory coronavirus 2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. The epidemic accelerated in Philadelphia, PA, in the spring of 2020, with the city experiencing a first peak of infections on 15 April, followed by a decline through midsummer. Here, we investigate spread of the epidemic in the first wave in Philadelphia using full-genome sequencing of 52 SARS-CoV-2 samples obtained from 27 hospitalized patients collected between 30 March and 17 July 2020. Sequences most commonly resembled lineages circulating at earlier times in New York, suggesting transmission primarily from this location, though a minority of Philadelphia genomes matched sequences from other sites, suggesting additional introductions. Multiple genomes showed even closer matches to other Philadelphia isolates, suggestive of ongoing transmission within Philadelphia. We found that all of our isolates contained the D614G substitution in the viral spike and belong to lineages variously designated B.1, Nextstrain clade 20A or 20C, and GISAID clade G or GH. There were no viral sequence polymorphisms detectably associated with disease outcome. For some patients, genome sequences were determined longitudinally or concurrently from multiple body sites. In both cases, some comparisons showed reproducible polymorphisms, suggesting initial seeding with multiple variants and/or accumulation of polymorphisms after infection. These results thus provide data on the sources of SARS-CoV-2 infection in Philadelphia and begin to explore the dynamics within hospitalized patients. Understanding how SARS-CoV-2 spreads globally and within infected individuals is critical to the development of mitigation strategies. We found that most lineages in Philadelphia had resembled sequences from New York, suggesting infection primarily but not exclusively from this location. Many genomes had even nearer neighbors within Philadelphia, indicating local spread. Multiple genome sequences were available for some subjects and in a subset of cases could be shown to differ between time points and body sites within an individual, indicating heterogeneous viral populations within individuals and raising questions on the mechanisms responsible. There was no evidence that different lineages were associated with different outcomes in patients, emphasizing the importance of individual-specific vulnerability.
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http://dx.doi.org/10.1128/mBio.03456-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829343PMC
January 2021

Heightened resistance to host type 1 interferons characterizes HIV-1 at transmission and after antiretroviral therapy interruption.

Sci Transl Med 2021 01;13(576)

Laboratory of Molecular Immunology, Rockefeller University, New York, NY 10065, USA.

Type 1 interferons (IFN-I) are potent innate antiviral effectors that constrain HIV-1 transmission. However, harnessing these cytokines for HIV-1 cure strategies has been hampered by an incomplete understanding of their antiviral activities at later stages of infection. Here, we characterized the IFN-I sensitivity of 500 clonally derived HIV-1 isolates from the plasma and CD4 T cells of 26 individuals sampled longitudinally after transmission or after antiretroviral therapy (ART) and analytical treatment interruption. We determined the concentration of IFNα2 and IFNβ that reduced viral replication in vitro by 50% (IC) and found consistent changes in the sensitivity of HIV-1 to IFN-I inhibition both across individuals and over time. Resistance of HIV-1 isolates to IFN-I was uniformly high during acute infection, decreased in all individuals in the first year after infection, was reacquired concomitant with CD4 T cell loss, and remained elevated in individuals with accelerated disease. HIV-1 isolates obtained by viral outgrowth during suppressive ART were relatively IFN-I sensitive, resembling viruses circulating just before ART initiation. However, viruses that rebounded after treatment interruption displayed the highest degree of IFNα2 and IFNβ resistance observed at any time during the infection course. These findings indicate a dynamic interplay between host innate responses and the evolving HIV-1 quasispecies, with the relative contribution of IFN-I to HIV-1 control affected by both ART and analytical treatment interruption. Although elevated at transmission, host innate pressures are the highest during viral rebound, limiting the viruses that successfully become reactivated from latency to those that are IFN-I resistant.
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http://dx.doi.org/10.1126/scitranslmed.abd8179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923595PMC
January 2021

Gene coexpression networks reveal novel molecular endotypes in alpha-1 antitrypsin deficiency.

Thorax 2021 02 10;76(2):134-143. Epub 2020 Dec 10.

Department of Medicine, Yale University School of Medicine, New Haven, Connecticut, USA.

Background: Alpha-1 antitrypsin deficiency (AATD) is a genetic condition that causes early onset pulmonary emphysema and airways obstruction. The complete mechanisms via which AATD causes lung disease are not fully understood. To improve our understanding of the pathogenesis of AATD, we investigated gene expression profiles of bronchoalveolar lavage (BAL) and peripheral blood mononuclear cells (PBMCs) in AATD individuals.

