2019_ABDULWHHAB_MA_PhD.pdf (9.59 MB)
Use of Face-Mask Sampling as a Means of Characterising the Microbiota Exhaled from Human Respiratory Tract in Health and Disease
thesisposted on 2020-02-04, 14:05 authored by Mohamad T. Abdulwhhab
Introduction: Facemasks carrying a gelatine sampling matrix have been used to sample exhalations from TB patients. The aim here was to further develop this approach by evaluating molecular detection, quantitation and capture of signals in healthy volunteers and in HIV-uninfected patients with suspected Pneumocystis jirovecii pneumonia (PJP).
Methods: DNA isolation was optimised with NaOH or collagenase to dissolve gelatine followed by bead beating. Assays for selected marker and degradative proteins were established as were six 16SrDNA-directed qPCRs with different phylogenetic specificities. Mask samples were taken from healthy volunteers over 15, 30 and 60 minutes with four different respiratory efforts. Pathogen detection was tested in patients with suspected PJP (MtLSU target) and a subset of samples were subjected to microbiome profiling.
Results: In healthy volunteers, capture of exhaled bacteria appeared maximal at 15 minutes while accumulation continued up to 60 for proteins. Reading-out loud produced most SP-A, albumin and α-amylase. P. jirovecii was detected in 7/20 patients diagnosed and 3/19 patients suspected but not diagnosed with PJP. The median captured signal was 8.59 x 104 (IQR= 3.01 x 105 – 1.81 x 104) MtLSU copies/mask. Blood β-D-Glucan results correlated with the mask results (Spearman r=0.65; p<0.0001) while other relevant clinical indices did not. Microbiome results provided further evidence for different microbial outputs between individuals and with different respiratory efforts and samples. However, the sampling system provided a high background 16S signal.
Conclusion: The mask approach has been significantly improved towards sampling material exhaled from the upper and lower respiratory tracts with evidence that different breathing activities produce different yields. Sampling in suspected PJP adds diagnostic likelihood in cases with positive β-D-Glucan results and reinforces evidence that P. jirovecii is an airborne infection. Microbiome studies revealed several differences, but conclusions were limited by high background signals. Mask sampling is promising non-invasive investigative and diagnostic tool.
Date of award2019-12-16
Author affiliationDepartment of Respiratory Sciences
Awarding institutionUniversity of Leicester