Amplified parallel antigen rapid test for point-of-care salivary detection of SARS-CoV-2 with improved sensitivity.

In the ongoing COVID-19 pandemic, simple, rapid, point-of-care tests not requiring trained personnel for primary care testing are essential. Saliva-based antigen rapid tests (ARTs) can fulfil this need, but these tests require overnight-fasted samples; without which independent studies have demonstrated sensitivities of only 11.7 to 23.1%. Herein, we report an Amplified Parallel ART (AP-ART) with sensitivity above 90%, even with non-fasted samples. The virus was captured multimodally, using both anti-spike protein antibodies and Angiotensin Converting Enzyme 2 (ACE2) protein. It also featured two parallel flow channels. The first contained spike protein binding gold nanoparticles which produced a visible red line upon encountering the virus. The second contained signal amplifying nanoparticles that complex with the former and amplify the signal without any linker. Compared to existing dual gold amplification techniques, a limit of detection of one order of magnitude lower was achieved (0.0064 ng·mL-1). AP-ART performance in detecting SARS-CoV-2 in saliva of COVID-19 patients was investigated using a case-control study (139 participants enrolled and 162 saliva samples tested). Unlike commercially available ARTs, the sensitivity of AP-ART was maintained even when non-fasting saliva was used. Compared to the gold standard reverse transcription-polymerase chain reaction testing on nasopharyngeal samples, non-fasting saliva tested on AP-ART showed a sensitivity of 97.0% (95% CI: 84.7-99.8); without amplification, the sensitivity was 72.7% (95% CI: 83.7-94.8). Thus, AP-ART has the potential to be developed for point-of-care testing, which may be particularly important in resource-limited settings, and for early diagnosis to initiate newly approved therapies to reduce COVID-19 severity.

Prognostic significance of invasion depth in oral tongue squamous cell carcinoma.

PURPOSE: Staging for tongue carcinoma does not consider its depth of invasion. We aim to determine the prognostic significance of invasion depth in tongue cancer. MATERIALS AND METHODS: Retrospective review of patients with tongue carcinoma who underwent curative surgery between 2002 and 2005; 67 patients were analyzed. Tumors were divided into 2 groups: group A = invasion depth <4 mm; group B = invasion depth ≥4 mm. Recurrence and survival rates were calculated for each group and compared. RESULTS: The local recurrence rate in group B was significantly higher (10 vs. 29.7%, p = 0.048). Group A patients had a superior 5-year overall survival (68.8 vs. 41.6%, p = 0.012), disease-specific survival (67.1 vs. 41.1%, p = 0.026) and local recurrence-free survival (89.5 vs. 65.4%, p = 0.035). Five-year regional recurrence, locoregional recurrence and distant recurrence-free survival rates were not significantly different between the 2 groups (p = 0.390, p = 0.173 and p = 0.207). The impact of invasion depth on survival was maintained on multivariate analysis (p = 0.031). CONCLUSION: Invasion depth is an important prognostic indicator in tongue cancer.

In-vivo optical detection of cancer using chlorin e6--polyvinylpyrrolidone induced fluorescence imaging and spectroscopy.

BACKGROUND: Photosensitizer based fluorescence imaging and spectroscopy is fast becoming a promising approach for cancer detection. The purpose of this study was to examine the use of the photosensitizer chlorin e6 (Ce6) formulated in polyvinylpyrrolidone (PVP) as a potential exogenous fluorophore for fluorescence imaging and spectroscopic detection of human cancer tissue xenografted in preclinical models as well as in a patient. METHODS: Fluorescence imaging was performed on MGH human bladder tumor xenografted on both the chick chorioallantoic membrane (CAM) and the murine model using a fluorescence endoscopy imaging system. In addition, fiber optic based fluorescence spectroscopy was performed on tumors and various normal organs in the same mice to validate the macroscopic images. In one patient, fluorescence imaging was performed on angiosarcoma lesions and normal skin in conjunction with fluorescence spectroscopy to validate Ce6-PVP induced fluorescence visual assessment of the lesions. RESULTS: Margins of tumor xenografts in the CAM model were clearly outlined under fluorescence imaging. Ce6-PVP-induced fluorescence imaging yielded a specificity of 83% on the CAM model. In mice, fluorescence intensity of Ce6-PVP was higher in bladder tumor compared to adjacent muscle and normal bladder. Clinical results confirmed that fluorescence imaging clearly captured the fluorescence of Ce6-PVP in angiosarcoma lesions and good correlation was found between fluorescence imaging and spectral measurement in the patient. CONCLUSION: Combination of Ce6-PVP induced fluorescence imaging and spectroscopy could allow for optical detection and discrimination between cancer and the surrounding normal tissues. Ce6-PVP seems to be a promising fluorophore for fluorescence diagnosis of cancer.

