18f-fluorodeoxysorbitol positron emission tomography for noninvasive detection of invasive mold infections in patients

Abstract

Methods for detecting a mold infection in a subject, comprising administering 18F-fluorodeoxysorbitol (18F-FDS) to the subject and taking an image, e.g., a positron emission tomography (PET) and computed tomography (CT) image, are disclosed. The mold infection can include, for example, an Aspergillus, a non-Aspergillus (galactomannan-negative), or an azole-resistant (Aspergillus calidoustus) mold infection, or other clinically relevant molds.

Description

18F-FLUORODEOXYSORBITOL POSITRON EMISSION TOMOGRAPHY FORNONINVASIVE DETECTION OF INVASIVE MOLD INFECTIONS IN PATIENTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No. 63 / 699,572, filed September 26, 2024 and U.S. Provisional Application No. 63 / 763,992 filed February 27, 2025, which are incorporated herein by reference in their entireties.FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant HL131829, AI153349, and AI145435 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Invasive mold infections are an increasing global public health concern, affecting multiple organ systems (e.g., lung, brain), and associated with mortality rates as high as 85%1-3. The rising incidence of invasive mold infections is attributed to the expanding population of immuno suppressed patients3. The COVID- 19 pandemic has also led to a dramatic increase in invasive mold infections, particularly pulmonary aspergillosis and mucormycosis4,5. Aspergillus spp. remain the major cause of invasive mold infections in hematologic cancer patients and transplant recipients. Other opportunistic molds, however, which are even more challenging to diagnose and treat6, are on the rise due to several reasons, including wide-spread Aspergillus- targeted antifungal prophylaxis7,8. In 2022, the World Health Organization (WHO) categorized Aspergillus and Mucorales molds as Critical and High Priority Group pathogens, respectively9. Prompt identification of invasive mold infections and initiation of appropriate treatment is critical for improving patient outcomes. Establishing a definitive diagnosis is challenging, however, as it often requires invasive procedures (e.g., bronchoalveolar lavage, biopsy) to obtain a relevant clinical sample, and the slow turnaround times with fungal cultures, result in further delays. Additionally, biomarkers such as galactomannan have limited sensitivity and specificity for diagnosis of invasive aspergillosis and are unable to detect most other invasive mold infections. Importantly, all clinically available imaging tools such as radiography, ultrasonography, computed143844.601_P18516-01tomography (CT), and magnetic resonance imaging (MRI) rely on structural changes in anatomy or tissue morphology that arc often delayed relative to the disease process, arc non-specific, and reflect a combination of the infection and the host inflammatory response10 12. There have been several efforts to develop pathogen- specific imaging approaches for Aspergillus12'19, although none have been prospectively studied in patients (other than a case report20), or applied to other mold infections. Therefore, there is an urgent need for sensitive, whole-body, noninvasive tools to diagnose invasive mold infections.SUMMARY

[0004] The presently disclosed subject matter provides a method for detecting a mold infection in a subject, the method comprising administering18F-fluorodeoxy sorbitol (18F-FDS) to the subject and taking an image. In some embodiments, the present invention provides18F-FDS for use in imaging a mold infection in a subject in thereof.

[0005] In some aspects, the present disclosure provides methods for detecting a clinical mold infection in a subject, the method comprising administering18F-fluorodeoxysorbitol (18F-FDS) to the subject and imaging the18F-fluorodeoxysorbitol (18F-FDS) in the subject to detect uptake of the18F-fluorodeoxysorbitol (18F-FDS) by mold thereby indicating the presence or absence a clinical mold infection.

[0006] In some embodiments, the subject is suspected of having a mold infection or being at risk of a mold infection. In some embodiments, the absence of uptake of18F-FDS in the image is indicative of the absence of a mold infection by more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more species of mold. In some embodiments, the methods can be used as a preliminary screen to identify infection by more than one species of mold. In some embodiments, the methods further comprise the step of invasively sampling the mold infection after first detecting the presence of uptake of18F-FDS by mold in the subject so that the species of the mold can be determined.

[0007] In some aspects, the present disclosure provides methods for monitoring a response of a subject to a treatment for a mold infection comprising: administering a treatment for a mold infection to the subject; administering18F-fluorodeoxy sorbitol (18F-FDS) to the subject; and imaging the18F-Iluorodeoxysorbitol (18F-FDS) in the subject to detect uptake of the18F- fluorodeoxysorbitol (18F-FDS) by mold thereby providing an indication of the response of the subject to the treatment. In some embodiments, the steps of administering18F-fluorodeoxysorbitol (18F-FDS) to the subject and imaging the18F-fluorodeoxysorbitol (18F-FDS) to detect uptake of243844.601_P18516-01the18F-fluorodeoxysorbitol (18F-FDS) by mold in the subject are repeated two or more times to provide two or more images. In some embodiments, the two or more images arc compared to monitor response to the treatment. In some embodiments, a decrease in18F-fluorodeoxysorbitol (18F-FDS) uptake as evidenced by a comparison of the two or more images is evidence of a positive response to the treatment. In some embodiments, the treatment is an antibiotic treatment or an antifungal treatment.

[0008] In some embodiments of any of the methods described above, the imaging comprises positron emission tomography-computed tomography (“PET-CT”) imaging.

[0009] In some embodiments, the subject is a human.

[0010] In some embodiments, the mold infection is selected from an Aspergillus mold infection, a non-Aspergillus (galactomannan-negative) mold infection, and an azole-resistant (Aspergillus calidouslus) mold infection.

[0011] In some embodiments, the methods further comprise testing the subject for galactomannan status before, during or after administration of18F-FDS to the subject. In some embodiments, the subject is galactomannan negative and the uptake of18F-FDS is indicative of a non-Aspergillus (galactomannan-negative) mold infection.

[0012] In some embodiments, the non-Aspergillus (galactomannan-negative) mold infection is selected from the group consisting of a Rhizopus spp. mold infection, a Mucor spp. mold infection, a Lichtheimia corymbifera mold infection, a Syncephalastrum racemosum mold infection, a Cunninghamella bertholletiae mold infection, and a Cladophialophora bantiana mold infection and a Fusarium solani mold infection, and combinations thereof. In some embodiments, the non- Aspergillus (galactomannan-negative) mold infection is selected from the group consisting of a Rhizopus spp. mold infection, a Mucor spp. mold infection, and a Fusarium solani mold infection, and combinations thereof. In some embodiments, the non-Aspergillus (galactomannan-negative) mold infection is a Mucor spp. mold infection.

[0013] In some embodiments of any of the foregoing methods, the mold infection is selected from an Aspergillus spp. mold infection, a Rhizopus spp. mold infection, a Mucor spp. mold infection, a Lichtheimia corymbifera mold infection, a Syncephalastrum racemosum mold infection, a Cunninghamella bertholletiae mold infection, a Cladophialophora bantiana mold infection, and a Fusarium solani mold infection, and combinations thereof. In some embodiments, the subject has two or more of an Aspergillus spp. mold infection, a Rhizopus spp. mold infection, a Mucor343844.601_P18516-01spp. mold infection, a Lichtheimia corymbifera mold infection, a Syncephalastrum racemosum mold infection, a Cunninghamella bertholletiae mold infection, a Cladophialophora bantiana mold infection and a Fusarium solani mold infection, and the method allows simultaneous detection of mold infections by two or more different mold species. In some embodiments, at least one of mold infections is an Aspergillus spp. mold infection and at least one of the mold infections is a Mucor spp. mold infection.

[0014] In some embodiments of any of the methods described above, the imaging allows localization of the mold infection to an ar ea of the body and / or determination of the extent of the mold infection in an area of the body.

[0015] In some embodiments, the area of the body is selected from the group consisting of the brain, lungs and the nasopharyngeal pathway.

[0016] In some embodiments, the mold infection is selected from an invasive pulmonary mold infection, a cerebral mold infection, and a nasopharyngeal mold infection.

[0017] In some embodiments, the mold infection comprises a rhinosinusal infection due to infection by an Aspergillus ssp., Rhizopus ssp., and / or Mucor ssp.

[0018] In some embodiments, the subject is immunocompromised.

[0019] In some embodiments, the subject has cancer.

[0020] In some embodiments, the imaging comprises taking a whole-body image.

[0021] In some embodiments, the imaging distinguishes a mold infection from a sterile inflammation and / or cancer.

[0022] In some embodiments, the subject has a gram-negative bacterial infection, has been previously diagnosed with a gram-negative bacterial infection, is suspected of having a gramnegative bacterial infection, or being at risk of a gram-negative bacterial infection.

[0023] In some embodiments, the imaging distinguishes a mold infection from an infection with a gram-negative bacteria.

[0024] In some embodiments, the methods further comprises the step of diagnosing or identifying a mold infection when the subject also has an infection by a gram-negative bacteria, has been previously diagnosed with an infection by a gram-negative bacteria, or is suspected of being at risk by infection with a gram-negative bacteria. In some embodiments, the gram negative bacteria is a Pseudomonas spp. In some embodiments, the Pseudomonas spp. is P. aeruginosa.443844.601_P18516-01

[0025] In some embodiments, the mold infection of any of the foregoing methods is not an infection by Aspergillus fumigatus .

[0026] In some embodiments, the methods comprise the step of distinguishing a mold infection from a bacterial infection in an infective tissue lesion having a volume in the subject by determining the volume of the infective tissue lesion occupied by an infective biomass by image analysis of the results of the imaging step, wherein a high volume of occupation of the lesion by the biomass is indicative of a mold infection and a low volume of occupation of the lesion by the biomass is indicative a bacterial infection. In some embodiments, a high volume of occupation of the lesion is defined by a biomass occupation of the lesion of greater than 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, or 10.0%. In some embodiments, the image analysis comprises automated segmentation of the image. In some embodiments, a low volume of occupation of the lesion by the biomass is less than 0.95%, 0.9%, 0.8%, 0.5%, or 0.3%.

[0027] Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Drawings as best described herein below.BRIEF DESCRIPTION OF THE FIGURES

[0028] Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

[0029] FIG. 1A, FIG. IB, and FIG. 1C show the clinical study design. FIG. 1A, Consort diagram for study. Patients with confirmed invasive mold-related fungal infections and controls with confirmed inflammatory or oncologic disease (without infection) were prospectively enrolled. FIG. IB, Location of invasive mold infections and other pathologies. FIG. 1C, 18F-FDS PET showing cranial (patient 1 , left) and pulmonary (patient 4, right) mold infections with the lesions marked by red arrows.

