2.1.

Participants and Regulatory Approval

Spectroscopy data were collected from subjects enrolled in a phase 1 clinical trial examining the safety and feasibility of MB PDT performed at the time of percutaneous abscess drainage (ClinicalTrials.gov Identifier: NCT02240498). The primary results of this clinical trial are reported separately.⁸ Of the 18 subjects treated in this trial, optical spectroscopy data were collected from the final 13 subjects as described below. For the first five subjects treated, funding was not available to construct the spectroscopy system described in Sec. 2.2 and perform spectroscopy measurements. All study procedures were approved by the Research Subjects Review Board at the University of Rochester Medical Center, and written informed consent was provided by all subjects.

2.2.

Diffuse Reflectance Spectroscopy System

Spatially resolved diffuse reflectance data at the abscess wall were collected using an optical spectroscopy system that was described in detail previously,¹⁸ as shown in Fig. 1(a). Briefly, this system consists of an optical switch (FSM 1x2, Piezosystem Jena, Inc., Hopedale, Massachusetts, United States) that routes either broadband white light (HL-2000-HP-FHSA, Ocean Optics, Inc., Largo, Florida, United States) or laser light at 640 nm (OBIS FP 1193841, Coherent, Inc., Santa Clara, California, United States) to the source fiber on the optical probe displayed in Figs. 1(b) and 1(c). Light transmitted by each of the eight detector fibers is sequentially routed to a spectrometer (QE Pro, Ocean Optics) for detection via another optical switch (FSM 1x8, Piezosystem Jena, Inc.). Detected spectra were corrected for dark background and integration time, and were each divided by a corresponding calibration measurement made using an integrating sphere (3P-GPS-020-SF, Labsphere, Inc., North Sutton, NH) incorporated into the system housing. Details of the calibration procedure are reported elsewhere.¹⁸ While we have examined the fluorescence data collected by this system pre-clinically,¹⁹ the focus of the current study is the analysis of diffuse reflectance data.

Fig. 1

(a) Image of spectroscopy system showing fiber connections for source and detector fibers, port for integrating sphere calibration, LabVIEW interface, and the laser used for clinical PDT. (b) Image of fiber optic probe, with distal tip indicated by red arrow. (c) Image of fiber optic probe distal face showing the $200\mu \mathrm{m}$ diameter fibers used for delivery and detection of light. The source fiber is labeled “S,” detector fibers are labeled “D,” and outside diameter is abbreviated as “OD.”

The fiber optic probe used for delivery of incident light and detection of diffuse reflectance (Pioneer Optics Company, Bloomfield, Connecticut, United States) is shown in Fig. 1(b). This flexible probe has an outside diameter of 2 mm, to allow it to be inserted through the standard of care drainage catheter used for percutaneous abscess drainage, as described in Sec. 2.3. To determine the source–detector separations necessary for determination of optical properties from measured spectra, images were captured of the probe face using a stereomicroscope with a large working distance (SMZ1500, Nikon Instruments, Inc., Melville, New York, United States). These images were captured with and without a United States Air Force target (USAF 1951 1X, Edmund Optics, Inc., Barrington, New Jersey, United States) in the frame to translate pixel coordinates to distance. These source-detector separations ranged from 320 to $1310\mu \mathrm{m}$, as shown in Fig. 1(c).

As described by Bridger et al., these source-detector separations were used to generate a Monte Carlo lookup table for extraction of optical properties from measured spectra.¹⁸ This pre-clinical validation demonstrated root mean square errors of 0.26 to $0.52\mu \mathrm{M}$ for MB concentration recovery and 0.86 to $2.7{\mathrm{cm}}^{-1}$ for reduced scattering coefficient (${\mu }_s^{\prime }$) recovery. This prior publication used a lower resolution color camera to determine fiber positions, whereas the current results utilize the more precise positions determined by microscopic evaluation. Otherwise, the procedure was identical to the previously reported multispectral fitting method. Based on visual inspection of measured spectra, absorption spectra were fit as a superposition of oxy- and deoxy-hemoglobin, MB monomer, and MB dimer absorption spectra. MB dimer absorption was included as MB dimerizes at high concentrations and there is an affiliated change in the absorption spectrum.²⁸ Reduced scattering spectra were assumed to follow a power-law relationship of the form ${\mu }_s^{\prime }=a{(\lambda /{\lambda }_0)}^{-b}$, where $\lambda$ corresponds to wavelength in nm and ${\lambda }_0$ is a normalization wavelength set to 665 nm. Quality of fitting is reported as the sum of root squared error (SRSE) across detector fibers between measured and simulated reflectance spectra.

