Assessing the performance of a Loop Mediated Isothermal Amplification (LAMP) assay for the detection and subtyping of high-risk suptypes of Human Papilloma Virus (HPV) for Oropharyngeal Squamous Cell Carcinoma (OPSCC) without DNA purification.

BACKGROUND: Oropharyngeal Squamous Cell Carcinoma (OPSCC) is increasing in incidence despite a decline in traditional risk factors. Human Papilloma Virus (HPV), specifically subtypes 16, 18, 31 and 35, has been implicated as the high-risk etiologic agent. HPV positive cancers have a significantly better prognosis than HPV negative cancers of comparable stage, and may benefit from different treatment regimens. Currently, HPV related carcinogenesis is established indirectly through Immunohistochemistry (IHC) staining for p16, a tumour suppressor gene, or polymerase chain reaction (PCR) that directly tests for HPV DNA in biopsied tissue. Loop mediated isothermal amplification (LAMP) is more accurate than IHC, more rapid than PCR and is significantly less costly. In previous work we showed that a subtype specific HPV LAMP assay performed similar to PCR on purified DNA. In this study we examined the performance of this LAMP assay without DNA purification. METHODS: We used LAMP assays using established primers for HPV 16 and 18, and new primers for HPV 31 and 35. LAMP reaction conditions were tested on serial dilutions of plasmid HPV DNA to confirm minimum viral copy number detection thresholds. LAMP was then performed directly on different human cell line samples without DNA purification. RESULTS: Our LAMP assays could detect 105, 103, 104, and 105 copies of plasmid DNA for HPV 16, 18, 31, and 35, respectively. All primer sets were subtype specific, with no cross-amplification. Our LAMP assays also reliably amplified subtype specific HPV DNA from samples without requiring DNA isolation and purification. CONCLUSIONS: The high risk OPSCC HPV subtype specific LAMP primer sets demonstrated, excellent clinically relevant, minimum copy number detection thresholds with an easy readout system. Amplification directly from samples without purification illustrated the robust nature of the assay, and the primers used. This lends further support HPV type specific LAMP assays, and these specific primer sets and assays can be further developed to test for HPV in OPSCC in resource and lab limited settings, or even bedside testing.

Technical note: Evaluation of tined endoscopically placed mucosal leads for temporary gastric neurostimulation.

OBJECTIVE: To compare larger diameter corkscrew-tined leads with previously described intracardiac pacing leads for temporary gastric neurostimulation in a canine model. MATERIALS AND METHODS: Two mongrel dogs underwent gastroscopy under general anesthesia, with endoscopic placement of two cardiac leads (1 mm tine diameter, 4 mm depth) placed sequentially in 1) transverse configuration in the distal antrum mucosa; 2) longitudinal (1 cm apart) configuration in gastric corpus. Stomach was then stimulated with maximal parameters to induce neutrally mediated contraction. Procedures were then repeated with larger leads (5 mm tine diameter, 8 mm length). Gastric contractions were measured with serosal strain transducers. RESULTS: Leads were placed endoscopically without difficulty. Neither lead type punctured through to the serosa of the stomach. Neither cardiac nor larger leads were capable of eliciting any gastric contractile activity with endoscopic placement either in the transverse or longitudinal orientations. DISCUSSION: While successful on the serosal side, both the cardiac leads and the larger alternative leads failed to produce stomach contraction when implanted mucosally. This may be due to the elastic nature of the mucosa, which was observed to twist around both types of leads significantly, hindering proper penetration into the muscularis. CONCLUSION: These results suggest that the current concept of temporary gastric electrical neurostimulation via a mucosal approach must be reevaluated, as the procedure most likely does not accurately mimic electrical stimulation in the muscularis.

Percutaneous endoscopically assisted transenteric full-thickness gastric biopsy: initial experience in humans.

BACKGROUND: GI neuromuscular diseases (GINMD) can cause severe dysmotility and symptoms. Full-thickness biopsy specimens may help diagnose these disorders histologically. OBJECTIVE: To assess a novel percutaneous endoscopically assisted transenteric (PEATE) biopsy method for obtaining full-thickness gastric tissue in patients with suspected GINMD. DESIGN: Prospective proof-of-concept case series. SETTING: Tertiary care gastroenterology unit. PATIENTS: Ten patients (8 women, mean [standard deviation] age 43 [10] years) with gastroparesis-like symptoms (mean [standard deviation] gastroparesis cardinal symptom index 3.28 [1.46] out of 5) and/or clinical findings suggestive of a gastric GINMD. INTERVENTIONS: All patients underwent PEATE biopsy during standard gastroscopy as an outpatient procedure. Tissue was stained for histology and immunohistochemistry of gut wall elements. Interstitial cells of Cajal (ICC) counts were compared with archived normal gastric tissue from control gastrectomies. MAIN OUTCOME MEASUREMENTS: Biopsy success, complications, histopathological findings according to the London Classification of GINMD. RESULTS: Full-thickness antral tissue suitable for analysis was obtained in 9 in 10 patients (90%). PEATE biopsy was well tolerated by all patients without complications. Histology suggested GINMD in 4 of 9 cases (44%), with possible degenerative leiomyopathy in 2, probable inflammatory leiomyopathy in 1, and abnormal ICC networks (>50% reduction in ICC counts) in 1 patient. LIMITATIONS: PEATE biopsy specimen size is smaller than a standard laparoscopic full-thickness biopsy. CONCLUSIONS: PEATE full-thickness gastric biopsy is a simple and safe method of assessing histopathological abnormalities in gastric GINMD without the need for laparoscopy or general anesthesia.