Paleoserological detection of Coronavirus antigens in dental calculus of human remains dating from the beginning of the 19th century, French Ardennes.

OBJECTIVE: Vanishing viral RNA restricts our ability to detect ancient pathogens, so, we used paleo serological approaches to trace the dynamics of the Coronavirus in ancient populations. MATERIALS AND METHODS: We investigated 10 ancient dental calculus samples collected from a cemetery dated to the beginning of the 19th century and excavated in Charleville-Mézières. After paleoserum samples were extracted from dental calculus, paleoserology using mini-line-blot incorporating one alpha-Coronavirus (Coronavirus 229 E) and two beta-Coronavirus (Coronavirus OC 43, SARS-CoV-2) antigens and controls was completed by an automated Western blotting assay. RESULTS: Once appropriate controls had validated the data, mini-line-blot detected antibodies against the two beta-Coronavirus antigens in individuals US1300 and US1339, automated Western blotting confirming one beta-Coronavirus antigen for individual US1300 and an additional individual US1326. DISCUSSION: Combing mini-line blot and automated Western blot assays made it possible to detect immunoreactive immunoglobulin tracing circulation of Coronavirus in France at the very beginning of the 19th century.

Introducing clinical nanorachaeaology: Isolation by co-culture of Nanopusillus massiliensis sp. nov.

BACKGROUND: Nanoarchaeota, obligate symbiont of some environmental archaea with reduced genomes, have been described in marine thermal vent environments, yet never detected in hosts, including humans. METHODS: Here, using laboratory tools geared towards the detection of nanoarchaea including PCR-sequencing, WGS, microscopy and culture. RESULTS: We discovered a novel nanoarchaea, Nanopusillus massiliensis, detected in dental plate samples by specific PCR-based assays. Combining fluorescent in situ hybridization (FISH) with scanning electron microscopy disclosed close contacts between N. massiliensis and the archaea Methanobrevibacter oralis in these samples. Culturing one sample yielded co-isolation of M. oralis and N. massiliensis with a 606,935-bp genome, with 23.6% GC encoded 16 tRNA, 3 rRNA and 942 coding DNA sequences, of which 400 were assigned to clusters of orthologous groups. CONCLUSION: The discovery of N. massiliensis, made publicly available in collection, extended our knowledge of human microbiota diversity, opening a new field of research in clinical microbiology here referred to as clinical nanoarchaeology.

Chemical & Nano-mechanical Study of Artificial Human Enamel Subsurface Lesions.

White lesions represent an early phase of caries formation. 20 human sound premolars were subjected to pH cycling procedure to induce subsurface lesions (SLs) in vitro. In addition, 2 teeth with naturally developed white spot lesions (WSLs) were used as references. All specimens characterized by confocal Raman microscopy being used for the first time in examining white & subsurface lesions and providing a high resolution chemical and morphological map based on phosphate peak intensity alterations at 960 cm-1. Nanoindentation technique was used to measure Hardness (H) and Young's modulus (E) of enamel. Phosphate map of examined samples exhibited presence of intact surface layer (ISL) followed by severe depletion in (PO43-) peak in the area corresponding to the body of the lesion. In all examined groups, the mechanical properties of enamel were decreased in lesion area and found to be inversely related to penetration depth of indenter owing to enamel hierarchical structure. By combining the above two techniques, we linked mechanical properties of enamel to its chemical composition and ensured that the two methods are highly sensitive to detect small changes in enamel composition. Further work is required to bring these two excellent tools to clinical application to perceive carious lesions at an early stage of development.

Laboratory Studies of Nonlinear Optical Signals for Caries Detection.

Multiphoton confocal microscopy and nonlinear spectroscopy are used to investigate the caries process in dentin. Although dentin is a major calcified tissue of the teeth, its organic phase comprises type I collagen fibers. Caries drive dentin demineralization and collagen denaturation. Multiphoton microscopy is a powerful imaging technique: the biological materials are transparent to infrared frequencies and can be excited to penetration depths inaccessible to 1-photon confocal microscopy. The laser excitation greatly reduces photodamage to the sole focal region, and the signal-to-noise ratio is improved significantly. The method has been used to follow pathologic processes involving collagen fibrosis or collagen destruction based on their 2-photon excited fluorescence (2PEF) emission and second harmonic generation (SHG). Combining multiphoton imaging with nonlinear spectroscopy, we demonstrate that both 2PEF and SHG intensity of human dentin are strongly modified during the tooth caries process, and we show that the ratio between SHG and 2PEF signals is a reliable parameter to follow dental caries. The ratio of the SHG/2PEF signals measured by nonlinear optical spectroscopy provides valuable information on the caries process, specifically on the degradation of the organic matrix of dentin. The goal is to bring these nonlinear optical signals to clinical application for caries diagnosis.

Use of new minimum intervention dentistry technologies in caries management.

Preservation of natural tooth structure requires early detection of the carious lesion and is associated with comprehensive patient dental care. Processes aiming to detect carious lesions in the initial stage with optimum efficiency employ a variety of technologies such as magnifying loupes, transillumination, light and laser fluorescence (QLF® and DIAGNOdent® ) and autofluorescence (Soprolife® and VistaCam®), electric current/impedance (CarieScan(®) ), tomographic imaging and image processing. Most fluorescent caries detection tools can discriminate between healthy and carious dental tissue, demonstrating different levels of sensitivity and specificity. Based on the fluorescence principle, an LED camera (Soprolife® ) was developed (Sopro-Acteon, La Ciotat, France) which combined magnification, fluorescence, picture acquisition and an innovative therapeutic concept called light-induced fluorescence evaluator for diagnosis and treatment (LIFEDT). This article is rounded off by a Soprolife® illustration about minimally or even non-invasive dental techniques, distinguishing those that preserve or reinforce the enamel and enamel-dentine structures without any preparation (MIT1- minimally invasive therapy 1) from those that require minimum preparation of the dental tissues (MIT2 - minimally invasive therapy 2) using several clinical cases as examples. MIT1 encompasses all the dental techniques aimed at disinfection, remineralizing, reversing and sealing the caries process and MIT2 involves a series of specific tools, including microburs, air abrasion devices, sonic and ultrasonic inserts and photo-activated disinfection to achieve minimal preparation of the tooth. With respect to minimally invasive treatment and prevention, the use of lasers is discussed. Furthermore, while most practices operate under a surgical model, Caries Management by Risk Assessment (CaMBRA) encourages a medical model of disease prevention and management to control the manifestation of the disease, or keep the oral environment in a state of balance between pathological and preventive factors. Early detection and diagnosis and prediction of lesion activity are of great interest and may change traditional operative procedures substantially. Fluorescence tools with high levels of magnification and observational capacity should guide clinicians towards a more preventive and minimally invasive treatment strategy.