2.3.

Clinical Data Collection

All subjects first had their abscess drained using standard of care image-guided percutaneous drainage, as described in the main trial report.⁸ Aspirated fluid was processed by the Clinical Microbiology Laboratory, including identification of isolates and determination of antibiotic susceptibility. Growth of isolates was scored on a semi-quantitative scale ranging from 0 (no growth) to 4+ (heavy growth). The standard of care procedure utilized CT guidance for 16 subjects and ultrasound guidance for 2 subjects. Immediately following drainage, the cavity was first flushed twice with sterile saline. The disinfected optical probe was then removed from the bag and the proximal fiber ends connected to the spectroscopy system. The distal end was then held in place at the entrance port of the system integrating sphere by the interventional radiologist. Calibration spectra were captured at each detector fiber for both broadband and laser illumination, with an integration time of 100 ms for broadband illumination and 50 ms for laser illumination. These calibration spectra were used to correct patient data as described in Sec. 2.2.

In order to measure patient optical properties, the interventional radiologist advanced the optical probe through the standard of care drainage catheter until it made gentle contact with the abscess wall. Surface contact was insured through tactile feedback by the study doctor, and by monitoring the signal detected at the closest detector fiber in real time. As described by Chen et al.,²⁹ detected reflectance increases as the probe approaches the surface before rapidly decreasing when surface contact is made. This is due to the transition from specular to diffuse reflectance.

Once surface contact was made, diffuse reflectance and fluorescence spectra were captured at each detector fiber sequentially. For each fiber, an initial integration time of 50 ms was used. Assuming a linear relationship between integration time and detected signal, the maximum magnitude of the detected signal was used to calculate the integration time that would result in 50% of the dynamic range of the spectrometer being utilized. This was chosen to compromise between signal to noise ratio and measurement time. A measurement was then performed at this calculated integration time. Dark spectra were also collected at this point as described in Sec. 2.2 using the same integration times by disabling the source switch. If the captured spectra were not of sufficient quality, the probe was withdrawn and replaced by the study doctor, and spectroscopy was repeated. This measurement was meant to capture native abscess wall optical properties prior to MB infusion and is referred to as the “pre-MB” measurement elsewhere.

Following this, 0.1% MB (BPI Labc, LLC, Largo, Florida, United States) was infused into the abscess cavity and incubated for 10 min to allow for bacterial uptake. A 10 min incubation interval was selected based on prior reports of in vitro PDT efficacy against multiple bacterial isolates from human abscess aspirates using this incubation time.⁷ This short interval was also selected to improve specificity of MB uptake, based on the rapid internalization of cationic photosensitizers by bacteria relative to host cells.³⁰ MB was then aspirated and the cavity was flushed twice with sterile saline for all subjects. The optical probe was then re-inserted through the drainage catheter into gentle contact with the abscess wall, and measurements were repeated as described above. This measurement was meant to capture the uptake of MB and is referred to as the “post-MB” measurement throughout.

After the completion of optical spectroscopy measurements, the optical probe was removed and PDT was performed as described in the main clinical trial report.⁸ This consisted of an infusion of a lipid emulsion to scatter treatment light through the irregular abscess cavity, insertion of a sterile optical fiber, and delivery of laser light at 665 nm.

2.4.

Data Processing

As described above, collected spectra were corrected for dark background and integration time, and divided by corresponding spectra collected at the entrance port of the system integrating sphere. Optical property spectra were extracted using the Monte Carlo lookup table approach described in Sec. 2.2. Whereas pre-clinical validation of the system used all eight detector fibers, high-quality data could not be collected at all detector fibers for all subjects in clinical measurements. This was mainly due to poor surface contact between specific detector fibers and the abscess wall, even after repositioning of the optical probe. Additionally, for subjects with high MB uptake, spectra collected at larger source-detector separations did not have sufficient signal to noise ratio for analysis due to major attenuation by MB absorption. In cases where spectra at individual detectors were not usable, these detected fibers were excluded from the optical property inversion. As we have shown previously, utilization of fewer source-detector separations does not significantly impact the accuracy of optical property recovery, as long as data from at least 4-5 detector fibers are included.^31,32 This was achievable for all but the first subject, resulting in 13 pre-MB measurements and 12 matched post-MB measurements.