Improved formulation of photosensitizer chlorin e6 polyvinylpyrrolidone for fluorescence diagnostic imaging and photodynamic therapy of human cancer.

An improved formulation of the photosensitizer chlorin e6 (Ce6) in combination with the hydrophilic polymer polyvinylpyrrolidone (PVP) was investigated for its potential clinical applications in fluorescence diagnosis and photodynamic therapy (PDT) of cancer. This study reports the comparative preclinical biodistribution and efficacy of Ce6 delivered with or without PVP versus dimethyl sulfoxide (DMSO). The safety and fluorescence pharmacokinetics of Ce6-PVP in humans was also accessed. Biodistribution results showed that Ce6-PVP had higher tumor to normal tissue ratio compared to the other formulations. The sensitivity and specificity derived from the area under the receiver operating characteristics curves showed that the formulations were able to discriminate tumor from peritumoral muscle in the following order: Ce6-PVP > Ce6 > Ce6-DMSO. In vitro PDT results showed that Ce6-PVP was found to induce selective phototoxicity in leukemic cells compared to peripheral mononuclear blood cells. In addition, in vivo light irradiation at 1h after Ce6-PVP was found to induce greater tumor necrosis without causing animal toxicity. In patients, preferential accumulation of Ce6-PVP was observed in angiosarcoma lesions compared to normal skin following intravenous administration. In conclusion, PVP significantly enhanced the Ce6 concentration in tumors compared with Ce6 alone and increased the therapeutic index of PDT without any side effects in animal model. No serious adverse events were observed in human as well.

Immune response against angiosarcoma following lower fluence rate clinical photodynamic therapy.

Tumor response to photodynamic therapy (PDT) is dependent on treatment parameters used. In particular, the light fluence rate may be an important determinant of the treatment outcome. In this clinical case report, we describe the response of angiosarcoma to PDT carried out using different fluence rates and drug and light doses. A patient with recurrent multifocal angiosarcoma of the head and neck was recruited for PDT. A new generation chlorin-based photosensitizer, Fotolon, was administered at a dose of 2.0 to 5.7 mg/kg. The lesions were irradiated with 665 nm laser light for a light dose of 65 to 200 J/cm2 delivered at a fluence rate of 80 or 150 mW/cm2. High dose PDT carried out at a high fluence rate resulted in local control of the disease for up to a year; however, the disease recurred and PDT had to be repeated. PDT of new lesions carried out at a lower fluence rate resulted in tumor eradication. More significantly, it also resulted in spontaneous remission of neighboring and distant untreated lesions. Repeat PDT carried out on a recurrent lesion at a lower fluence rate resulted in eradication of both treated and untreated lesions despite the lower total light dose delivered. Immunohistochemical examination of biopsy samples implies that PDT could have activated a cell-mediated immune response against untreated lesions. Subsequent histopathological examination of the lesion sites showed negative for disease. Our clinical observations show that lower fluence rate PDT results in better outcome and also indicate that the fluence rate, rather than the total light dose, is a more crucial determinant of the treatment outcome. Specifically, lower fluence rate PDT appears to activate the body's immune response against untreated lesions.

Novel delivery of Chlorin e6 using anti-EGFR antibody tagged virosomes for fluorescence diagnosis of oral cancer in a hamster cheek pouch model.

PURPOSE: Overexpression of epidermal growth factor receptor (EGFR) is observed in oral squamous cell carcinoma (OSCC) and is associated with increased proliferation, metastasis and therapeutic resistance. We aim to develop a novel drug delivery system comprised of a photosensitizer Chlorin e6 (Ce6) that is encapsulated in a viral envelope and tagged with anti-EGFR antibody to target OSCC. METHODS: Ce6 was encapsulated in both virosomes (Ce6-Vir) and virosomes tagged with anti-EGFR antibody (Ce6-Vir-EGFR'). In vitro studies were conducted to assess the cellular uptake and bioavailability of the photosensitizer in OSCC cells. Ce6 alone or in constructs was then administered in a hamster cheek pouch model and fluorescence imaging and spectroscopy was performed. RESULTS: In vitro results showed that the uptake of Ce6-Vir-EGFR' was lower than that for Ce6-Vir and Ce6 possibly due to its large size. Nevertheless, in vivo results showed significant tumor specificity of Ce6-Vir-EGFR' compared to Ce6. The tumor to normal mucosa ratio showed that Ce6-Vir-EGFR' can successfully target OSCC lesions and therefore shows potential for use in fluorescence diagnosis of OSCC. CONCLUSIONS: Both the virosome-Ce6 constructs were internalized by OSCC cells and successfully used for fluorescence imaging. Tagging with anti-EGFR antibody further improved the targeting ability toward OSCC.

