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Structural and Functional Characteristics of the Microbiome in Deep-Dentin Caries

Liu, G; Wu, C; Abrams, W R; Li, Y
Dental caries is a cariogenic bacteria-mediated, fermentable carbohydrate-driven dynamic disease. The new ecological hypothesis for dentin caries suggests that an alteration in the microbial community and the presence of specific metabolic pathway genes contribute to the initiation and progression of caries. This study aimed to determine the structural and functional characteristics of a microbial community of human deep-dentin carious lesions. Sixteen deep-dentin carious lesions were obtained from the first permanent molars of 8 patients aged 9 to 18 y. Shotgun metagenomic sequencing was used to measure the microbial composition and abundance at the phylum, class, order, family, genus, and species levels. Functional analysis of the DNA sequencing data set was also performed and compared among different layers of the lesions using DIAMOND software against the Kyoto Encyclopedia of Genes and Genomes database. This study found that in the deep-dentin carious lesions, Actinobacteria (35.8%) and Firmicutes (31.2%) were the most prevalent phyla, followed by Bacteroidetes (13.6%), Proteobacteria (3.6%), and Fusobacteria (2.5%). The microbial composition varied among the individuals, but there were no significant differences in the distribution of the relative microbial abundance between the superficial layers and the deep layers. Although 14.5% of the top 10 taxa were identified as Lactobacillus at the genus level, only 25% of the deep-dentin carious samples showed Lactobacillus as the most abundant genus. Other abundant taxa included Actinomyces (10.5%), Olsenella (9.4%), Prevotella (8.8%), Propionibacterium (7.2%), Streptococcus (3.9%), Selenomonas (3.7%), Corynebacterium (1.9%), Leptotrichia (1.4%), and Parascardovia (1.1%). The most abundant pathway identified in the KEGG database was the metabolic pathway containing 101,427 annotated genes, which consisted of 51.4% of all annotated genes. The carbohydrate metabolism pathway, amino acid metabolism, and membrane transport were the functional traits of the level 2 pathways. These findings suggest that the potent interaction within the microbial communities in deep-dentin carious lesions may play a fundamental role in caries etiology.
PMID: 32196394
ISSN: 1544-0591
CID: 4353842

Detection of Zika virus using reverse-transcription LAMP coupled with reverse dot blot analysis in saliva

Sabalza, Maite; Yasmin, Rubina; Barber, Cheryl A; Castro, Talita; Malamud, Daniel; Kim, Beum Jun; Zhu, Hui; Montagna, Richard A; Abrams, William R
In recent years, there have been increasing numbers of infectious disease outbreaks that spread rapidly to population centers resulting from global travel, population vulnerabilities, environmental factors, and ecological disasters such as floods and earthquakes. Some examples of the recent outbreaks are the Ebola epidemic in West Africa, Middle East respiratory syndrome coronavirus (MERS-Co) in the Middle East, and the Zika outbreak through the Americas. We have created a generic protocol for detection of pathogen RNA and/or DNA using loop-mediated isothermal amplification (LAMP) and reverse dot-blot for detection (RDB) and processed automatically in a microfluidic device. In particular, we describe how a microfluidic assay to detect HIV viral RNA was converted to detect Zika virus (ZIKV) RNA. We first optimized the RT-LAMP assay to detect ZIKV RNA using a benchtop isothermal amplification device. Then we implemented the assay in a microfluidic device that will allow analyzing 24 samples simultaneously and automatically from sample introduction to detection by RDB technique. Preliminary data using saliva samples spiked with ZIKV showed that our diagnostic system detects ZIKV RNA in saliva. These results will be validated in further experiments with well-characterized ZIKV human specimens of saliva. The described strategy and methodology to convert the HIV diagnostic assay and platform to a ZIKV RNA detection assay provides a model that can be readily utilized for detection of the next emerging or re-emerging infectious disease.
PMID: 29401479
ISSN: 1932-6203
CID: 2946992

Rapid diagnosis of Zika virus through saliva and urine by Loop-mediated isothermal amplification (LAMP)

Castro, Talita; Sabalza, Maite; Barber, Cheryl; Abrams, William; Da Costa, Antonio Charlys; De Pádua Milagres, Flavio Augusto; Braz-Silva, Paulo Henrique; Malamud, Daniel; Gallottini, Marina
Background: Zika virus (ZIKV) is a single-stranded RNA virus and member of the Flaviviridae family. Recent studies have reported that saliva can be an important alternative to detect ZIKV. Saliva requires less processing than blood greatly simplifying the assay. Loop-mediated Isothermal Amplification (LAMP) is a rapid assay that detects nucleic acids, including ZIKV RNA. Aim: The aim of this study was to evaluate the efficacy of saliva and urine to diagnose ZIKV infection in subjects during the acute phase, through ZIKV RNA detection by LAMP. Method: A total of 131 samples (68 saliva and 63 urine samples) from 69 subjects in the acute phase of ZIKV infection, and confirmed positive for ZIKV by blood analysis through real time-PCR, were collected and analyzed by Reverse Transcriptase Loop-mediated Isothermal Amplification (RT-LAMP). Results: From the 68 saliva samples, 45 (66.2%) were positive for ZIKV with an average time to positivity (Tp) of 13.5 min, and from the 63 urine samples, 25 (39.7%) were positive with the average Tp of 15.8 min. Saliva detected more samples (p = 0.0042) and had faster Tp (p = 0.0176) as compared with urine. Conclusion: Saliva proved to be a feasible alternative to diagnose ZIKV infection during the acute phase by LAMP.
PMID: 30202506
ISSN: 2000-2297
CID: 3277962