Methods: We performed RNA-Seq on RNA extracted from matched BAL and PBMC samples isolated from 89 subjects enrolled in the Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) study. Subjects were stratified by genotype and augmentation therapy. Supervised and unsupervised differential gene expression analyses were performed using Weighted Gene Co-expression Network Analysis (WGCNA) to identify gene profiles associated with subjects' clinical variables. The genes in the most significant WGCNA module were used to cluster AATD individuals. Gene validation was performed by NanoString nCounter Gene Expression Assay.

Result: We observed modest effects of AATD genotype and augmentation therapy on gene expression. When WGCNA was applied to BAL transcriptome, one gene module, ME31 (2312 genes), correlated with the highest number of clinical variables and was functionally enriched with numerous immune T-lymphocyte related pathways. This gene module identified two distinct clusters of AATD individuals with different disease severity and distinct PBMC gene expression patterns.

Conclusions: We successfully identified novel clusters of AATD individuals where severity correlated with increased immune response independent of individuals' genotype and augmentation therapy. These findings may suggest the presence of previously unrecognised disease endotypes in AATD that associate with T-lymphocyte immunity and disease severity.
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http://dx.doi.org/10.1136/thoraxjnl-2019-214301DOI Listing
February 2021

ICTV Virus Taxonomy Profile: .

J Gen Virol 2021 01 27;102(1). Epub 2020 Nov 27.

Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.

Viruses in the family have a circular genome of 3.0 kb with three open reading frames. The packaged genome is inferred to be single-stranded DNA by analogy to related viruses. Redondoviruses were discovered through metagenomic sequencing methods in samples from human subjects and are inferred to replicate in humans. Evidence of redondovirus infection is associated with periodontitis and critical illness, but redondoviruses have not been shown to be the causative agent of any diseases. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family , which is available at ictv.global/report/redondoviridae.
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http://dx.doi.org/10.1099/jgv.0.001526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8116785PMC
January 2021

Pathogenesis of Aging and Age-related Comorbidities in People with HIV: Highlights from the HIV ACTION Workshop.

Pathog Immun 2020 17;5(1):143-174. Epub 2020 Jun 17.

Department of Medicine; University of California; San Francisco, California.

People with HIV (PWH) experience accentuated biological aging, as defined by markers of inflammation, immune dysfunction, and the epigenetic clock. They also have an elevated risk of multiple age-associated comorbidities. To discuss current knowledge, research gaps, and priorities in aging and age-related comorbidities in treated HIV infection, the NIH program staff organized a workshop held in Bethesda, Maryland in September 2019. This review article describes highlights of discussions led by the Pathogenesis/Basic Science Research working group that focused on three high priority topics: immunopathogenesis; the microbiome/virome; and aging and senescence. We summarize knowledge in these fields and describe key questions for research on the pathogenesis of aging and age-related comorbidities in PWH. Understanding the drivers and mechanisms underlying accentuated biological aging is a high priority that will help identify potential therapeutic targets to improve healthspan in older PWH.
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http://dx.doi.org/10.20411/pai.v5i1.365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7449259PMC
June 2020

A Summary of the Fifth Annual Virology Education HIV Microbiome Workshop.

AIDS Res Hum Retroviruses 2020 11 7;36(11):886-895. Epub 2020 Sep 7.

Department of Epidemiology, The George Washington University, Washington, District of Columbia, USA.

In October of 2019, researchers and community members from around the world met at the NIH for the fifth annual International Workshop on Microbiome in HIV. New research was presented on the role of the microbiome on chronic inflammation and vaccine design, interactions of genetics, environment, sexual practice and HIV infection with the microbiome and the development and clinical trials of microbiome-based therapeutic approaches intended to decrease the probability of HIV acquisition/transmission or ameliorate sequelae of HIV. The keynote address by Dr. Jacques Ravel focused on his work on the vaginal microbiome and efforts to improve the analysis and resolution of microbiome data.
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http://dx.doi.org/10.1089/AID.2020.0121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7869876PMC
November 2020

The effect of varenicline on mood and cognition in smokers with HIV.