In vivo Biocompatibility, Biodistribution and Therapeutic Efficiency of Titania Coated Upconversion Nanoparticles for Photodynamic Therapy of Solid Oral Cancers.

Despite the advantages of using photodynamic therapy (PDT) for the treatment of head and neck tumors, it can only be used to treat early stage flat lesions due to the limited tissue penetration ability of the visible light. Here, we developed near-infrared (NIR) excitable upconversion nanoparticle (UCN) based PDT agent that can specifically target epithelial growth factor receptor (EGFR) overexpressing oral cancer cells, in a bid to widen the application of PDT against thick and solid advanced or recurrent head and neck cancers. In vivo studies using the synthesized anti-EGFR-PEG-TiO2-UCNs following systemic administration displayed no major sub-acute or long term toxic effects in terms of blood biochemical, hematological or histopathological changes at a concentration of 50 mg/kg. NIR-PDT even in the presence of a 10 mm tissue phantom placed over the xenograft tumor, showed significant delay in tumor growth and improved survival rate compared to conventional chlorin-e6 (Ce6) PDT using 665 nm red light. Our work, one of the longest study till date in terms of safety (120 d), PDT efficacy (35 d) and survival (60 d), demonstrates the usefulness of UCN based PDT technology for targeted treatment of thick and bulky head and neck tumors.

Loss of parafibromin immunoreactivity is a distinguishing feature of parathyroid carcinoma.

PURPOSE: A reliable method for diagnosing parathyroid carcinoma has remained elusive over the years, resulting in its under-recognition and suboptimal therapy. Obtaining an accurate diagnosis has become an even more pressing matter with recent evidence that germline HRPT2 gene mutations are found in patients with apparently sporadic parathyroid carcinoma. There is a high prevalence of HRPT2 gene mutations and biallelic inactivation in parathyroid carcinoma. We hypothesize that loss of parafibromin, the protein product of the HRPT2 gene, would distinguish carcinoma from benign tissue. EXPERIMENTAL DESIGN: We generated a novel antiparafibromin monoclonal antibody and performed immunostaining on 52 definite carcinoma specimens, 6 equivocal carcinoma specimens, 88 benign specimens, and 9 hyperparathyroidism-jaw tumor (HPT-JT) syndrome-related adenomas from patients with primary hyperparathyroidism from nine worldwide centers and one national database. RESULTS: We report that the loss of parafibromin nuclear immunoreactivity has 96% sensitivity [95% confidence interval (CI), 85-99%] and 99% specificity (95% CI, 92-100%) in diagnosing definite carcinoma. Inter-observer agreement for evaluation of parafibromin loss was excellent, with unweighted kappa of 0.89 (95% CI, 0.79-0.98). Two equivocal carcinomas misclassified as adenomas were highlighted by parafibromin immunostaining. One of these tumors has since recurred, satisfying criteria for a definite carcinoma. Similarly, eight of nine HPT-JT syndrome-related adenomas showed absent nuclear immunoreactivity. CONCLUSIONS: Parafibromin is a promising molecular marker for diagnosing parathyroid carcinoma. The similar loss of parafibromin immunoreactivity in HPT-JT syndrome-related adenomas suggests that this is a pivotal step in parathyroid tumorigenesis.

Titania coated upconversion nanoparticles for near-infrared light triggered photodynamic therapy.

Because of the limited penetration depth of visible light that generally excites most of the available photosensitizers (PSs), conventional photodynamic therapy (PDT) is limited to the treatment of superficial and flat lesions. Recently, the application of deep penetrating near-infrared (NIR) light excitable upconversion nanoparticles (UCNs) in conjunction with PDT has shown to have clear potential in the treatment of solid tumors due to its ability to penetrate thick tissue. However, various constructs developed so far have certain limitations such as poor or unstable PS loading, reducing their therapeutic efficacy and limiting their application to solution or cell-based studies. In this work, we present a method to fabricate uniform core-shell structured nanoconstruct with a thin layer of photocatalyst or PS-titanium dioxide (TiO2) stably coated on individual UCN core. Our design allows controllable and highly reproducible PS loading, preventing any leakage of PS compared to previously developed nanoconstructs, thus ensuring repeatable PDT results. Further surface modification of the developed nanoconstructs with polyethylene glycol (PEG) rendered them biocompatible, demonstrating good therapeutic efficacy both in vitro and in vivo.

