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Additional problems with ANR microscopy were attributable to the spectrofluorometric properties of the dyes.

Established texts state Auramine O labels mycolic acids in the mycobacterial cell wall32 but direct evidence for this is lacking. Fluorescence is enhanced on binding to DNA and RNA and some acid-fast staining may be due to nucleic acid labelling33. The total emission spectrum of auramine O is wide (500-700nm) and overlaps with Nile red. Labelling of nucleic acids may create the impression of ‘beads’ within the organism which are difficult to discriminate from LBs emitting fluorescence at similar wavelengths.

Furthermore, Nile red is solvatochromatic and binds different lipid structures with shifting excitation and emission spectra according to the position, shape and intensity of the solvent. When hydrophilic membrane phospholipids are stained, entire cells emit a diffuse red fluorescence (λ>610nm) and intracellular LBs may be obscured. Alternatively, staining of hydrophobic intra-cytoplasmic TAG results in emission of yellow-gold fluorescence (λ≈528nm)563 with increased spectral overlap with Auramine O (Figure 5.7). Nile red is also prone to photo-bleaching (fade of fluorescence on exposure to light) which can cause loss of labelling during microscopy and imaging564.

a

For 50ml of dithiothreitol-lipase (1mg/ml), 3.75ml dithiothreitol concentrate (Oxoid) was added to 50mg lipase from Candida rugosa (Sigma) and 46.25ml mycobacterial free distilled water. 1ml of this preparation could be incubated with an equal volume of sputum for 1 hour.

141

Figure 5.6 Improvements to staining protocol for LB microscopy

A-C: M smegmatis stained with NR after lipase treatment at different concentrations. LBs are visible in all samples. D and E: A sputum sample from a smear positive TB patient. Without lipase

treatment, the background matrix on the WG filter is severe and no bacilli can be seen. After lipase (1mg/ml) treatment, the background is mild and LB positive bacilli are seen through it. F: Merged ALTR (1:200) image of two LB positive bacilli viewed with FITC and TRITC filters. G and H: Black and white images of an ALTR (1:200) LB positive bacillus viewed with FITC and TRITC. Different staining patterns indicate labelling of different cellular components by auramine (G) and LTR (H).

142 Excitation (dotted line) and emission (solid line) spectra for Nile red are at shorter wavelengths for hydrophobic (gold) than hydrophilic (red) environments. Auramine O spectra are shown below the x- axis.

The LipidTOXTM neutral stains (Invitrogen) are a new set of fluorescence probes with more specific binding to neutral lipids and greater photostability than Nile Red. LipidTOXTM Red neutral has good spectral characteristics (λexcitation>577m, λemission>609nm) for combination

with auramine O (Figure 5.8), so an AuramineO/LipidTOX Red neutral (ALTR) technique was optimised and compared with ANR.

1:50, 1:200 and 1:1000 dilutions of LTR were prepared in PBS. A sputum sample from patient 2 was processed with dithiothreitol-lipase (1mg/ml) and four batches of three heat- fixed 10µl smears were prepared. The first batch was ANR stained in the usual manner. Batches 2, 3 and 4 were stained with Auramine O and differentiated with 0.5% acid-alcohol, then labelled respectively with one of the three LTR dilutions for 20 minutes before

washing and counterstaining with KMNO4. Stained slides were blinded and taken from the

BSL-3 laboratory to the darkroom for reading within 24 hours. LB counts and the severity of background Nile red/LTR staining were recorded. Results are shown in Table 5.5.

Differences in LB counts between ANR staining and each of the ALTR preparations were analysed by two-sample tests and differences in background staining were assessed by Fisher’s exact test.

Figure 5.7 Difficulties associated with spectrochromatic properties of Nile red

Nile red + TAG

Nile red + Phospholipids

Auramine O excitation Auramine emission Wavelength (nm) Fl u o resc ence (arb itrary un its) 350 450 550 650 750

At long λ: Diffuse Nile red staining of membrane phospholipids obscures LBs

At shorter λ: Nile red stained LBs are difficult to discriminate from ‘beaded’ auramine O stained nucleic acids

143 Stain LB count (%), mean(SD) Change in mean LB count from ANRa, % (p-valuea) Background Change in background from ANR, p-valueb Mild (n, %) Moderate (n, %) Severe (n, %) ANR 62.4 (9.3) - 5 6 1 - ALTR (1:50) 92.4 (7.9) 30.0 (<0.001) 5 6 1 1 ALTR (1:200) 81.3 (10.8) 18.9 (<0.001) 6 3 3 0.32 ALTR (1:1000) 40.8 (16.5) -21.6 (<0.001) 9 3 0 0.21

Table 5.5 LB background sputum matrix staining with ANR or ALTR microscopy

a

Statistical analysis by two-sample t-test

b

Statistical analysis by Fisher’s exact test

Images from ALTR slides prepared using 1:50 and 1:200 dilutions of LTR were similar to ANR images (Figure 5.6F, G and H) but had higher LB counts (p<0.001 in both cases). This reflects less photo-bleaching with LTR. LB counts were lower on slides prepared using the 1:1000 dilution of LTR, suggesting that this preparation was too weak. No significant difference in staining of the background matrix was detected between ANR and ALTR.

On the basis of these experiments, the ALTR method using a 1:200 dilution of LTR replaced ANR microscopy for the clinical study.

After altering the fluorescence stains for LB microscopy, the choice of microscope filters was reviewed. Whilst the ‘long-pass’ filters employed until this point produced reasonable images, narrower ‘band-pass’ filters are more selective in the wavelengths of light they allow to pass, improving image detail, reducing the risk of bleed-through between dyes and minimising fluorescence of the background matrix33. A fluorescein isothiocyanate (FITC) and tetramethylrhodamine (TRITC) filter-set (Figure 5.8) was found to be optimal for ALTR staining and was used in the clinical study.

144 Excitation (dotted line) and emission (solid line) spectra are shown in green and red for auramine O and LTR respectively. A good fit is demonstrated between these dyes and the FITC-TRITC filter set, and the use of bandpass filters reduces the effect of spectral overlap previously seen with ANR and long-pass filters.