[0030] FIG. 2A and FIG. 2B show quantification of18F-FDS PET signal in all patients with mold infections. FIG. 2A, To quantify the PET signal, spherical volumes of interest (VOIs) were drawn at the sites of pathology (red circle) and unaffected sites (yellow circle) of the same tissue to calculate the target-to-nontarget tissue ratio (TNT). FIG. 2B, Median TNT for all nine enrolled543844.601_P18516-01patients are shown. Data were derived from nine patients, four with invasive mold infections (patient 4 had pulmonary and cerebral lesions) and five with inflammatory or oncologic diagnoses. Data are shown as median and interquartile range (IQR). Statistical analysis was performed using a two-tailed Mann-Whitney U test. L, liver; H, heart.

[0031] FIG. 3A, FIG. 3B, and FIG. 3C show thoracic18F-FDS PET / CT imaging in patients. FIG. 3A, Sagittal CT (left) with the corresponding transverse CT (middle) and18F-FDS PET / CT (right) of a representative patient with confirmed invasive pulmonary mold infection (patient 3). FIG. 3B, Representative control patient with interstitial lung disease (control 1). Pulmonary lesions are marked with arrows. FIG. 3C, TNT measurements derived from three patients with invasive pulmonary mold infections, and four control patients with n = 3 VOIs per patient. Data are shown as median ± IQR. Statistical analyses were performed using a two-tailed Mann-Whitney U test. A, aorta; H, heart; K, kidney; L, liver.

[0032] FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D show cranial18F-FDS PET imaging in patients. FIG. 4A, Representative transverse (upper) and coronal (lower) CT (left) and18F-FDS PET / CT (right) images from a patient with confirmed invasive cerebral mold infection (patient 1). FIG. 4B, Representative control patients without cerebral disease (control 2). FIG. 4C, Representative transverse MRI and18F-FDS PET / MRI images from patient 1. The research PET was co-registered with the MRI performed for clinical reasons. FIG. 4D, TNT measurements derived from two patients with invasive cerebral mold infections, and the five control patients without cerebral pathology with n = 2 VOIs per patient (patient 4 with mold infection has 1 VOI only). Data are shown as median ± IQR. Statistical analyses were performed using a two-tailed Mann- Whitney U test.

[0033] FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, FIG. 5G, and FIG. 5H show18F-FDS uptake by clinically relevant molds. FIG. 5A, Methodology for18F-FDS in vitro uptake. Briefly, molds were co-incubated with18F-FDS for two hours, centrifuged to form a pellet and then washed.18F-FDS uptake in the pellet was measured as becquerel (Bq) and corrected for protein (mg). Heat killed (HK) molds were generated by exposing them to 70°C for 60 min. FIG. 5B- FIG. 5D,18F-FDS uptake by live and HK Aspergillus fumigatus (ATCC 1022), Rhizopus oryzae, and Mucor circinelloides. The x axis (time) in panel b is in min. FIG. 5e- FIG. 5G, Competitive inhibition of18F-FDS uptake with increasing concentrations of cold (unlabeled) sorbitol in Aspergillus, Rhizopus, and Mucor. FIG. 5H,18F-FDS uptake (at 120 min.) by the643844.601_P18516-01reference A. fumigatus (ATCC 1022) and 30 random clinical isolates representing a wide range of clinically relevant molds including Lichtheimia corymbiform, Syncephalastrum racemosum, Cunninghamella bertholletiae, Cladophialophora bandana (clinical isolate from patient 4), and Fusarium solani. All assays were performed in triplicate. Data are shown as median ± IQR.

[0034] FIG. 6A, FIG. 6B, FIG. 6C, and FIG. 6D show tissue histology from human and animal studies. FIG. 6A, Schematic showing the tissue volume occupied by mold and bacterial infections. FIG. 6B, Methodology for quantifying the area and volume affected by mold infections in tissues. Automated segmentation of fungi is shown in red (middle panel). FIG. 6C, Representative Grocott methenamine silver (GMS) stained histopathology slides from nine tissue samples from eight unique patients with pulmonary, cerebral, or rhinosinusal invasive Aspergillus infections. FIG. 6D, Mold and bacterial biomass expressed as % of the infected lesion volume for infections in human and mouse tissues. Human data are derived from nine tissue samples from eight unique patients with pulmonary, cerebral or rhinosinusal invasive Aspergillus infections from current studies (multiple slides per patient), tissue slides from four patients with bacterial infections (digital library supported by the National Library of Medicine), five slides each from Aspergillus, Rhizopus and Mucor infected mouse tissues from current studies, and four slides from E. coli infected mouse tissues. Data are shown as median ± IQR. Statistical analyses were performed using a two-tailed Mann- Whitney U test.

[0035] FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E, FIG. 7F, FIG. 7G, and FIG. 7H show a mouse model of invasive cerebral aspergillosis. Representative MIP (left) and coronal (right)18F-FDS PET / CT from FIG. 7A, uninfected, FIG. 7B, LPS-induced systemic inflammation, and FIG. 7C, Aspergillus x c mice are shown. Lesions are marked with arrows. FIG. 7d,18F-FDS PET SUVr (normalized to tissues from uninfected animals) derived from eight uninfected mice, seven mice with LPS-induced systemic inflammation, and seven mice with Aspergillus infection are shown. FIG. 7E, Ex vivo quantification of18F-FDS uptake (percent injected dose [%ID] / g) in postmortem brain tissues derived from three uninfected mice, four mice with LPS-induced systemic inflammation, and three mice with Aspergillus infection are shown. FIG. 7F, Representative GMS-stained brain tissues from Aspergillus-infected mice show Aspergillus hyphae. Scale bars is 100 pm. FIG. 7G, Representative coronal (upper left), transverse (middle left) and sagittal (lower left) MIP and coronal (upper right) and transverse (lower right) CT segmentation. FIG. 7H, Representative coronal (upper) and transverse (lower) MRI T1 (left), T2743844.601_P18516-01(middle), and18F-FDS PET / MRI (right, T1 MRI) from Aspergillus-infected mice. Cerebral lesions arc marked with arrows. Aspergillus fumigatus (ATCC 1022) was used for the studies. Data arc shown as median ± IQR. Statistical analyses were performed using a two-tailed Mann- Whitney U test. LPS, lipopolysaccharide. SUVr, standardized uptake value ratio.

[0036] FIG. 8 shows PET imaging in the patient with breast cancer. Sagittal CT (left) with the corresponding18F-FDG PET / CT (middle) and18F-FDS PET / CT (right) images of a patient with breast cancer (control 5). The lesion is marked with arrows. L, liver; SUV, standardized uptake value; TNT, target-to-non-target ratio.

[0037] FIG. 9 shows thoracic18F-FDS PET / CT in patients with invasive pulmonary mold infections. Sagittal CT (left) and the corresponding transverse CT (middle) and18F-FDS PET / CT (right) are shown with the arrows marking the infected lesions. A, aorta; H, heart; TNT, target-to- non-target ratio.

[0038] FIG. 10 shows thoracic18F-FDS PET / CT in patients with inflammatory pathologies. Sagittal CT (left) and the corresponding transverse CT (middle) and18F-FDS PET / CT (right) are shown with the arrows marking the lesions. A, aorta; H, heart; TNT, target-to-non-target ratio.

[0039] FIG. 11A and FIG. 11B show cranial imaging in patient 4 with invasive cerebral mold infection. The PET active lesion is in the medial aspect of the right frontal cortex. FIG. 11 A, Representative transverse (upper) and coronal (lower) CT (left) and18F-FDS PET / CT (right) images. FIG. 11B, Representative transverse MRI (left) and18F-FDS PET / MRI (right) images. The research PET was co-registered with the MRI performed for clinical reasons. S, sagittal sinus; TNT, target-to-non-target ratio.

[0040] FIG. 12 shows cranial18F-FDS PET / CT in control patients. Transverse CT (left) and18F- FDS PET / CT (right) are shown. S, sagittal sinus; TNT, target-to-non-target ratio.

[0041] FIG. 13 shows18F-FDS uptake in mammalian cells, molds and E. coli.18F-FDS uptake in live and lysed mammalian cells (THP-I macrophages), reference (ATCC) Aspergillus fumigatus, Rhizopus spp., Mucor spp., and E. coli (Enterobacterales bacteria) after two hours of incubation. Data are shown as median ± IQR.

[0042] FIG. 14A, FIG. 14B, and FIG. 14C show18F-FDS metabolism in Aspergillus. FIG. 14A, Schematic representation of incorporation of18F-FDS in Aspergillus via the phosphotransferase system and SdhA (sorbitol dehydrogenase). After phosphorylation,18F-FDS-6-phosphate, gets trapped intracellularly until F-18 decays to oxygen for further metabolism by SdhA. FIG. 14B,843844.601_P18516-01Methodology for evaluating18F-FDS metabolism in Aspergillus fumigatus (ATCC 1022) or E. coli (ATCC 25922). FIG. 14C, Radio-thin layer chromatography (radio-TLC) studies of the cytoplasmic fraction from the lysed pellet demonstrates intracellular conversion of18F-FDS into the major metabolite. The x axis indicates the travel distance in millimeters. At least three radio- TLC runs were performed for each compound.

[0043] FIG. 15 shows histopathology from infected tissues. Representative slides are derived from nine tissue samples from eight unique patients with pulmonary, cerebral or rhinosinusal invasive mold infections and mouse tissues from current studies. Grocott methenamine silver (GMS) and Gram stains are used for fungi and bacteria, respectively. Bacterial infections (humans) are from a digital library (Infectious Diseases Images eMicrobes). Bacteria or mold are marked with arrows. Scale bars = 50 pm.

[0044] FIG. 16 shows tissue histology from human and animal studies. Quantification of the area occupied by mold and bacterial infections in human and mouse tissues expressed as percentage of total area. Human data are derived from nine tissue samples from eight unique patients with pulmonary, cerebral or rhinosinusal invasive Aspergillus infections from the current study (multiple slides per patient), tissue slides from four patients with bacterial infections (digital library supported by the National Library of Medicine), five slides each from Aspergillus, Rhizopus and Mucor infected mouse tissues from the current study, and four slides from E. coli infected mouse tissues. Data are shown as median ± IQR. Statistical analyses were performed using a two-tailed Mann-Whitney U test.