Psychopharmacology (Berl) 2020 Apr 14;237(4):1223-1231. Epub 2020 Jan 14.

Department of Psychiatry, Perelman School of Medicine, University of Pennsylvania, 3535 Market Street, Suite 4100, Philadelphia, PA, USA.

Rationale: Barriers to smoking cessation, including negative affect and cognitive dysfunction, may contribute to high smoking rates among people living with HIV/AIDS (PLWH). Varenicline may help PLWH quit smoking by improving mood and cognition, yet this has not been explored.

Objectives: The goal of this study was to evaluate the effect of varenicline on mood and cognition among PLWH enrolled in a smoking cessation clinical trial.

Methods: In this secondary analysis of a varenicline trial (NCT01710137), we assessed mood (depression, anxiety) and cognition (attention, working memory) at weeks 0 (baseline), 1, 3, and 12 (end-of-treatment, EOT). Primary outcomes were changes in mood and cognition from baseline to EOT. Secondarily, mood and cognition were evaluated as predictors of biochemically confirmed 7-day point-prevalence abstinence at EOT.

Results: Overall, 173 subjects (87 varenicline, 86 placebo) were included. At EOT, varenicline reduced anxiety (P < 0.001), vs. placebo (P = 0.31; interaction P = 0.05). Across both treatment arms, reductions in anxiety from baseline to EOT were associated with a higher likelihood of abstinence (OR = 1.3, 95% CI 1.1 to 1.6, P = 0.01). There were no significant treatment by time interactions for cognition or depression.

Conclusions: These data suggest that varenicline operates, at least in part, by reducing anxiety. Anxiety should be an intervention target for smokers with HIV interested in quitting.
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http://dx.doi.org/10.1007/s00213-020-05451-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7125016PMC
April 2020

A Summary of the Fourth Annual Virology Education HIV Microbiome Workshop.

AIDS Res Hum Retroviruses 2020 05 9;36(5):349-356. Epub 2020 Mar 9.

Division of Gerontology, Department of Internal Medicine, Rush University Medical Center, Chicago, Illinois, USA.

Each year, a growing international collection of researchers meets at the NIH to share and discuss developments in the microbiome HIV story. This past year has seen continued progress toward a detailed understanding of host-microbe interactions both within and outside the field of HIV. Commensal microbes are being linked to an ever-growing list of maladies and physiologic states, including major depressive disorder, chronic kidney disease, and Parkinson disease. PubMed citations for "microbiome" are growing at an exponential rate with over 11,000 in 2018. Various microbial taxa have been associated with HIV infection, and some of these taxa associated with HIV infection have also been associated with systemic markers of inflammation in HIV infected individuals. Causality remains unclear however as environmental and behavioral factors may drive HIV risk, inflammation, and gut enterotype. Much of the work currently being done addresses potential mechanisms by which gut microbes influence immune and inflammatory pathways. No portion of the microbiome landscape has grown as rapidly as study of the interplay between gut microbes and response to cancer immunotherapy. As Dr. Wargo discussed in her keynote address, this area has opened the door to better understanding on how commensal microbes interact with the human immune system.
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http://dx.doi.org/10.1089/AID.2019.0197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7360108PMC
May 2020

Evidence for Persistent Monocyte and Immune Dysregulation After Prolonged Viral Suppression Despite Normalization of Monocyte Subsets, sCD14 and sCD163 in HIV-Infected Individuals.

Pathog Immun 2019 17;4(2):324-362. Epub 2019 Dec 17.

Department of Medicine; University of Pennsylvania Perelman School of Medicine; Philadelphia, Pennsylvania.

Background: People living with HIV on antiretroviral therapy (HIV/ART) experience excess non-AIDS comorbidities, and also remain at increased risk for certain infections and viral malignancies. Monocytes/macrophages are central to many of these comorbidities, and elevated plasma cytokines and immune activation during untreated infection are often incompletely reversed by ART and are also associated with comorbidities.

Methods: We investigated monocyte surface markers, gene expression, and plasma cytokines in 11 HIV-infected older individuals (median 53 years) who started therapy with low CD4 counts (median 129 cells/µl), with elevated hsCRP (≥ 2mg/L) despite long-term ART (median 7.4 years), along with matched controls.