Management of orocutaneous fistulas using a vacuum-assisted closure system.

BACKGROUND: The vacuum-assisted closure (VAC) system has been used to manage complicated wounds. The purpose of this study was to describe a novel technique in using the VAC system for orocutaneous fistulas. METHODS: A retrospective study was performed on 10 patients treated at the National Cancer Centre, Singapore, who developed postoperative orocutaneous fistulas. Hydrogum dental paste was used as a sealant together with the VAC system to close the fistulas. We used either the RENASYS or VAC ATS system with 50 mm Hg to 125 mm Hg continuous suction. RESULTS: The 10 patients developed 11 fistulas. The median age of this cohort was 67 years (range, 33-80 years). Nine patients had successful closure of their fistulas with VAC therapy whereas 1 patient had unsuccessful VAC therapy and required flap reconstruction. The median time to fistula closure was 19 days (range, 6-36 days). The median time to radiotherapy after surgery was 46 days (range, 26-62 days). CONCLUSION: VAC therapy is an effective treatment option for orocutaneous fistulas.

Photolon™ --photosensitization induces apoptosis via ROS-mediated cross-talk between mitochondria and lysosomes.

The localization of photosensitizers in the subcellular compartments during photodynamic therapy (PDT) plays a major role in the cell destruction; therefore, the aim of this study was to investigate the intracellular localization of Chlorin e6-PVP (Photolon™) in malignant and normal cells. Our study involves the characterization of the structural determinants of subcellular localization of Photolon, and how subcellular localization affects the selective toxicity of Photolon towards tumor cells. Using confocal laser scanning microscopy (CLSM) and fluorescent organelle probes; we examined the subcellular localization of Photolon™ in the murine colon carcinoma CT-26 and normal fibroblast (NHLC) cells. Our results demonstrated that after 30 min of incubation, the distribution of Photolon was localized mainly in the cytoplasmic organelles including the mitochondria, lysosomes, Golgi apparatus, around the nuclear envelope and also in the nucleus but not in the endo-plasmic reticulum whereas in NHLC cells, Photolon was found to be localized minimally only in the nucleus not in other organelles studied. The relationship between subcellular localization of Photolon and PDT-induced apoptosis was investigated. Apoptotic cell death was judged by the formation of known apoptotic hallmarks including, the phosphatidylserine externalization (PS), PARP cleavage, a substrate for caspase-3 and the formation of apoptotic nuclei. At the irradiation dose of 1 J/cm2, the percentage of apoptotic cells was 80%, respectively. This study provided substantial evidence that Photolon preferentially localized in the subcellular organelles in the following order: nucleus, mitochondria, lysosomes and the Golgi apparatus and subsequent photodamage of the mitochondria and lyso-somes played an important role in PDT-mediated apoptosis CT-26 cells. Our results based on the cytoplasmic organelles and the intranuclear localization extensively enhance the efficacy of PDT with appropriate photosensitizer and light dose and support the idea that PDT can contribute to elimination of malignant cells by inducing apoptosis, which is of physiological significance.

Early diagnosis of oral cancer based on the surface plasmon resonance of gold nanoparticles.

The high mortality rate in cancer such as oral squamous cell carcinoma is commonly attributed to the difficulties in detecting the disease at an early treatable stage. In this study, we exploited the ability of gold nanoparticles to undergo coupled surface plasmon resonance and set up strong electric fields when closely-spaced to improve the molecular contrast signal in reflectance-based imaging and also to enhance the Raman signal of bioanalytes in cancer. Colloidal gold nanoparticles were synthesized and conjugated to anti-epidermal growth factor receptor (EGFR) for imaging. A self-assembled surface enhanced Raman scattering (SERS)-active gold nanoparticle monolayer film was also developed as a biosensing surface using a simple drop-dry approach. We have shown that gold nanoparticles could elicit an optical contrast to discriminate between cancerous and normal cells and their conjugation with antibodies allowed them to map the expression of relevant biomarkers for molecular imaging under confocal reflectance microscopy. We have also shown that the SERS spectra of saliva from the closely-packed gold nanoparticles films was differentiable between those acquired from normal individuals and oral cancer patients, thus showing promise of a simple SERS-based saliva assay for early diagnosis of oral cancer.