[0045] FIG. 17 shows a18F-FDS PET dose response curve in the mouse model of pulmonary aspergillosis. Pulmonary18F-FDS PET SUVr (normalized to tissues from uninfected animals) derived from three mice (without immunosuppression) infected with low dose, five mice infected with low dose, four mice infected with medium dose, and four mice infected with high dose Aspergillus are shown. The infection burden is the number of conidia instilled per mouse. Aspergillus fumigatus (ATCC 1022) was used for the studies. Data are shown as median ± IQR. Statistical analyses were performed using a two-tailed Mann- Whitney U test. SUVr, standardized uptake value ratio.

[0046] FIG. 18A, FIG. 18B, FIG. 18C, FIG. 18D, FIG. 18E, FIG. 18F, FIG. 18G, FIG. 18H, and FIG. 181 show a mouse model of invasive pulmonary aspergillosis. Representative MIP (left), transverse (upper) and coronal (lower) CT (middle) and18F-FDS PET / CT (right) from FIG. 18 A,943844.601_P18516-01uninfected, FIG. 18B, LPS-induced pulmonary sterile inflammation, and FIG. 18C, Aspergillus- infcctcd mice arc shown. Lesions arc marked with arrows. Representative transverse (upper) and coronal (lower) CT (left) and18F-FDG PET / CT (right) from FIG. 18d, uninfected, FIG. 18e, LPS- induced pulmonary sterile inflammation, and FIG. 18f, Aspergillus-infected mice are shown. Lesions are marked with arrows. FIG. 18G,18F-FDS PET SUVr (normalized to tissues from uninfected animals) derived from five uninfected mice, seven mice with LPS-induced pulmonary sterile inflammation, and five mice with Aspergillus infection are shown. FIG. 18H,18F-FDG PET SUVr derived from three uninfected mice, ten mice with LPS-induced pulmonary sterile inflammation, and twelve mice with Aspergillus pulmonary infection are shown. FIG. 181, Representative GMS-stained lung tissues from Aspergillus nfecte mice show Aspergillus hyphae. Scale bars from left to right: 2 mm, 500 pm, and 50 pm. Aspergillus fumigatus (ATCC 1022) was used for the studies. Data are shown as median ± IQR. Statistical analyses were performed using a two-tailed Mann-Whitney U test. H, heart; LPS, lipopolysaccharide; MIP, maximum intensity projection; SUVr, standardized uptake value ratio.

[0047] FIG. 19A, FIG. 19B, FIG. 19C, FIG. 19D, FIG. 19E, and FIG. 19F show18F-FDS PET / CT in mice with rhinosinusal mold infections. Representative MIP (left), coronal (upper) and sagittal (lower) CT (middle) and18F-FDS PET / CT (right) from FIG. 19A, Aspergillus, FIG. 19B, Rhizopus, and FIG. 19C, Mucor-infected mice are shown. Lesions are marked with arrows. FIG. 19D,18F-FDS PET SUVr (normalized to tissues from uninfected animals) derived from eight uninfected mice, eight mice with LPS-induced systemic inflammation, eight mice with Aspergillus infection, six mice with Rhizopus infection, and eight mice with Mucor infection are shown. FIG. 19E, Ex vivo quantification of18F-FDS uptake (percent injected dose [%ID] / g) in postmortem nasopharyngeal tissue derived from four mice per group are shown. FIG. 19F, Representative GMS-stained nasopharyngeal tissues from Aspergillus, Rhizopus and Mucor-infected mice (left to right) show fungal hyphae. Scale bar is 50 pm. Aspergillus fumigatus (ATCC 1022), Rhizopus oryzae and Mucor circinelloid.es were used for the studies. Data are shown as median ± IQR (on a log scale for panel d). Statistical analyses were performed using a two-tailed (paned d) or one- tailed (panel c) Mann-Whitney U test. LPS, lipopolysaccharide; MIP, maximum intensity projection; SUVr, standardized uptake value ratio.

[0048] FIG. 20A, FIG. 20B, FIG. 20C, FIG. 20D and FIG. 20E provide representative MIP (left), transverse (upper) and coronal (lower) CT (middle) and18F-FDS PET / CT (right) from pulmonary1043844.601_P18516-01infection by Aspergillus-mfected. mice (Fig. 20A) and Pseudomonas-infected. mice (FIG. 20B). Lesions arc marked with arrows. FIG. 20C shows18F-FDS uptake in live and lysed mammalian cells (THP-1 macrophages), reference (ATCC) Aspergillus fumigatus, Rhizopus spp.. Mucor spp., E. coli (Enterobacterales bacteria, ATCC) and Pseudomonas aeruginosa (ATCC) after two hours of incubation. Fig. 20d shows18F-FDS PET SUVr (normalized to tissues from uninfected animals) derived from five uninfected mice, seven mice with LPS-induced pulmonary sterile inflammation, five mice with Aspergillus infection and 8 mice with Pseudomonas pulmonary infection are shown. FIG. 20E shows Pseudomonas pulmonary bacterial burden [logio colony-forming units (CFU)]. Data are shown as median ± IQR for panel c-d and as mean ± SD for panel e. Statistical analyses were performed using a two-tailed Mann-Whitney U test. H, heart; LPS, lipopolysaccharide; SUVr, standardized uptake value ratio.

[0049] FIG. 21A, FIG. 21B and FIG. 21C show representative MIP, coronal (upper) and sagittal (lower)18F-FDS PET / CT from Aspergillus-infected mice with rhinosinusal infection (FIG. 21 A), without treatment and after 1 week of voriconazole treatment (FIG. 21B). Lesions are marked with arrows. FIG. 21C shows18F-FDS PET SUVr (normalized to tissues from uninfected animals) derived from untreated and treated mice with rhinosinusal Aspergillus infection. Data are shown as median ± IQR. Statistical analyses were performed using a two-tailed Mann-Whitney U test. SUVr, standardized uptake value ratio.

[0050] FIG. 22 shows whole-body18F-FDS PET in patients with invasive mold infections. Coronal18F-FDS PET maximum intensity projections and the corresponding CT images from all patients with invasive mold infections are provided. Note the small pulmonary cavitary lesions in patients 3 and 4. Patient 4 with invasive pulmonary infection due to Cladophialophora bantiana was noted to have a cerebral lesion diagnosed incidentally on the 18F- FDS PET. Arrows indicate the infected lesions, that were independently reported by the clinical radiologist on the CT performed for clinical reasons in the patients. Brain lesions are not visible on the low-radiation dose research CT for patients 1 and 4.

[0051] FIG. 23A, FIG. 23B, FIG. 23C, FIG. 23D, and FIG. 23E provide data related to18F-FDS PET / CT in a mouse model of Pseudomonas pneumonia. Representative MIP (left), transverse (upper) and coronal (lower) CT (middle) and18F-FDS PET / CT (right) from pulmonary infection by 23 A, Aspergillus-infected mice (same as panel FIG. 18C) and 23B, Pseudomonas-infected mice are shown. Lesions are marked with arrows. 23C, 18F- FDS uptake in live and lysed mammalian1143844.601_P18516-01cells (THP-1 macrophages), reference (ATCC) Aspergillus fumigatus, Rhizopus spp., Mucor spp., E. coli (Entcrobactcralcs bacteria, ATCC) and Pseudomonas aeruginosa (ATCC) after two hours of incubation (some of these data also shown in FIG. 22). 23d,18F-FDS PET SUVr (normalized to tissues from uninfected animals) derived from uninfected mice (n = 5), mice with LPS-induced pulmonary sterile inflammation (n = 7), mice with Aspergillus infection (n = 5) and mice with Pseudomonas pulmonary infection (n = 7) are shown (some of these data also shown in panel Sl lg). 23E, Pseudomonas pulmonary bacterial burden [loglO colony-forming units (CFU)]. Data are shown as median ± IQR for panel c-d (on a log scale for panel d) and as mean ± SD for panel E. Statistical analyses were performed using a two-tailed Mann- Whitney U test. H, heart; LPS, lipopolysaccharide; SUVr, standardized uptake value ratio.DETAILED DESCRIPTION

[0052] The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the inventions are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0053] Mold infections are growing public health concern, especially in immunocompromised patients. Such patients are often subject to infection by multiple pathogens, included gramnegative bacteria such as Pseudomonas spp. In fact, co-inf ection by various mold organisms and Pseudomonas spp. is a common presentation in immunocompromised patients.18F-FDS has been previously identified as being useful for imaging of bacterial infection caused by E. coli (See 20150250906) and has been shown to distinguish infection by the gram-positive bacteria Staphylococcus aureus. However, as just discussed, many patients are presented that have infections caused by both mold and gram-negative bacteria. The methods of the present invention solve the problem of distinguishing between infection by gram-negative bacteria and mold by a non-invasive imaging method utilizing18F-FDS. The methods of the present invention also apply to the detection of mold infection by multiple different species of in a subject. Furthermore, the1243844.601_P18516-01methods of the present invention may be utilized to monitor the effective of treatment for mold infection in a subject.

[0054] Accordingly, in some embodiments, the presently disclosed subject matter provides a method for detecting a mold infection in a subject, the method comprising administering18F- fluorodeoxy sorbitol (18F-FDS) to the subject and taking an image. In some preferred embodiments, a therapeutically effective amount of18F-FDS is provided to the subject for imaging purposes.

[0055] In some embodiments, the presently described subject matter provides a method for monitoring the effectiveness of a treatment for a mold infection in a subject comprising administering an antifungal treatment and then monitoring the course of mold infection in the patient by administering18F-fluorodeoxysorbitol (18F-FDS) to the subject and taking an image. Two or more images may be taken over the course of the treatment and compared to determine the effectiveness of the treatment. In some preferred embodiments, a reduction in18F-FDS uptake at a previously identified site of infection in the subject is indicative of an effective treatment.

[0056] In particular embodiments, the image is a positron emission tomography (PET) and computed tomography (CT) image.

[0057] In some embodiments, the mold infection is selected from an Aspergillus, a non- Aspergillus (galactomannan-negative), or an azole-resistant (Aspergillus calidouslus) mold infection. In certain embodiments, the mold infection is selected from Aspergillus spp., Rhizopus spp., Mucor spp., Lichtheimia corymbifera, Syncephalastrum racemosum, Cunninghamella bertholletiae , Cladophialophora bantiana and Fusarium solani.