2.5.

Statistical Analysis

Continuous values are summarized across subjects as mean ± standard deviation, whereas categorical values are summarized as proportions with 95% confidence intervals. Demographic information was compared between the 13 subjects that received optical spectroscopy and the five treated prior to construction of the optical spectroscopy system using the Mann–Whitney test for continuous variables and Fisher’s exact test for categorical values. Extracted optical properties at 665 nm and ${\mathrm{SO}}_2$ were compared between pre- and post-MB measurements using the Wilcoxon signed-rank test. Correlation between MB concentration and change in ${\mathrm{SO}}_2$ was calculated using the Spearman correlation coefficient. Optical properties were compared between abscess locations using the Kruskal–Wallis test, with Dunn’s test for pairwise comparisons. The Mann–Whitney test was used to compare MB concentration between those with heavy bacterial growth reported for their standard of care abscess aspirate (4+) to those with less growth ($<4$). A $p$ value less than 0.05 was considered significant, and all analyses were performed in GraphPad PRISM (v6, GraphPad Software, Inc., San Diego, California, United States) and MATLAB (R2022b, The Mathworks, Inc., Natick, Massachusetts, United States).

3.1.

Subject Demographics

Subject demographics for all subjects that received PDT, as well as separated by whether optical spectroscopy measurements were made, are included in Table 1. As can be seen, there were no significant differences in demographics between subjects that did and did not receive optical spectroscopy. For the 13 subjects described in this report, 38.5% were male and the average age was $56.9\pm 20.3$ years.

Table 1

Subject demographics for overall sample and separated by whether spectroscopy was performed.

3.2.

Representative Data from Two Subjects

Representative diffuse reflectance spectra for two subjects are shown in Fig. 2, with fully corrected data shown as open circles and best fit spectra shown as solid lines. As can be seen in Figs. 2(a) and 2(c), pre-MB data were accurately fit with a super-position of oxy- and deoxy-hemoglobin absorption and power-law reduced scattering. Post-MB data, as shown in Figs. 2(b) and 2(d), were well fit by the addition of MB monomer and dimer spectra.

Fig. 2

Corrected diffuse reflectance data (open circles) and fits (solid line) for detector fibers 1, 2, 4, and 5 for subject 3 (a) pre-MB $(\mathrm{SRSE}=1.18\times {10}^{15})$ and (b) post-MB $(\mathrm{SRSE}=1.13\times {10}^{15})$, and subject 8 (c) pre-MB ($\mathrm{SRSE}=1.32\times {10}^{15})$ and (d) post-MB ($\mathrm{SRSE}=1.36\times {10}^{15}$). Colors and symbols indicate different detector fibers.

These fits were used to extract the optical property spectra shown in Fig. 3. As can be seen, there was variability between subjects in both pre-MB optical properties and MB uptake. Figures 3(a) and 3(c) demonstrate large differences in absorption spectra between subjects for both pre-MB and post-MB conditions. In both subjects, oxy- and deoxy-hemoglobin were the main absorbers.

Fig. 3

Recovered (a) absorption (${\mu }_a$) and (b) reduced scattering (${\mu }_s^{\prime }$) spectra for subject 3 shown in Figs. 2(a) and 2(b). Recovered (c) ${\mu }_a$ and (d) ${\mu }_s^{\prime }$ spectra for subject 8 shown in Figs. 2(c) and 2(d). Blue and red lines represent pre-MB and post-MB conditions, respectively.

In the post-MB condition for subject 3, oxy- and deoxy-hemoglobin remained the dominant absorbers. For subject 8, MB monomer and dimer dominated the post-MB absorption spectra, with some contributions from oxy- and deoxy- hemoglobin. Figures 3(b) and 3(d) illustrate notable differences in reduced scattering spectra between subjects for pre-MB and post-MB conditions.

