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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

The effect of a novel oral care protocol in decreasing the expression of cytokines in head and neck cancer patients receiving chemoradiation [Meeting Abstract]

Vasconcelos, R; Moya, J; Gabinskiy, M; Nightingale, K; Queiroz, E; Malamud, D; Barber, C; Howard, C; Kerr, R; Sanfilippo, N; Corby, P
Introduction Oral mucositis (OM) is one of the most debilitating adverse effects in patients undergoing cancer treatment. Physiologically, chemotherapy (CT) and radiotherapy (RT) evoke a profound inflammatory response, resulting in mucosal injury, which can result in an increased susceptibility to infection. Objectives The objective of this pilot study was to asses the effects of a novel oral care protocol on OM severity and to evaluate salivary cytokines in head and neck cancer patients undergoing RT or CT/RT at the NYU Langone Laura and Isaac Perlmutter Cancer Center. Methods A total of ten participants were included in this study, and randomized to an InterventionGroup (IG), or ControlGroup (CG). Subjects assigned to the CG received a standard of care oral hygiene on a bi-weekly basis. Subjects assigned to the IG received the Oral Mucosal Deterging and Dental Prophylaxis (OMDP) protocol weekly, which consisted of a periodontal surface debridement and cleansing and deterging of the oral mucosa surfaces. Results Salivary inflammatory biomarkers, noted in levels of IFN-gamma, IL10, IL12p70, IL13, TNFalpha and IL-6 had a significant increase in the CG and reduced or stayed the same under the IG. Although not statistically significant, a tendency of pain decrease was observed in the IG and difficulty in swallowing was statistically significant when both groups were compared (p = 0,016). Conclusions These results suggest that overall inflammation was consistently higher as compared to baseline in the CG, providing encouragement for the effectiveness of the oral care protocol as a coadjutant treatment for this population
ISSN: 1433-7339
CID: 3141662

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

The gut and oral microbiome in HIV disease: a workshop report

Moyes, D L; Saxena, D; John, M D; Malamud, D
Recent years have seen a massive expansion in our understanding of how we interact with our microbial colonists. The development of new, rapid sequencing techniques such as pyrosequencing and other next-generation sequencing systems have enabled us to begin to characterise the constituents of our diverse microbial communities, revealing the astonishing genetic richness that is our microbiome. Despite this, our ignorance of how these communities change over the course of an HIV infection is profound. Whilst some steps have been made to characterise the HIV microbiome at selected sites, these reports are still limited and much remains to be done. It has become apparent, however, that host-microbiota interactions are perturbed during HIV infections, with microbial translocation of potential pathogens linked to a variety of different HIV complications, including more rapid progression of disease. The use of probiotics and prebiotics has been investigated as treatments to alleviate symptoms for a variety of conditions, and is now being proposed for the treatment of symptoms associated with HIV. However, this is a new area of investigations and many questions remain unanswered. What we know about both of these topics is a drop in the ocean compared with what we need to know. In this article, we report on a workshop where these two major under-investigated research areas were presented, and future directions explored and discussed.
PMID: 27109284
ISSN: 1601-0825
CID: 2092162

Innate immunity in HIV-1 infection: epithelial and non-specific host factors of mucosal immunity- a workshop report

Nittayananta, W; Weinberg, A; Malamud, D; Moyes, D; Webster-Cyriaque, J; Ghosh, S
The interplay between HIV-1 and epithelial cells represents a critical aspect in mucosal HIV-1 transmission. Epithelial cells lining the oral cavity cover subepithelial tissues, which contain virus-susceptible host cells including CD4(+) T lymphocytes, monocytes/macrophages, and dendritic cells. Oral epithelia are among the sites of first exposure to both cell-free and cell-associated virus HIV-1 through breast-feeding and oral-genital contact. However, oral mucosa is considered to be naturally resistant to HIV-1 transmission. Oral epithelial cells have been shown to play a crucial role in innate host defense. Nevertheless, it is not clear to what degree these local innate immune factors contribute to HIV-1 resistance of the oral mucosa. This review paper addressed the following issues that were discussed at the 7th World Workshop on Oral Health and Disease in AIDS held in Hyderabad, India, during November 6-9, 2014: (i) What is the fate of HIV-1 after interactions with oral epithelial cells?; (ii) What are the keratinocyte and other anti-HIV effector oral factors, and how do they contribute to mucosal protection?; (iii) How can HIV-1 interactions with oral epithelium affect activation and populations of local immune cells?; (iv) How can HIV-1 interactions alter functions of oral epithelial cells?
PMID: 27109285
ISSN: 1601-0825
CID: 2092172

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