[0058] In some embodiments, the methods further comprise testing the subject for galactomannan status before, during or after administration of18F-FDS to the subject. In some embodiments, the subject is galactomannan negative and the uptake of18F-FDS is indicative of a non-Aspergillus (galactomannan-negative) mold infection. In some embodiments, the non-Aspergillus (galactomannan-negative) mold infection is selected from the group consisting of a Rhizopus spp. mold infection, a Mucor spp. mold infection, a Lichtheimia corymbifera mold infection, a Syncephalastrum racemosum mold infection, a Cunninghamella bertholletiae mold infection, and a Cladophialophora bantiana mold infection and a Fusarium solani mold infection, and combinations thereof. In some embodiments, the non-Aspergillus (galactomannan-negative) mold infection is selected from the group consisting of a Rhizopus spp. mold infection, a Mucor spp. mold infection, and a Fusarium solani mold infection, and combinations thereof. In some1343844.601_P18516-01embodiments, the non-Aspergillus (galactomannan-negative) mold infection is a Mucor spp. mold infection.

[0059] In particular embodiments, the mold infection is selected from an invasive pulmonary infection and a cerebral infection.

[0060] In other embodiments, the mold infection comprises a rhinosinusal infection due to Aspergillus, Rhizopus, and / or Mucor.

[0061] In certain embodiments, the subject is immunocompromised. In particular embodiments, the subject has cancer.

[0062] In certain embodiments, the image is a whole-body image.

[0063] In particular embodiments, the image can distinguish a mold infection from a sterile inflammation or cancer.

[0064] In particular embodiments, the image can distinguish a mold infection from infection by a gram- negative bacteria. In some embodiments, the gram negative bacteria is a Pseudomonas spp. In some embodiments, the Pseudomonas spp. is P. aeruginosa.

[0065] In some embodiments, the methods are used to provide a preliminary screen for mold infection in a patient. The methods of the disclosure are particularly suited to early detection of mold infections in a patient because the methods of the disclosure can be identified multiple species of mold in a single step. In some embodiments, the subject is suspected of having a mold infection or being at risk of a mold infection. In some embodiments, the absence of uptake of18F- FDS in the image is indicative of the absence of a mold infection by more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more species of mold. In some embodiments, the methods can be used as a preliminary screen to identify infection by more than one species of mold, for example the methods are useful for the simultaneous screening for infection by multiple mold species and, for example, allow screening for infection by 2, 3, 5, 6, 7, 8, 9, 10 or more different species of mold in a single imaging step. In some embodiments, the methods further comprise the step of invasively sampling the mold infection after first detecting the presence of uptake of18F-FDS by mold in the subject so that the species of the mold can be determined.

[0066] In some embodiments, the present disclosure provides methods for distinguishing between mold and bacterial infection in an infectious legion in a subject. In these embodiments,18F-FDS is administered to the subject and an image is taken as described above. The image in then analyzed to determine the volume of the lesion and the portion of the lesion that is occupied by the1443844.601_P18516-01an infectious biomass which is preferably visualized by18F-FDS uptake. In some embodiments, the image analysis comprises automated segmentation of the image to determine the volume of the lesion and the portion of the lesion occupied by the infective biomass. In some embodiments, a high volume of occupation of the lesion by the infective biomass is indicative of a mold infection and a low volume of occupation of the lesion by the infective biomass is indicative a bacterial infection. In some embodiments, a high volume of occupation of the lesion is defined by a biomass occupation of the lesion of greater than 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, or 10.0% (or any value in the range of 1.0 to 10.0%). In some embodiments, a low volume of occupation of the lesion by the biomass is less than 0.95%, 0.9%, 0.8%, 0.5%, or 0.3% (or any value therein).

[0067] The phrase “in one embodiment” or “in some embodiments” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

[0068] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references, i.e., “one or more,” unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. Likewise, the term “include” and its grammatical variants are intended to be nonlimiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.

[0069] As used herein, the term “therapeutically effective amount” means that18F-FDS is administered to a subject in vivo in an amount that is sufficient to effectively target the infectious organisms of interest and which can be detected in the subject over a reasonable time frame. In other embodiments, the term “therapeutically effective amount” means that18F-FDS is placed in contact with the infectious organisms of interest in vitro and which can be detected in the organisms over a reasonable time frame.1543844.601_P18516-01

[0070] Tn accordance with an embodiment, the present invention provides a pharmaceutical composition comprising18F-FDS and a pharmaceutically acceptable carrier.

[0071] In another embodiment, the present invention provides a pharmaceutical composition comprising18F-FDS, a pharmaceutically active compound, and a pharmaceutically acceptable carrier.

[0072] In accordance with an embodiment, the present invention provides a pharmaceutical composition comprising18F-FDS and / or derivatives thereof; wherein the composition includes a pharmaceutically and physiologically acceptable carrier, in an amount effective for use in a medicament, and most preferably for use as a medicament for use in the detection of the presence of mold organisms in a mammalian host, wherein the pharmaceutical composition is administered to the host, and after allowing a sufficient period of time for the mold to take up the labeled substrate, a determination is made whether the mold is present in the host by detecting the labeled substrate in the body of the mammalian host.

[0073] In accordance with a further embodiment, the present invention provides a pharmaceutical composition comprising18F-FDS and / or derivatives thereof; wherein the composition includes a pharmaceutically and physiologically acceptable carrier, in an amount effective for use in a medicament, and most preferably for use as a medicament for determination of the efficacy of an antibiotic and / or antifungal therapy in a mammalian host with a mold and / or bacterial infection, wherein the pharmaceutical composition is administered to the host, and after allowing a sufficient period of time for the infectious organism to take up the labeled substrate, the amount of infectious organisms (e.g., mold and or gram-negative bacteria) present in the host are determined by detecting the labeled substrate in the body of the mammalian host, followed by subjecting the mammalian host to antibiotic and / or antifungal treatment for a selected period of time; repeating the administration of the pharmaceutical composition and after allowing a sufficient period of time for the infectious organism to take up the labeled substrate, the amount infectious organisms present in the host are again determined by detecting the labeled substrate in the body of the mammalian host; and assessing whether the infection has been reduced after treatment with the antibiotic and / or antifungal treatment by comparing the amount of labeled substrate present before treatment to the amount of labeled substrate present after treatment, wherein when the amount of labeled substrate present after treatment is less, a determination is made that the antibiotic and / or antifungal treatment of is effective. In preferred embodiments, the methods of the present invention1643844.601_P18516-01are able to distinguish between infection by mold and by gram-negative bacteria in the host. In especially preferred embodiments, the methods of the present invention arc able to distinguish between infection by mold and by Pseudomonas spp. in the host.

[0074] In a further embodiment, the medicament further comprises a second therapeutic agent or the methods comprise administering a second therapeutic agent to the subject. In some embodiments, the therapeutic agent is an anti-infective agent, such as antihelmintics, antianaerobics, antibiotics, aminoglycoside antibiotics, antifungal antibiotics, cephalosporin antibiotics, macrolide antibiotics, miscellaneous antibiotics, penicillin antibiotics, quinolone antibiotics, sulfonamide antibiotics, tetracycline antibiotics, antimycobacterials, antituberculosis antimycobacterials, antiprotozoals, antimalarial antiprotozoals, antiviral agents, anti-retroviral agents, scabicides, and urinary anti-infectives.

[0075] A therapeutic agent and a biologically active agent are used interchangeably herein to refer to a chemical or biological compound that induces a desired pharmacological and / or physiological effect, wherein the effect may be prophylactic or therapeutic. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of those active agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the terms “active agent,” “pharmacologically active agent” and “drug” are used, then, it is to be understood that the invention includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs etc. The active agent can be a biological entity, such as a virus or cell, whether naturally occurring or manipulated, such as transformed.

[0076] In accordance with an embodiment of the present invention, the amount of time of18F-FDS to the infectious organisms (e.g., mold and / or gram-negative bacteria) in the subject should be sufficiently long to effect uptake of the labeled substrate in the infectious organisms in the subject. The time for the desired effect varies with dosage, target, age and other factors known to those of skill in the art. Generally, the time of exposure of the labeled substrates to the infectious organisms should range from about 1 hour to about 120 hours, preferably from about 1 hour to about 24 hours, more preferably from about 1 hour to about 12 hours.

[0077] With respect to labeled substrates described herein (i.e.,18F-FDS), the carrier can be any of those conventionally used, and is limited only by physico-chemical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration.1743844.601_P18516-01The carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well- known to those skilled in the art and arc readily available to the public. It is preferred that the carrier be one which is chemically inert to the active agent(s), and one which has little or no detrimental side effects or toxicity under the conditions of use. Examples of the carriers include soluble carriers such as known buffers which can be physiologically acceptable (e.g., phosphate buffer) as well as solid compositions such as solid-state carriers or latex beads.

[0078] The carriers or diluents used herein may be solid carriers or diluents for solid formulations, liquid carriers or diluents for liquid formulations, or mixtures thereof.

[0079] Solid earners or diluents include, but are not limited to, gums, starches (e.g., corn starch, pregelatinized starch), cellulosic materials (e.g., microcrystalline cellulose), acrylates (e.g., poly methylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.

[0080] For liquid formulations, pharmaceutically acceptable carriers may be, for example, aqueous or non-aqueous solutions, or suspensions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcoholic / aqueous solutions, cyclodextrins, emulsions or suspensions, including saline and buffered media.

[0081] Parenteral vehicles (for subcutaneous, intravenous, intraarterial, or intramuscular injection) include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Formulations suitable for parenteral administration include, for example, aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.

[0082] Intravenous vehicles include, for example, fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid earners, particularly for injectable solutions.

[0083] The choice of carrier will be determined, in pail, by the particular labeled substrate, as well as by the particular method used to administer the composition. Accordingly, there are a variety1843844.601_P18516-01of suitable formulations of the pharmaceutical composition of the invention. The following formulations for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal and interperitoneal administration are exemplary, and are in no way limiting. More than one route can be used to administer the labeled substrates of the present invention, and in certain instances, a particular route can provide a more immediate and more effective response than another route.

[0084] Injectable formulations are in accordance with the invention. The requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Trissei, 15th ed., pages 622-630 (2009)).

[0085] As used herein the term “pharmaceutically active compound” or “therapeutically active compound” means a compound useful for the treatment or modulation of a disease or condition in a subject suffering therefrom. Examples of pharmaceutically active compounds can include any drugs known in the art for treatment of disease indications. Particular examples of a pharmaceutically active compounds are antifungal agents and antibiotic agents.

