Gingivitis

Gingival tissue biopsies were obtained from five healthy adult volunteers with no gingival inflammation. The gingival specimens were completely de-epithelialized with a scalpel, for the exclusion of the most of the keratinocytes resident in the gingival. In brief, the connective tissues were grinded and then washed several time with PBS (LiStarFish, Milan, Italy) and subsequently cultured using TheraPEAK™MSCGM-CD™ BulletKit serum free, chemically defined (MSCGM-CD) medium for the growth of human MSCs (Lonza, Basel, Switzerland). The medium was changed twice a week, and cells spontaneously migrating from the explant fragments after reaching about 80% of confluence, were trypsinized using Triple Select (LiStar Fish) (Diomede et al., 2014 (link)). On day 6, colonies of 50 or more cells were scored as colony-forming unit fibroblasts (CFU).

Information regarding socio-demographic characteristics and general health-related variables, such as history of AIDS-related illnesses and current medications, were collected using a questionnaire administered during the study visit.
An extra-oral examination of the major salivary glands, and oral mucosal examination were performed by both a CTU examiner (non-OHS) and an OHS on each participant. Both examiners recorded their findings including descriptors of lesions with respect to location, color, and character, and a presumptive diagnosis. Examiners were blinded to each other’s findings. Oral disease endpoints explored included PC; EC; AC; HL; herpes labialis; recurrent intra-oral herpes simplex; warts; recurrent aphthous stomatitis; necrotizing gingivitis/periodontitis; necrotizing stomatitis; KS; non-Hodgkin’s lymphoma; squamous cell carcinoma; and salivary gland disease (as defined by presence/absence of parotid enlargement). A 5-minute unstimulated whole saliva (UWS) flow rate was recorded, and collected. A 1-minute oral rinse/throat wash using 10 mL of sterile saline was also collected. Both saliva and throat wash specimens were processed, frozen in aliquots at minus 80°C at the site laboratory, and shipped to the UNC-CH specimen bank unit. Before, the throat wash was processed at the sites, 2.5 mL was extracted and cultured for the presence of Candida. A blood draw was performed at the time of the visit for CD4+ cell count and plasma HIV-1 viral load to be measured. The CD4+ cell count and the HIV-1 viral load assay were performed in a CLIA certified laboratory for US sites, and in a laboratory certified for protocol testing by the DAIDS Immunology Quality Assurance (IQA) Program for the Haiti site. Plasma HIV-1 viral load were performed utilizing the Abbott Realtime HIV-1 Assay.

Between February and April 2018, a convenience sample of 62 children aged between birth and 59 months of age was recruited in Korle Bu Teaching Hospital, Ghana. Recruitment was carried out in accordance with University College London Ethics application 4050/003, and proposals submitted to the Ethical and Protocol Review Committee of the College of Health Sciences, University of Ghana as CHS-Et/M.5-P1.11/2017–2018. Written consent was given by the parents of the participants. Additional information regarding the ethical, cultural, and scientific considerations specific to inclusivity in global research is included in the (S1 Checklist).
Participants were only recruited when they were clinically stable, where a blood test was requested by their attending doctor as part of their standard clinical care, and when urgent care would not be delayed by the acquisition of the images for this study. Participants were excluded if they were receiving oxygen, had received a blood transfusion in the last 24 hours, or were otherwise deemed to be very ill or unstable. Participants were excluded when conditions such as uveitis or gingivitis that affected the colouration of the imaged mucosa were recorded. Before participation, the research nurse/doctor explained the purpose of the study, and answered any questions which the parent or child raised. After all questions were answered, parents were provided with written consent forms prior to image acquisition.
For each participant, blood haemoglobin concentration was measured with a HemoCue Hb 301 point-of-care anaemia screening device (HemoCue AB, Ängelholm, Sweden) in accordance with the manufacturer’s guidelines, after which they were immediately imaged by a trained clinician. The imaging was carried out using the back-facing camera on a Samsung Galaxy S8 smartphone with a custom mobile application which captured and stored raw, lossless (Adobe Digital Negative) camera images. Using raw images means that there was minimal post-sensor processing. There were 3 pairs of images taken: (1) an image of the sclera and surrounding skin, (2) an image of the folded-over lower eyelid, and (3) an image of the lower lip. Each pair of images consisted of an image taken with the smartphone camera flash turned on, and another taken automatically afterwards with the smartphone camera flash turned off. The flash was diffused using a polymer diffuser in order to ensure the flash brightness fell within safe limits.

Upon confirmation of eligibility for enrollment in the study, clinical periodontal measurements including probing pocket depth (PPD) (mm), clinical attachment loss (CAL) (mm), plaque index (PI) [26 (link)], gingival index (GI) [27 (link)], and bleeding on probing (BOP) (presence/absence) (%) [28 (link)] were recorded from all participants (test and control) during their visit to the Periodontology Department at timepoint 2. Clinical periodontal measurements were performed at six sites on each tooth (mesio-buccal, mid-buccal, disto-buccal, mesio-lingual, mid-lingual, and disto-lingual locations), except for third molars, using a manual periodontal probe (Williams, Hu-Friedy, Chicago, IL, USA) by a single trained examiner (AS). Intra-examiner agreement was determined for CAL. The intra-examiner reproducibility was determined through repeated examinations of 10 subjects with a one-hour interval (k = 0.95).
Diagnosis of periodontal disease was based on clinical and radiographic criteria proposed by 2017 World Workshop on the Classifications of Periodontal and Peri-implant Disease and Conditions [29 (link)]. Individuals with a BOP < 10% without attachment loss and radiographic bone loss were considered to have periodontal health [30 (link)]. Individuals presenting with a BOP ≥ 10%, and PPD ≤ 3 mm without attachment loss and radiographic bone loss were considered gingivitis [31 (link)]. The criteria for patients with periodontitis were (1) interdental CAL detectable at ≥ 2 non-adjacent teeth or (2) buccal or oral CAL ≥ 3 mm with PPD > 3 mm detectable at ≥ 2 teeth [32 (link)]. The periodontal examiner was not blind to the test or control status.