Results: Frequency of monocyte subsets (based on CD14/CD16/CD163), were not different from controls, but surface expression of CD163 was increased ( = 0.021) while PD1 was decreased ( = 0.013) along with a trend for higher tissue factor ( = 0.096). As a group, HIV/ART participants had elevated plasma CCL2 (MCP-1; = 0.0001), CXCL9 (MIG; = 0.04), and sIL2R ( = 0.015), which were correlated, while sCD14 was not elevated. Principal component analysis of soluble markers revealed that 6/11 HIV/ART participants clustered with controls, while 5 formed a distinct group, driven by IL-10, CCL11, CXCL10, CCL2, CXCL9, and sIL2R. These individuals were significantly older than those clustering with controls. Transcriptomic analysis revealed multiple genes linked to immune functions including inflammation, immune cell development, and cell-cell signaling that were downregulated in HIV/ART monocytes and distinct from patterns in untreated subjects.

Conclusions: Long-term ART-treated individuals normalize monocyte subsets but exhibit immune dysregulation involving both aberrant inflammation and monocyte dysfunction, as well as inter-individual heterogeneity, suggesting complex mechanisms linking monocytes and HIV/ART comorbidities.
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http://dx.doi.org/10.20411/pai.v4i2.336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6930814PMC
December 2019

Bidirectional Associations among Nicotine and Tobacco Smoke, NeuroHIV, and Antiretroviral Therapy.

J Neuroimmune Pharmacol 2020 12 13;15(4):694-714. Epub 2019 Dec 13.

Department of Psychiatry, University of Pennsylvania, 3535 Market Street, Suite, Philadelphia, PA, 4100, USA.

People living with HIV (PLWH) in the antiretroviral therapy (ART) era may lose more life-years to tobacco use than to HIV. Yet, smoking rates are more than twice as high among PLWH than the general population, contributing not just to mortality but to other adverse health outcomes, including neurocognitive deficits (neuroHIV). There is growing evidence that synergy with chronic inflammation and immune dysregulation that persists despite ART may be one mechanism by which tobacco smoking contributes to neuroHIV. This review will summarize the differential effects of nicotine vs tobacco smoking on inflammation in addition to the effects of tobacco smoke components on HIV disease progression. We will also discuss biomarkers of inflammation via neuroimaging as well as biomarkers of nicotine dependence (e.g., nicotine metabolite ratio). Tobacco smoking and nicotine may impact ART drug metabolism and conversely, certain ARTs may impact nicotine metabolism. Thus, we will review these bidirectional relationships and how they may contribute to neuroHIV and other adverse outcomes. We will also discuss the effects of tobacco use on the interaction between peripheral organs (lungs, heart, kidney) and subsequent CNS function in the context of HIV. Lastly, given the dramatic rise in the use of electronic nicotine delivery systems, we will discuss the implications of vaping on these processes. Despite the growing recognition of the importance of addressing tobacco use among PLWH, more research is necessary at both the preclinical and clinical level to disentangle the potentially synergistic effects of tobacco use, nicotine, HIV, cognition and immune dysregulation, as well as identify optimal approaches to reduce tobacco use. Graphical Abstract Proposed model of the relationships among HIV, ART, smoking, inflammation, and neurocognition. Solid lines represent relationships supported by evidence. Dashed lines represent relationships for which there is not enough evidence to make a conclusion. (a) HIV infection produces elevated levels of inflammation even among virally suppressed individuals. (b) HIV is associated with deficits in cognition function. (c) Smoking rates are higher among PLWH, compared to the general population. (d) The nicotine metabolite ratio (NMR) is associated with smoking behavior. (e) HIV and tobacco use are both associated with higher rates of psychiatric comorbidities, such as depression, and elevated levels of chronic stress. These factors may represent other mechanisms linking HIV and tobacco use. (f) The relationship between nicotine, tobacco smoking, and inflammation is complex, but it is well-established that smoking induces inflammation; the evidence for nicotine as anti-inflammatory is supported in some studies, but not others. (g) The relationship between tobacco use and neurocognition may differ for the effects of nicotine (acute nicotine use may have beneficial effects) vs. tobacco smoking (chronic use may impair cognition). (h) Elevated levels of inflammation may be associated with deficits in cognition. (i) PLWH may metabolize nicotine faster than those without HIV; the mechanism is not yet known and the finding needs validation in larger samples. We also hypothesize that if HIV-infection increases nicotine metabolism, then we should observe an attenuation effect once ART is initiated. (j) It is possible that the increase in NMR is due to ART effects on CYP2A6. (k) We hypothesize that faster nicotine metabolism may result in higher levels of inflammation since nicotine has anti-inflammatory properties.
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http://dx.doi.org/10.1007/s11481-019-09897-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7383218PMC
December 2020

Upper Respiratory Dysbiosis with a Facultative-dominated Ecotype in Advanced Lung Disease and Dynamic Change after Lung Transplant.