[0086] The term “antibiotic agent” as well as words stemming therefrom, as used herein, generally includes pharmaceutically or therapeutically active compounds that work by interfering with the metabolism of the infectious organism. Antibacterial antibiotics are commonly classified based on their mechanism of action, chemical structure, or spectrum of activity. Most target bacterial functions or growth processes. Those that target the bacterial cell wall (penicillins and cephalosporins) or the cell membrane (polymixins), or interfere with essential bacterial enzymes (quinolones and sulfonamides) have bactericidal activities. Those that target protein synthesis (aminoglycosides, macrolides, and tetracyclines) are usually bacteriostatic. Further categorization is based on their target specificity. “Narrow-spectrum” antibacterial antibiotics target specific types of bacteria, such as gram-negative or gram-positive bacteria, whereas broad- spectrum antibiotics affect a wide range of bacteria.

[0087] “Antifungal agents” generally includes pharmaceutically or therapeutically active compounds that work by interfering with the metabolism of fungi. Suitable antifungal agents include, but are not limited to voriconazole, fluconazole, isavuconazol, itraconazole, vivjoa, caspofungin, anidulafungin, micafungin, amphotericin B (including amphotericin B deoxycholate,1943844.601_P18516-01liposomal amphotericin B, and amphotericin B lipid complex), nystatin, 5-fluorocytosine, kctoconazolc, miconazole, clotrimazole, posaconazolc, griscofulvin, iodoquinol, and clioquinol.

[0088] For purposes of the invention, the amount or dose of18F-FDS that is administered should be sufficient to effectively target the infectious organisms in vivo, such that the uptake of the labeled substrates can be detected, in the subject over a reasonable time frame. The dose will be determined by the efficacy of the particular labeled substrate formulation and the location of the infectious organisms in the subject, as well as the body weight of the subject to be treated.

[0089] The dose of the18F-FDS will also be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular labeled substrate. Typically, an attending physician will decide the dosage of the labeled substrates with which to treat each individual subject, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, compound to be administered, route of administration, and the severity of the condition being treated. By way of example, and not intending to limit the invention, the dose of the labeled substrates of the present invention can be about 0.001 to about 1000 mg / kg body weight of the subject being treated, from about 0.01 to about 100 mg / kg body weight, from about 0.1 mg / kg to about 10 mg / kg, and from about 0.5 mg to about 5 mg / kg body weight. In another embodiment, the dose of the labeled substrates of the present invention can be at a concentration from about 1 nM to about 10,000 nM, preferably from about 10 nM to about 5,000 nM, more preferably from about 100 nM to about 500 nM.

[0090] In another embodiment, the term “administering” means that18F-FDS is introduced into a subject, preferably a subject receiving treatment for a disease, and the18F-FDS is allowed to come in contact with the one or more disease related infectious organisms in vivo.

[0091] As used herein, the term “treat,” as well as words stemming therefrom, includes diagnostic and preventative as well as disorder remitative treatment.

[0092] As used herein, the term “mammalian host” or “subject” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals a e from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order2043844.601_P18516-01Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.

[0093] In a further embodiment,18F-FDS can be used in combination with one or more additional therapeutically active agents which are known to be capable of treating conditions or diseases discussed above. For example, the described labeled substrates of the present invention could be used in combination with one or more known therapeutically active agents, to treat a disease or condition.

[0094] As used herein, the term “detection” “imaging” or “radiodetection” means the use of certain properties of isotopes and the energetic particles emitted from radioactive material to diagnose or treat various medical conditions. In addition, the term “scintigraphy” means a diagnostic test in which a two-dimensional image of a body having a radiation source is obtained through the use of radioisotopes. A radioactive chemical is injected intravenously into the patient which then concentrates in the target cells or organ of interest. By placing a camera that senses radioactivity over the body, an image of the target cells or organ of interest can be created. The particles can be detected by suitable devices such as gamma cameras, positron emission tomography (PET) machines, single photon emission computed tomography (SPECT) machines and the like.

[0095] In accordance with some embodiments, the term “imaging” or “detection” can also include detection of photons as from a fluorescent dye on the labeled substrate.

[0096] In accordance with another embodiment, the present invention provides a method for detection of the location of infectious organisms (e.g., mold and / or gram-negative bacteria) in the body of a mammalian host comprising: a) administering to the host a therapeutically effective amount of18F-FDS; b) allowing a sufficient period of time for the infectious organism to take up the18F-FDS; and c) determining the location of the infectious organisms present in the host by detecting the18F-FDS in the body of the mammalian host.

[0097] In accordance with an embodiment, the present invention provides a method of diagnosing, locating, and assessing the efficacy of treatment of an infectious disease (e.g., a mold infection) in a patient comprising administering to a subject suspected of having said disease,18F-FDS prepared according to the above methods, which is selectively taken up by an infectious organism, obtaining a diagnostic image of the subject, determining the location of labeled substrate taken up by the infectious organisms in the subject, and correlating the location of the sequestered labeled substrate2143844.601_P18516-01with the location of the infectious organisms in the subject. When the detectable moiety is a positron emitter, such as18F, the spectroscopy can be, for example, SPECT, PET, gamma scintigraphy, or MRI.

[0098] In accordance with a further embodiment, the present invention provides a method for the determination of the efficacy of an antifungal therapy in a mammalian host with a mold infection comprising: a) administering to the host a therapeutically effective amount of18F-FDS; b) allowing a sufficient period of time for the infectious organism to take up the18F-FDS; c) determining the amount infectious organisms are present in the host by detecting the18F-FDS in the body of the mammalian host; d) subjecting the mammalian host to antifungal treatment for a selected period of time; e) repeating steps a)-c) one or more times; f) assessing whether the mold infection has been reduced after d) by comparing the amount of18F-FDS present before d) to the amount of18F- FDS present after d), wherein when the amount of18F-FDS present after d) is less, than a determination is made that the antifungal treatment of d) is effective.EXAMPLES

[0099] The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are only intended for the purposes of illustration and are not to be construed as limiting in any manner to make compounds of the disclosure by other methods.EXAMPLE 1

[0100] Overview

[0101] Invasive mold infections are a major cause of mortality in immunosuppressed and cancer patients. Diagnosis is challenging, requiring invasive procedures or reliance on fungal biomarkers with limited sensitivity and an inability to detect non-Aspergillus molds. In this Example, we perform whole-body18F-fluorodeoxysorbitol (18F-FDS) positron emission tomography (PET) and computed tomography (CT) in nine prospectively enrolled patients with high- suspicion of invasive2243844.601_P18516-01mold infections (eventually confirmed using culture or molecular assays) or other pathologies (NCT05611892).18F-FDS PET / CT is safe and can rapidly detect and localize invasive pulmonary and cerebral infections due to Aspergillus, non-Aspergillus (galactomannan-negative), or azole- resistant (Aspergillus ccilidoustus) molds, and differentiate them from sterile inflammation or cancer. Moreover,18F-FDS selectively and rapidly accumulates intracellularly in a wide range of clinically relevant molds, including azole-resistant molds, via a saturable process. Tissue histology from eight patients with invasive mold infections demonstrates that lesional biomass occupied by molds is approximately 50-fold higher than for bacterial infections. In animals,18F-FDS PET / CT is able to detect and localize pulmonary and cerebral aspergillosis, as well as rhinosinusal infections due to Aspergillus, Rhizopus, and Mucor, confirming the clinical data.18F-FDS PET represents a promising, noninvasive diagnostic tool for the detection and localization of invasive mold infections throughout the body.

[0102] Synopsis

[0103] Invasive mold infections are increasingly prevalent in immunosuppressed patients, affecting multiple organ systems (e.g., lung, brain), and associated with mortality as high as 85%. While Aspergillus remain a major cause, other mold infections, with similar clinical presentation, are on the rise. Prompt identification of invasive mold infections and initiation of appropriate treatment is critical for improving patient outcomes. Definitive diagnosis remains challenging, however, often requiring invasive procedures (e.g., bronchoalveolar lavage, biopsy). Further, biomarkers such as galactomannan have limited sensitivity and specificity for Aspergillus and are unable to detect most other molds. Importantly, all currently available imaging tools are nonspecific, and unable to differentiate infection from other pathologies. Although pathogenspecific imaging approaches for Aspergillus have been described in rodent models, they have not been evaluated in prospective clinical studies, or applied to other mold infections. Here, we present a novel PET tracer,18F-fluorodeoxysorbitol (18F-FDS, sorbitol radioanalog), for rapid, noninvasive, whole-body detection and localization of invasive infections due to a wide range of clinically relevant molds, in prospectively enrolled patients.18F-FDS PET can be easily synthesized and incorporated into current clinical practice for these patients:

[0104] (1) We performed whole-body18F-FDS PET / CT, in accordance with U.S FDA guidance, in nine prospectively enrolled patients with high- suspicion of invasive mold infections (eventually confirmed using culture or molecular assays) or other pathologies (NCT05611892).18F-FDS2343844.601_P18516-01PET / CT was safe and rapidly (same day) detected and localized invasive pulmonary and cerebral infections due to Aspergillus, non-Aspergillus (galactomannan-ncgativc), and azoic -resistant molds (up to 7 days after starting antifungals), and differentiate them from sterile inflammation or cancer, with high specificity.18F-FDS is easily synthesized from commercial18F- fluorodeoxy glucose (18F- FDG) (globally available), by a simple kit at room temperature, allowing on-demand synthesis and global availability. As imaging studies are already the standard of care for patients suspected of invasive mold infections,18F-FDS PET can be easily incorporated into the clinical workflow.

[0105] Interestingly,18F-FDS PET identified a cerebral lesion in a patient with Cladophialophora bantiana (galactomannan-negative), previously missed on the brain MRI, suggesting a level of sensitivity superior to the best available imaging tools. C. bantiana has a high propensity for brain dissemination and is associated with high mortality due to diagnostic delays (median 115 days).

[0106] (2) While18F-FDS was developed to image infections due to the Enterobacterales group of Gram negative bacteria (e.g., E. coli, Salmonella, and the like), a study by Kim et al., Nat Commun, 2022 described the utility of18F-FDS PET to detect and monitor invasive pulmonary aspergillosis in mouse models. Kim et al., however, could not explain why Aspergillus infections could be visualized in vivo using18F-FDS PET, even though the in vitro uptake of18F-FDS is lower in molds compared to E. coli. Without wishing to be bound to any one particular theory, it was thought that differences in the biomass occupied by molds versus bacteria within infectious lesions may explain this paradox. Using tissue samples from another cohort of eight unique patients with pulmonary, cerebral, or rhinosinusal invasive Aspergillus from the Johns Hopkins Hospitals, we demonstrate that Aspergillus occupy approximately 50-fold higher biomass than bacteria. Similar findings were observed in tissues obtained from our animal infections with Aspergillus, Rhizopus, Mucor and E. coli. This highlights a new insight, with major implications for developing novel pathogen- specific imaging tracers. While18F-FDS PET will detect acute, high burden (7 log 10 colony forming units / mL) Enterobacterales (bacterial) infections, Enterobacterales bacteria are rarely in the differential for presentations consistent with invasive mold infections and patient characteristics, response to empiric antibiotics can easily distinguish them from mold infections.