3.3.

Native Abscess Wall Optical Properties

Native, pre-MB abscess wall optical properties at 665 nm across subjects are tabulated in Table 2. Substantial inter-patient variability in native abscess-wall optical properties and oxygen saturation were observed (Fig. 4). Means and standard deviations for absorption coefficient at 665 nm (${\mu }_{a,665}$) were $0.15\pm 0.1{\mathrm{cm}}^{-1}$ (range: 0.03 to $0.36{\mathrm{cm}}^{-1}$), and $8.45\pm 2.37{\mathrm{cm}-}^1$ (range: 4.8 to $13.2{\mathrm{cm}}^{-1}$) for reduced scattering coefficient at 665 nm (${\mu }_{s,665}^{\prime }$). Oxygen saturation (${\mathrm{SO}}_2$) was found to be $58.83\%\pm 35.78\%$ (range: 5.6% to 100%).

Table 2

Extracted optical properties at 665 nm and oxygen saturation for each subject, before and after addition of methylene blue (MB). MB concentration ([MB]) is also included for post-MB measurements.

Fig. 4

Extracted (a) absorption coefficients (${\mu }_a$) at 665 nm, (b) reduced scattering coefficients (${\mu }_s^{\prime }$) at 665 nm, and (c) oxygen saturation (${\mathrm{SO}}_2$) for all subjects for pre-MB and post-MB conditions. Note that (a) is on a log scale, and (b) and (c) are on linear scales.

3.4.

Methylene Blue Uptake and Changes in Extracted Optical Properties

Measured MB concentrations across subjects are tabulated in Table 2. Mean and standard deviation for this was $71.83\pm 108.22\mu \mathrm{M}$ (range: $0-311\mu \mathrm{M}$), which reveals substantial inter-patient variation. As expected, there was a significant increase in ${\mu }_a$ at 665 nm for post-MB measurements ($p=0.001$), due to the presence of MB absorption [Fig. 4(a)]. There was also an apparent decrease in ${\mu }_s^{\prime }$ at 665 nm [$p=0.005$, Fig. 4(b)]. While there was a decrease in ${\mathrm{SO}}_2$ from pre- to post-MB values [median reduction = 28.8%, Fig. 4(c)], this difference was not significant ($p=0.11$). Additionally, this change in ${\mathrm{SO}}_2$ from pre- to post-MB measurement was not significantly correlated with measured MB concentration ($\rho =0.49$, $p=0.11$). There was also no significant difference in MB concentration between those subjects graded as having heavy growth on their standard of care abscess aspirate (4+ as reported by the Clinical Microbiology Laboratory) and those with less than heavy growth (4+: $32.2\pm 30.7\mu \mathrm{M}$, $<4$: $68.1\pm 120.8\mu \mathrm{M}$; $p=0.81$).

As the fiber optic probe was removed and reinserted between the pre- and post-MB data collection, some of this variability may also represent small differences in the exact placement of the probe face. Due to clinical time constraints, we were unable to perform repeated measurements to assess variability due to probe placement. This will be an important piece of future studies.

3.5.

Differences based on Abscess Location or Bacteria Present

For the 13 abscesses that were quantified optically, four were located in the pelvis, five were associated with the colon, and four were found in other locations. While sample sizes were small, we performed preliminary analysis to examine regional differences in optical properties. None of the quantities reported in Table 2 were found to be significantly different between these rough location groupings.

Of the bacterial species found in abscesses measured, the most common was Escherichia coli (E. coli, $n=3$). Other abscess cavities were found to contain a variety of other species. While abscess containing E. coli tended to have higher MB uptake than those with other bacterial species ($97.7\pm 114.7\mu \mathrm{M}$ versus $14.8\pm 22.7\mu \mathrm{M}$), this difference was not statistically significant ($\mathrm{p}=0.17$). Four of the 13 abscesses measured were found to contain antibiotic-resistant bacterial strains. MB uptake was not different between strains that were resistant or susceptible to antibiotics ($p=0.63$), indicating that PDT susceptibility in antibiotic-resistant strains would not be limited by MB concentration.