Ann Am Thorac Soc 2019 11;16(11):1383-1391

Department of Medicine.

The oropharyngeal microbiome is a primary source of lung microbiota, contributes to lower respiratory infection, and is also a driver of oral health. We sought to understand oropharyngeal microbial communities in advanced lung disease, community dynamics after lung transplantation, and ecological features of dysbiosis. Oropharyngeal wash samples were obtained from individuals with end-stage disease awaiting transplantation ( = 22) and longitudinally from individuals at 6 weeks, 3 months, and 6 months after transplantation ( = 33), along with healthy control subjects ( = 14). Bacterial 16S and fungal internal transcribed spacer rRNA regions were deep-sequenced, and bacterial community respiratory patterns were imputed from taxonomic composition. Healthy subjects' oropharyngeal microbiomes showed a gradient of community types reflecting relative enrichment of strictly anaerobic, aerobic, or facultative anaerobic bacteria. Patients with end-stage lung disease showed severe dysbiosis by both taxonomic composition and respiration phenotypes, with reduced richness and diversity, increased facultative and decreased aerobic bacteria, and absence of communities characterized by obligate aerobes. In patients at 6 weeks and 3 months post-transplant, richness and diversity were intermediate between healthy and pretransplant subjects, with near-normal distribution of community types. However, by 6 months post-transplant, oropharyngeal wash resembled the low-diversity facultative-dominated profile of pretransplant subjects. Community ecotype correlated with abundance. End-stage lung disease is associated with marked upper respiratory tract dysbiosis involving both community structure and respiratory metabolism profiles of constituent bacteria. Dynamic changes occur after lung transplantation, with partial normalization early but later appearance of severe dysbiosis similar to pretransplant patients. Aberrant oropharyngeal communities may predispose to abnormal lung microbiota and infection risk both in advanced lung disease and after transplantation.
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http://dx.doi.org/10.1513/AnnalsATS.201904-299OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6945465PMC
November 2019

Molecular analysis of the endobronchial stent microbial biofilm reveals bacterial communities that associate with stent material and frequent fungal constituents.

PLoS One 2019 29;14(5):e0217306. Epub 2019 May 29.

Department of Medicine, Division of Pulmonary, Allergy and Critical Care, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

Endobronchial stents are increasingly used to treat airway complications in multiple conditions including lung transplantation but little is known about the biofilms that form on these devices. We applied deep sequencing to profile luminal biofilms of 46 endobronchial stents removed from 20 subjects primarily with lung transplantation-associated airway compromise. Microbial communities were analyzed by bacterial 16S rRNA and fungal ITS marker gene sequencing. Corynebacterium was the most common bacterial taxa across biofilm communities. Clustering analysis revealed three bacterial biofilm types: one low diversity and dominated by Corynebacterium; another was polymicrobial and characterized by Staphylococcus; and the third was polymicrobial and associated with Pseudomonas, Streptococcus, and Prevotella. Biofilm type was significantly correlated with stent material: covered metal with the Staphylococcus-type biofilm, silicone with the Corynebacterium-dominated biofilm, and uncovered metal with the polymicrobial biofilm. Subjects with sequential stents had frequent transitions between community types. Fungal analysis found Candida was most prevalent, Aspergillus was common and highly enriched in two of three stents associated with airway anastomotic dehiscence, and fungal taxa not typically considered pathogens were highly enriched in some stents. Thus, molecular analysis revealed a complex and dynamic endobronchial stent biofilm with three bacterial types that associate with stent material, a central role for Corynebacterium, and that both expected and unexpected fungi inhabit this unique niche. The current work provides a foundation for studies to investigate the relationship between stent biofilm composition and clinical outcomes, mechanisms of biofilm establishment, and strategies for improved stent technology and use in airway compromise.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0217306PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6541290PMC
February 2020
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