[0107] Given that non-Aspergillus molds, with similar clinical presentation, are on the rise (due to wide- spread Aspergillus-targeted antifungal prophylaxis), and current noninvasive biomarkers,2443844.601_P18516-01namely galactomannan are unable to detect non-Aspergillus molds, we evaluated18F-FDS uptake by a wide range of clinically relevant molds. We demonstrate rapid18F-FDS accumulation in 30 random clinical isolates including Aspergillus, Rhizopus, Mucor, other clinically relevant molds, including azole-resistant molds, but not by heat killed molds or mammalian cells, confirming that sorbitol uptake is a conserved process. Although, Aspergillus is known to metabolize sorbitol,18F- FDS (sorbitol) uptake by a range of relevant mold fungi is a new and important finding from our studies.

[0108] Finally, we developed several clinically relevant animal models of invasive pulmonary, rhinosinusal and cerebral mold infections, representing the major forms of human infections. In these animal studies,18F-FDS PET / CT was able to detect and localize pulmonary and cerebral aspergillosis, as well as rhinosinusal infections due to Aspergillus, Rhizopus, and Mucor, and differentiate these infections from LPS-induced sterile inflammation, confirming the results from the clinical data. The18F-FDS PET signal was proportional to the infection burden. Moreover, while18F-FDS PET was able to differentiate infection from sterile inflammation,18F-FDG PET, which measures inflammation and is non-specific, could not distinguish pulmonary aspergillosis versus sterile inflammation. To our knowledge, in vivo detection of ncm-Aspergillus mold infections as well as the use of rhinosinusal and cerebral models of invasive mold disease, including by non-Aspergillus molds, are novel and have not been reported previously.

[0109] In summary, the Example represents a major advance, and present a powerful, new, clinically-relevant, rapid and noninvasive diagnostic tool to detect and localize invasive mold infections anywhere in the body. This technology fills a wide gap in the diagnostic pathway for patients with suspected invasive mold infections and will help to improve patient outcomes for a disease associated with high mortality.

[0110] Introduction

[0111] We have previously described18F-fluorodeoxysorbitol (18F-FDS), a radioanalog of sorbitol (sugar alcohol), as a positron emission tomography (PET) tracer to selectively image infections due to the Enterobacterales group of bacteria (e.g., Escherichia coll)21,22. However, Aspergillus can also utilize sorbitol as a carbon source23,24, and a recent study has shown that18F-FDS PET can detect invasive pulmonary aspergillosis in animal models17. In this Example, we performed whole-body18F-FDS PET / CT in nine prospectively enrolled patients with either invasive mold infections or other pathologies (NCT05611892). We demonstrate that18F-FDS PET is safe and2543844.601_P18516-01can rapidly detect and localize invasive pulmonary and cerebral mold infections due to drug- susceptible or azole -resistant molds, and differentiate them from sterile inflammation and cancer. We also show that18F-FDS is selectively accumulates in a wide range of clinically relevant molds, but not by heat killed fungi or mammalian cells. Importantly,18F-FDS is rapidly metabolized intracellularly by molds, via a saturable process. Tissue histology from eight unique patients with pulmonary, cerebral or rhinosinusal invasive mold infections, demonstrates the lesional biomass occupied by molds is approximately 50-fold higher than for bacterial infections. In animal studies,18F-FDS PET / CT is able to detect and localize pulmonary and cerebral aspergillosis, as well as rhinosinusal infections due to several molds, confirming the clinical data. Importantly,18F-FDS can be easily synthesized from commercially available18F-fluorodeoxyglucose (18F-FDG)25, the most widely utilized PET tracer globally, by a simple one-step kit-based method, without the need for specialized radiosynthesis and purification facilities26, allowing on-demand synthesis and global availability of18F-FDS.

[0112] Results

[0113] l8F-FDS PET / CT selectively detects invasive mold infections

[0114] We prospectively enrolled nine newly identified patients with either invasive mold infections (n = 4) using the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium (EORTC / MSGERC) consensus Definitions (Table 1) or uninfected controls with other pathologies (n = 5) (NCT05611892).

[0115] Confirmatory diagnosis of mold infections was established in all four patients, using culture or molecular assays (FIG. 1, Table 2). Sterile inflammatory pathologies (n = 4) were determined clinically and / or by tissue biopsies and oncologic diagnosis (n = 1; breast cancer) was made via tissue biopsy (FIG. 1, Table 2)22. The median age was 59 years (range, 37 to 71 years), and 44% (4 of 9) were female. Importantly, the enrolled patients were representative of patients hospitalized in a tertiary care setting with several co-morbidities (Table 2). Whole-body18F-FDS PET / CT performed in accordance with U.S. FDA guidelines was safe and well tolerated in all patients.18F-FDS kinetics were consistent with prior studies22, demonstrating rapid renal and some hepatobiliary elimination and low background in unaffected brain and lung tissues. Patients with invasive mold infections had received up to seven days of antifungal treatments by the time of imaging. Despite this,18F-FDS PET / CT was able to detect and localize invasive pulmonary and2643844.601_P18516-01cerebral infections all patients with mold infections [Aspergillus, non-Aspergillus (galactomannan- ncgativc) molds, or azolc-rcsistant Aspergillus calidoustus)}.

[0116] To quantify the PET signal, spherical volumes of interest (VOIs) were drawn at the sites of pathology and unaffected sites of the same tissue to calculate the target-to-nontarget ratio (TNT).18F-FDS PET signal was significantly higher at the sites of invasive mold infections with a TNT of 5.79 [interquartile range (IQR), 3.18 to 15.20] versus sterile inflammatory pathologies or cancer with a TNT of 1.40 (IQR, 1.01 to 1.93) (P = 0.008) (FIG. 2). Importantly, while the18F- FDG PET clearly demonstrated the breast cancer lesion, no signal was noted on18F-FDS PET (FIG. 8). Using a TNT cutoff of 3.0,18F-FDS PET had a sensitivity of 100% [95% confidence interval (CI), 28 to 99%] and a specificity of 100% (95% CI, 48 to 100%).

[0117] For pulmonary infections,18F-FDS PET signal was significantly higher at infection sites with a TNT of 3.40 (IQR, 3.10 to 14.05) compared to sites with sterile inflammatory pathologies with a TNT of 1.79 (IQR, 1.57 to 2.17) (P <0.001) (FIG. 3, FIGS. 9-10). Similarly, the18F-FDS PET signal was significantly higher at the sites of invasive cerebral mold infections with a TNT of 6.39 (IQR, 5.19 to 16.92) (P = 0.007) (FIG. 4, FIGS. 11-12 and 22). Importantly,18F-FDS PET was able to identify a cerebral lesion in patient 4 with Cladophialophora bandana pulmonary infection, which was previously missed on a brain MRI performed for clinical reasons (FIG. 11). Thus, using a defined TNT cutoff of 3.00,18F-FDS PET was able to detect and localize invasive pulmonary and cerebral infections in all patients with mold infections [Aspergillus, non-Aspergillus (galactomannan-negative) molds, or azole -resistant Aspergillus calidoustus)}, despite having received up to seven days of antifungal treatments by the time of imaging. Moreover, lesions in all control patients had a TNT ratio of <3.

[0118] lsF-FDS uptake by clinically relevant molds

[0119] In vitro studies were performed to determine18F-FDS uptake by a wide range of clinically relevant molds (FIG. 5a). Rapid18F-FDS accumulation was noted in the live strains, whereas no uptake was noted in the heat killed controls (FIG. 5b-d) or in mammalian cells (THP-1 macrophages) (FIG. 13). Competitive inhibition tests conducted by co-incubation with increasing concentrations of unlabeled sorbitol decreased the18F-FDS uptake (FIG. 5g-e), suggesting a saturable mechanism. Finally, all the 30 random clinical isolates including Aspergillus spp., Rhizopus spp., Mucor spp., and other clinically relevant molds (Lichtheimia corymbifera, Syncephalastrum racemosum, Cunninghamella bertholletiae , Cladophialophora bandana, and2743844.601_P18516-01Fusarium solani), along with azole-resistant molds demonstrated robust18F-FDS uptake. These findings confirm that sorbitol uptake is conserved across a wide range of clinically relevant molds (FIG. 5h).

[0120] Finally, we performed studies to elucidate the metabolism of18F-FDS by molds. We coincubated actively growing Aspergillus fiimigatus with18F-FDS and analyzed the cytoplasmic fraction from the lysed fungal pellet by radio-thin layer chromatography (radio-TLC).18F-FDS is rapidly metabolized intracellularly by Aspergillus in a manner similar to the intracellular metabolism of18F-FDS in E. coli (FIG. 14).

[0121] Mold biomass occupies a significant proportion of the infected lesion

[0122] The in vitro uptake of18F-FDS is lower in molds compared to E. coli (FIG. 13). However, in vivo visualization of infections by18F-FDS PET is dependent on the total tracer uptake within the infected lesion.

[0123] We therefore compared the biomass within infectious lesions occupied by molds and bacteria (FIG. 6). Infected tissue samples from eight unique patients with pulmonary, cerebral or rhinosinusal invasive Aspergillus infections from the Johns Hopkins Hospitals (patient characteristics are outlined in Table 3), or publicly available digitized slides of bacterial infections in patients27, were analyzed. Tissues from animal infected with Aspergillus, Rhizopus, Mucor or E. coli were also analyzed. High power views are shown in FIG. 15. The percentage of the infected lesion volume occupied by the mold or bacteria was calculated using automated segmentation (FIG. 6, FIG. 16). Data from human tissues demonstrated that the biomass occupied by Aspergillus was 11.71% (IQR 7.85 to 13.03%), which is ~50-fold higher than for bacterial infections at 0.24% (IQR 0.10 to 0.38%) (P < 0.001). Similar findings were observed in tissues obtained from animal studies (P = 0.016).

[0124] Table 3. Patient characteristics for the tissue histology study.2843844.601_P18516-01

[0125] ALL, acute lymphocytic leukemia; BMT, bone marrow transplantation; DM, diabetes mellitus.