Zika Virus Specific Diagnostic Epitope Discovery

Sabalza, Maite; Barber, Cheryl A; Abrams, William R; Montagna, Richard; Malamud, Daniel
High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.
PMID: 29286404
ISSN: 1940-087x
CID: 2895352

Modulation of the orodigestive tract microbiome in HIV-infected patients

Saxena, D; Li, Y; Devota, A; Pushalkar, S; Abrams, W; Barber, C; Corby, P; Poles, M; Phelan, J; Malamud, D
More than 37 million people are living with human immunodeficiency virus 1 (HIV), and more people than ever received lifesaving antiretroviral therapy worldwide. HIV-1 infection disrupts the intestinal immune system, leading to microbial translocation and systemic immune activation. We investigated the impact of HIV-1 infection on the GI microbiome and its association with host immune activation. The data indicated that the microbiome was different in HIV-positive and HIV-negative individuals. The initial sequence analysis of saliva indicated that there were major differences in the phyla of Bacteroidetes, Firmicutes, Proteobacteria, and TM7. Phylum Tenericutes was only seen in HIV-positive saliva. At the family level, we identified differences in Streptococcacea, Prevotellaceae, Porphyromonadaceae, and Neisseriaceae, whereas data from various sites in GI tract indicated that Prevotella melaninigencia, Fusobacterium necrophorum, Burkholderia, Bradyrhizobium, Ralstonia, and Eubacterium biforme were predominant but differentially present at various sites. Furthermore, there was a decrease in seven proteins associated with the alternative complement pathway and an increase in 6 proteins associated with the lectin and classical complement pathways. The correlation with a shift in complement pathways suggests that compromised immunity could be responsible for the observed dysbiosis in the GI microbiome.
PMID: 27109275
ISSN: 1601-0825
CID: 2091922

Saliva and viral infections

Corstjens, Paul L A M; Abrams, William R; Malamud, Daniel
Over the last 10 years there have been only a handful of publications dealing with the oral virome, which is in contrast to the oral microbiome, an area that has seen considerable interest. Here, we survey viral infections in general and then focus on those viruses that are found in and/or are transmitted via the oral cavity; norovirus, rabies, human papillomavirus, Epstein-Barr virus, herpes simplex viruses, hepatitis C virus, and HIV. Increasingly, viral infections have been diagnosed using an oral sample (e.g. saliva mucosal transudate or an oral swab) instead of blood or urine. The results of two studies using a rapid and semi-quantitative lateral flow assay format demonstrating the correlation of HIV anti-IgG/sIgA detection with saliva and serum samples are presented. When immediate detection of infection is important, point-of-care devices that obtain a non-invasive sample from the oral cavity can be used to provide a first line diagnosis to assist in determining appropriate counselling and therapeutic path for an increasing number of diseases.
PMID: 26662485
ISSN: 1600-0757
CID: 2402452

A Rapid, Self-confirming Assay for HIV: Simultaneous Detection of Anti-HIV Antibodies and Viral RNA

Chen, Zongyuan; Zhu, Hui; Malamud, Daniel; Barber, Cheryl; Ongagna, Yhombi Yvon Serge; Yasmin, Rubina; Modak, Sayli; Janal, Malvin N; Abrams, William R; Montagna, Richard A
OBJECTIVE: We developed a microfluidic system to simultaneously detect host anti-HIV antibodies and viral RNA in the same specimen in order to satisfy two important diagnostic criteria, especially within resource-limited settings. First, the system can detect acute HIV infection and allow immediate confirmation of a seropositive screening result by detection of HIV RNA. It also addresses the well-known "seroconversion window" during early HIV infection when antibodies are not yet detectable and viral loads are at their highest. METHODS: We first developed and optimized two separate manual assays for the detection of host anti-HIV antibodies and viral RNA and then converted them to the microfluidic system. We optimized a commercially available serologic assay to run within the microfluidic device while we incorporated the isothermal LAMP assay to detect the presence of viral RNA. The microfluidic device and instrumentation were developed to simultaneously perform both assays without any user intervention. RESULTS: The finalized system consists of a disposable injection molded and film-laminated microfluidic CARD disposable device and a portable, software controlled instrument, which together can automatically perform all steps of both assays without any user intervention after the initial loading of samples and reagents. The microfluidic CARD cartridge has multiple microchannels, valves, pumps and reservoirs, which perform the immunoassay, isolates viral RNA for detection by magnetic bead based purification, and Reverse Transcriptase loop-mediated isothermal amplification (RT-LAMP). The microfluidic system was able to detect host anti-HIV antibodies and viral RNA in either a blood or saliva sample. CONCLUSION: The ability to detect antibodies and simultaneously confirm a seropositive HIV-RNA result provides healthcare workers with a complete and accurate appraisal of a patient's infection status in the earliest stages of the disease and represents an important tool for the "Test and Treat" and "Treatment as Prevention" approaches for controlling the HIV epidemic.
PMID: 26925300
ISSN: 2155-6113
CID: 2009662