[0126] l8F-FDS PET localizes fungal infections in animal models

[0127] Next, we developed several clinically relevant mouse models of Aspergillus, Rhizopus, and Mucor infections and evaluated the ability of18F-FDS PET to detect and localize the mold infections. Immunosuppressed mice were used for all studies. For pulmonary aspergillosis, mice were infected intratracheally with increasing doses of A. fumigatus conidia and the pulmonary18F- FDS PET signal was measured, demonstrating a PET signal proportional to the infection burden (FIG. 17). Thereafter, we evaluated18F-FDS PET in uninfected animals, animals with pulmonary aspergillosis and those with lipopolysaccharide (LPS)-induced sterile pulmonary inflammation (FIG. 18). Although substantial pulmonary disease was noted on CT and18F-FDG PET in mice with sterile inflammation, no18F-FDS PET signal was noted in these mice (FIG. 18b). However, mice with pulmonary aspergillosis demonstrated18F-FDS PET signal, which colocalized with the lesions noted on CT (FIG. 18c). The pulmonary18F-FDS PET signal was substantially higher in the mice with pulmonary aspergillosis versus those with sterile inflammation (FIG. 18g, P = 0.003). By contrast,18F-FDG PET, which measures inflammation and is not specific for infection, could not distinguish mice with pulmonary aspergillosis versus those with sterile inflammation (FIG. 18h, P = 0.338). Postmortem pulmonary tissue histology demonstrated Aspergillus hyphae in the mice with pulmonary aspergillosis (FIG. 18i). We also performed studies with Pseudomonas, that can cause pneumonia in immunosuppressed patients. The pulmonary18F-FDS PET signal was substantially higher in the mice with pulmonary aspergillosis versus those with Pseudomonas pneumonia (Fig. 23, P = 0.002). Importantly, the18F-FDS PET signal from P. aeruginosa pneumonic lesions were no different than those noted with sterile pulmonary2943844.601_P18516-01inflammation due to LPS. Consistent with prior studies32, in vitro uptake of! 8 F-FDS by Pseudomonas was substantially lower than the uptake by molds (P =0.003) (Fig. 23c).

[0128] Similar findings were observed in mouse models of rhinosinusal mold infections due to A. fumigatus, Rhizopus oryzae and Mucor circinelloid.es, where18F-FDS PET was able to detect and localize the infection sites; significantly higher PET signals were noted in mice with mold infections versus those with LPS-related systemic inflammation (FIG. 19, P < 0.002). Postmortem ex vivo biodistribution confirmed the imaging findings and tissue histology demonstrated fungal hyphae in the infected mice (FIG. 19e-f). Fig. 20a, Fig. 20b, Fig. 20c, Fig. 20d and Fig. 20e provide representative MIP (left), transverse (upper) and coronal (lower) CT (middle) and18F-FDS PET / CT (right) from pulmonary infection by A.ypergzVZw.y-infected mice and Pseudomonas- infected mice. Fig. 21a, Fig. 21b and Fig. 21c show representative MIP, coronal (upper) and sagittal (lower)18F-FDS PET / CT from Aspergillus-'mtcc cd mice with rhinosinusal infection (Fig. 21a), without treatment and after 1 week of voriconazole treatment (Fig. 21b).18F-FDS PET signal in voriconazole treated (for 1 week) mice was significantly low compared to untreated mice (P = 0.011). These results show that the imaging methods described herein can be used to monitor mold infection following treatment.

[0129] Finally, we developed a mouse model of invasive cerebral aspergillosis, utilizing direct intracerebral infection with A. fumigatus.18F-FDS PET was able to detect and localize the infection sites with significantly higher PET signal in mice with invasive cerebral aspergillosis compared to mice with LPS-related systemic inflammation (FIG. 7a-d, P < 0.001). Of note, the “ring enhancing” PET uptake pattern in mice (FIG. 7c) was similar to that noted in the patient with invasive cerebral aspergillosis (FIG. 4a) and likely represents a lack of blood supply and dead tissues in the necrotic core of the lesion, which is well described with invasive mold infections. Postmortem ex vivo biodistribution confirmed the imaging findings and tissue histology demonstrated fungal hyphae in the infected mice (FIG. 7e-f). Additionally, the18F-FDS PET signal colocalized with the lesions noted on the MR1 (FIG. 7h).

[0130] Discussion

[0131] In this example, we describe results from a prospective study evaluating18F-FDS PET / CT to specifically detect and localize invasive mold infections (and differentiate them from other pathologies) in a patient population representative of a tertiary care hospital setting within seven days of initiation of antifungal treatments. By applying a TNT cutoff of 3 as previously described22,3043844.601_P18516-0118F-FDS PET had a sensitivity of 100% and a specificity of 100% to diagnose invasive mold infections.

[0132] 18F-FDS-PET fills a wide gap in the diagnostic pathway for patients with suspected invasive mold infections. When infections occur in relatively inaccessible sites, minimally invasive clinical samples (blood, urine, stool, or cerebrospinal fluid) can often yield non-diagnostic results or are otherwise insensitive for deep-seated infections. Currently available imaging tools (e.g. radiography, ultrasonography, CT, MRI,18F-FDG PET) are non-specific, cannot differentiate an infection from other pathologies, such as sterile inflammation or cancer10'12’28,29. Moreover, these modalities rely on host responses to infection that could be significantly altered in immunocompromised hosts. Although major strides have been made in the development of microbial cell-free DNA detection for infections30,31, clinically available biomarkers, namely galactomannan and 1,3-beta-D-glucan, have limited sensitivity and specificity for Aspergillus spp. and galactomannan is unable to reliably detect non-Aspergillus molds. Additionally, biomarkers cannot provide spatial information on the location or extent of infection. Therefore, definitive diagnosis of invasive mold infections almost always requires invasive procedures such as bronchoalveolar lavage, or deep tissue biopsy, which is challenging, dangerous (e.g., brain biopsy), and delays the diagnosis. Because of these challenges, empiric antifungal treatment (prior to establishing a confirmatory diagnosis) is the norm in patients with suspected invasive mold infections. However, treatment can decrease the sensitivity of culture-based tests performed later. The development of rapid, whole-body, noninvasive, and specific diagnostics able to detect invasive mold infections in patients who are often immunosuppressed and receiving empiric treatment would be a crucial advancement in the field and could quickly become the clinical standard of care.

[0133] This Example supports that18F-FDS PET overcomes many of these challenges. In one exemplary case,18F-FDS PET was able to localize known pulmonary invasive mold infection due to C. bantiana in a patient with a negative galactomannan test.18F-FDS PET also identified previously undiagnosed cerebral involvement with C. banliana that was not detected on brain MRI performed for clinical reasons (FIG. 11). C. bantiana has a high propensity for dissemination to the brain and is associated with high mortality due to delays in diagnosis (median of 115 days)32. Early and accurate identification of cerebral infection has implications for treatment (the need for an antifungal agent with CNS penetration) and for prognostication. This example highlights many3143844.601_P18516-01potential advantages of18F-FDS PET including its utility in diagnosing and localizing infections due to diverse clinically important molds not otherwise detected by currently available biomarkers, and a level of sensitivity superior to the best currently available imaging tools.

[0134] Our data show that18F-FDS-PET can distinguish invasive mold infections from normal tissue and sterile inflammation in humans and rodents. This is due to low tissue background signal from18F-FDS in several tissues including the lungs and the brain, which are the two primary sites for mold infections. Unlike molds and some other pathogens, mammalian cells cannot metabolize sorbitol, and substitution of the hydroxyl group by fluorine at the 2-position in18F-FDS completely abrogates recognition by mammalian cells33. We have previously shown that cancer cell lines do not take up18F-FDS21, and that18F-FDS PET demonstrates low uptake in cancerous lesions in animal and human studies21,22. Some brain tumors or inflammatory pathologies have been reported to accumulate18F-FDS34"36, but these reports describe pituitary lesions (outside the blood-brain barrier), and / or measure18F-FDS uptake immediately after tracer injection (5 min in the case reports34,35and 60 min in animal studies36), and thus have a higher background signal. Utilizing dynamic18F-FDS PET over 120 min, we have demonstrated that while brain tumors can demonstrate initial PET uptake, this dissipates over time (after 60 min)21. Therefore, the findings previously noted in brain tumors or inflammatory pathologies are consistent with a non-specific, blood pool effect, consistent with capillary leak at the site of inflammation or tumor37. In our studies, imaging was performed 120-180 min after tracer injection with significant clearance of the background21,22,38. In fact, in the current studies, the TNT measurements for invasive mold infections were highest in the brain. For example, in patient 4, where imaging was performed 180 min after tracer injection, the18F-FDS PET derived TNT in the right frontal cortex lesion was 17 (FIG. 11). The corresponding head MRI (performed for clinical reasons), reported postoperative changes from prior surgeries and white matter changes, but did not demonstrate evidence of pathologic enhancement.

[0135] Differentiation of microbes by selective growth media, utilizing small molecules (mostly sugars and sugar alcohols), was historically a mainstay of clinical microbiology39. We (and others) have exploited this differential metabolism by mammalian cells versus pathogens40, to develop several pathogen- specific imaging approaches11,12. While we had originally developed18F-FDS, a radioanalog of sorbitol (sugar alcohol), to selectively image infections due to the Enterobacterales group of Gram negative bacteria21,22, a recent study by Kim et al described the utility of18F-FDS3243844.601_P18516-01PET to detect and monitor invasive pulmonary aspergillosis in animal models17. However, this study could not explain why Aspergillus infections could be visualized well in vivo using18F-FDS PET, even though the in vitro uptake of18F-FDS is approximately 8-fold lower in molds compared to E. coli for the same mass (FIG. 13). It was hypothesized that the low uptake of18F-FDS by Aspergillus is likely due to the need for induction of sorbitol dehydrogenase by sorbitol, which may not be achieved by PET tracers41. Our study disproves this hypothesis, demonstrating that Aspergillus rapidly accumulates18F-FDS without the need for induction by sorbitol, via a saturable process (FIG. 5). Using radio-TEC studies, we further demonstrate that18F-FDS is indeed rapidly metabolized intracellularly by A. fumigatus, which is identical to the intracellular metabolism of18F-FDS noted in E. coli (FIG. 14). Instead, we hypothesized that differences in the biomass occupied by molds versus bacteria within infectious lesions may explain the excellent PET signal noted in vivo. Our study confirmed this hypothesis. The biomass occupied by Aspergillus spp. in pulmonary, cerebral, or rhinosinusal infections is approximately 50-fold higher than for bacterial infections (FIG. 6). Similar findings were observed in tissues obtained from animal infections. These mechanistic findings have important implications for the development of novel pathogenspecific imaging tracers.