Rapid Point-of-Care Isothermal Amplification Assay for the Detection of Malaria without Nucleic Acid Purification

Modak, Sayli S; Barber, Cheryl A; Geva, Eran; Abrams, William R; Malamud, Daniel; Ongagna, Yhombi Serge Yvon
Malaria remains one of the most prevalent infectious diseases and results in significant mortality. Isothermal amplification (loop-mediated isothermal amplification) is used to detect malarial DNA at levels of ~1 parasite/microL blood in
PMID: 26819557
ISSN: 1178-6337
CID: 1929202

Periluminal Distribution of HIV-Binding Target Cells and Gp340 in the Oral, Cervical and Sigmoid/Rectal Mucosae: A Mapping Study

Patyka, Mariia; Malamud, Daniel; Weissman, Drew; Abrams, William R; Kurago, Zoya
Studies have shown that the transmission of HIV is most likely to occur via rectal or vaginal routes, and rarely through oral exposure. However, the mechanisms of virus entry at mucosal surfaces remain incompletely understood. Prophylactic strategies against HIV infection may be attainable once gaps in current knowledge are filled. To address these gaps, we evaluated essentially normal epithelial surfaces and mapped the periluminal distribution of CD4+ HIV target cells, including T cells and antigen-presenting cells, and an HIV-binding molecule gp340 that can be expressed by epithelial cells in secreted and cell-associated forms. Immunohistochemistry for CD4, CD16, CD3, CD1a and gp340 in human oral, rectal/sigmoid and cervical mucosal samples from HIV-negative subjects demonstrated that periluminal HIV target cells were more prevalent at rectal/sigmoid and endocervical surfaces lined by simple columnar epithelium, than at oral and ectocervical surfaces covered by multilayered stratified squamous epithelium (p<0.001). gp340 expression patterns at these sites were also distinct and strong in oral minor salivary gland acini and ducts, including ductal saliva, in individual rectum/sigmoid and endocervix periluminar columnar cells, and in ectocervix squamous cells. Only weak expression was noted in the oral non-ductal squamous epithelium. We conclude that periluminal HIV target cells, together with periluminal epithelial cell-associated gp340 appear to be most accessible for HIV transmission at rectal/sigmoid and endocervical surfaces. Our data help define vulnerable structural features of mucosal sites exposed to HIV.
PMID: 26172445
ISSN: 1932-6203
CID: 1669172

Oral vs salivary diagnostics [Meeting Abstract]

Marques, Joana; Corby, Patricia M; Barber, Cheryl A; Abrams, William R; Malamud, Daniel
The field of "salivary diagnostics" includes studies utilizing samples obtained from a variety of sources within the oral cavity. These samples include; whole unstimulated saliva, stimulated whole saliva, duct saliva collected directly from the parotid, submandibular/sublingual glands or minor salivary glands, swabs of the buccal mucosa, tongue or tonsils, and gingival crevicular fluid. Many publications state "we collected saliva from subjects" without fully describing the process or source of the oral fluid. Factors that need to be documented in any study include the time of day of the collection, the method used to stimulate and collect the fluid, and how much fluid is being collected and for how long. The handling of the oral fluid during and post-collection is also critical and may include addition of protease or nuclease inhibitors, centrifugation, and cold or frozen storage prior to assay. In an effort to create a standard protocol for determining a biomarker's origin we carried out a pilot study collecting oral fluid from 5 different sites in the mouth and monitoring the concentrations of pro-and anti-inflammatory cytokines detected using MesoScaleDiscovery (MSD) electrochemiluminesence assays. Our data suggested that 3 of the cytokines are primarily derived from the submandibular gland, while 7 of the cytokines come from a source other than the major salivary glands such as the minor salivary glands or cells in the oral mucosae. Here we review the literature on monitoring biomarkers in oral samples and stress the need for determining the blood/saliva ratio when a quantitative determination is needed and suggest that the term oral diagnostic be used if the source of an analyte in the oral cavity is unknown.
ISSN: 0277-786x
CID: 2341282