[0136] 18F-FDS PET also shows promise for the diagnosis and localization of non-Aspergillus molds, which may have a similar clinical presentation, are on the rise due to use of Aspergillus- targeted antifungal prophylaxis7, and are not detected by current noninvasive biomarkers (i.e. galactomannan)6. In addition to Aspergillus spp., we demonstrate rapid18F-FDS accumulation in a diverse group of clinical isolates, including azole-resistant molds, confirming that sorbitol uptake is a conserved process. Although, Aspergillus is known to metabolize sorbitol23,24,18F-FDS (sorbitol) uptake by a wide range of clinically relevant mold fungi is a new finding from our studies. Our mechanistic and observational findings are further supported by clinically relevant animal models of invasive pulmonary, rhinosinusal and cerebral mold infections, representing the major forms of human invasive mold infections2. To our knowledge, in vivo detection of non- Aspergillus mold infections as well as the use of rhinosinusal and cerebral models of invasive mold disease, including by non-Aspergillus molds, are novel and have not been reported previously.

[0137] 18F-FDS PET is a promising diagnostic tool that can be accessible to a wide range of institutions and patient populations. PET is now a routine clinical tool42, and is increasingly available both in high- and low-income and middle-income countries. Due to the short half-life of3343844.601_P18516-01most PET radionuclides, e.g., 109 min for F-l 8, the tracer has to be synthesized on-site or at a nearby location, requiring dedicated infrastructure (cyclotron) and trained personnel, thus limiting on-demand supply. However,18F-FDS can be synthesized rapidly from commercially available18F-FDG by a simple, one-step kit-based method26, allowing on-demand synthesis (at room temperature) and global availability of18F-FDS. Further, high specific activity (radioactivity per mass) is not required, as even low specific activity18F-FDS (20-50 Ci / mM) produces optimal imaging characteristics21,40. We believe this is a major advantage that will allow global, on-demand18F-FDS availability at any site with access to a PET scanner. Most patients suspected of invasive mold infections will undergo imaging studies such as CT or MRI as the current standard of care.18F-FDS PET / CT could be easily incorporated into the current clinical workflow for these patients. PET imaging typically takes 15-60 min, performed 60-120 min after tracer administration, and provides results on the same day. In fact, the18F-FDS PET / CT results in the current study were available much earlier than the conventional tests used to establish the confirmatory diagnosis for invasive mold infections. Newer technologies such as total-body PET scanners enable high- sensitivity imaging with dramatically improved signal-to-noise ratio and will further shorten the scan duration while increasing sensitivity43.

[0138] Although18F-FDS PET cannot distinguish aspergillosis from other mold infections (e.g. mucormycosis) an approach combining18F-FDS PET with galactomannan testing enables distinguishing between the two infections. Further, due to the need for invasive procedures, many mucormycosis cases remain undiagnosed or the diagnoses are significantly delayed. Therefore,18F-FDS PET could not only help in the early diagnosis, but when needed, also provide the rationale for an earlier invasive procedure as well as the best sites(s) to sample the infection. None of the imaged patients were neutropenic, which is also a major risk group for invasive mold infections. However, in prior human studies22, no correlation was found between the peripheral white blood cell or neutrophil counts and the18F-FDS PET signal in the infected patients, suggesting that18F-FDS PET is not dependent on host inflammatory cells. Additionally, all animal experiments in the current studies utilized neutropenic mice (induced with cyclophosphamide), which did not preclude18F-FDS PET to specifically detect, localize and monitor invasive fungal infections. Stringent criteria were utilized in this clinical study (i.e., prospective enrollment, eventual confirmation of invasive mold infection from the infection site, and inclusion of patients who received <7 days of antifungal treatment prior to the18F-FDS PET) strengthening the3443844.601_P18516-01reliability and clinical relevance of the results10. However, these criteria were also an impediment to patient recruitment leading to a small sample size for the clinical study. A validated PET tracer in clinical use would not be subject to such study restrictions, so it is believed that18F-FDS PET will ultimately have applications in many more patients for whom early diagnosis and localization can significantly alter the diagnostic pathway, treatment, and prognosis. Moreover, due to the ease of tracer synthesis and on-demand availability with the one-step, solid-phase cartridge system26, this technology could be practically implemented in most settings. Notably, Enterobacterales bacteria (e.g. E. coli, Salmonella, etc.) also metabolize sorbitol and acute [high burden, 7 logio colony forming units (CFU) / mL] infections would be visualized by 18F-FDS PET22. However, Enterobacterales bacteria are rarely in the differential for presentations consistent with invasive mold infections and patient characteristics, response to empiric antibiotics can easily distinguish Enterobacterales from mold infections. Similarly, while Pseudomonas pneumonia can present in this patient population, Pseudomonas do not accumulate18F-FDS, and no18F-FDS PET signal was noted in the mouse model of Pseudomonas pneumonia. In summary, we present data from a prospective clinical study evaluating whole-body18F-FDS PET / CT in nine patients with high- suspicion of invasive mold infections or other pathologies. We demonstrate that18F-FDS PET is safe, feasible, and can rapidly detect and localize invasive pulmonary and cerebral infections due to Aspergillus, non- Aspergillus, and azole-resistant molds, and differentiate them from sterile inflammation or cancer. Imaging data were confirmed by culture or molecular assays and observations in humans were replicated using clinically relevant animal models. While larger clinical studies are needed to validate these findings, our data strongly support the role of18F-FDS as an easily synthesizable PET tracer for noninvasive detection and localization of invasive mold infections throughout the body.

[0139] Methods[00140J All protocols were approved by the Johns Hopkins University Biosafety, Radiation Safety, Animal Care and Use (MO22M354) and Institutional Review Board (IRB00097331, IRB00071002) Committees.

[0141] 18F-FDS PET / CT in prospectively enrolled patients

[0142] 18F-FDS was synthesized as a sterile solution with high specific activity and high radiochemical purity by the Johns Hopkins PET Center. This prospective observational study was performed in accordance with the U.S. FDA Radioactive Drug Research Committee guidelines45,3543844.601_P18516-01with localization of18F-FDS PET signal to the infection sites as the primary outcome measure. This study was registered on clinicaltrials.gov (NCT05611892)44. Nine patients (four with newly diagnosed invasive mold infections, and five control patients with other pathologies22) were prospectively recruited from the Johns Hopkins Hospitals between October 2016 to July 2024 using the following criteria (Table 1).3643844.601_P18516-01

[0144] BAL, Bronchoalveolar lavage; CSF, cerebrospinal fluid; EIA, enzyme immunoassay; PCR, polymerase chain reaction.

[0145] Confirmatory diagnosis of mold infections was established in all four patients, using culture or molecular assays. Sterile inflammatory pathologies were determined clinically and / or by tissue biopsies, and oncologic diagnosis was made via tissue biopsy (Table 2).

[0146] Written informed consent was obtained from each subject, physical examination performed by a trained physician and screening laboratory tests performed and reviewed by the study principal investigator to confirm eligibility. On the day of imaging, a low-dose CT, selected to minimize the radiation exposure, was performed. An intravenous injection of18F-FDS (503 ± 191 MBq) was administered and PET was acquired at 120 min after tracer administration (except patient 4 where acquisition was at 180 min post-tracer injection) from the skull vertex to mid-thigh with 3 min per bed-position, using a Siemens Biograph PET / CT scanner. All patients were evaluated immediately after finishing the PET scan and 20-25 days after to assess early and delayed adverse events.

[0147] Human histology

[0148] Infected tissue samples from eight unique patients with confirmed pulmonary, cerebral or rhinosinusal invasive Aspergillus spp. infections from Johns Hopkins Hospitals were obtained (patient characteristics outlined in Table 3). Multiple random slides were obtained from each tissue sample, stained with GMS, digitally scanned and analyzed at 40x magnification. Additionally, publicly available digitized slides of bacterial infections in patients (case IDs: 963, 749, 1692, 1688) from Infectious Diseases Images eMicrobes Digital Library were analyzed27. The percentage of area occupied by the mold or bacteria was calculated using automated segmentation (ImageJ) after standardizing the area analyzed in each sample (FIG. 6, FIG. 16).

[0149] In vitro assays

[0150] A. fumigatus (ATCC 1022), or clinical isolates from the Johns Hopkins Hospitals clinical microbiology laboratory including Rhizopus oryzae and Mucor circinelloid.es were grown on potato dextrose agar (PDA) plates at 37°C and conidia were collected in sterile phosphate buffered3743844.601_P18516-01saline (PBS) with Tween 80 (0.01 %). A manual count of the conidia density (conidia / mL) was performed using a hcmocytomctcr. Fungal cultures were heat killed by exposure to 70°C heat for 60 min.18F-FDS (5 pCi / mL) was co-incubated with the fungi grown at 37°C with shaking and samples were collected at different time points followed by pelleting and washing thrice with PBS. Activity for each sample pellet was measured immediately using an automated gamma counter (PerkinElmer). Counts were corrected for background and decay and normalized to pellet weight (mg). Bacterial and mammalian cells (THP-1) studies were performed as described before21. All studies were performed in triplicate samples.

[0151] Metabolite studies

[0152] A. fumigatus (ATCC 1022) or E. coli (ATCC 25922) cultures were incubated with18F- FDS. Aliquots of 1 mL were pelleted by centrifugation and washed with PBS. The cells were lysed and the cytoplasmic fraction was separated by ultracentrifugation and filtered (0.22 pm syringe). The solute was analyzed on a radio-TLC (Eckert & Ziegler) using18F-FDS and18F-FDG as reference.

[0153] Az?zma / studies

[0154] Female BALB / c mice (6-8 weeks old, Charles River) were housed in pre-sterilized, filter- topped cages, with unrestricted access to both food and water [medicated to prevent bacterial superinfection prophylaxis with tetracycline (Sigma)]. All animals received anesthesia with 2% isoflurane and O2 mixture for infection inoculation, LPS-induced inflammation and imaging.

[0155] For the mold infection studies, mice were immunosuppressed using cortisone acetate (Sigma, 200 mg / Kg) administered subcutaneously at day five and one prior to mold infection and cyclophosphamide (Sigma, 150 mg / Kg) administered intraperitoneally at day four and one day before infection17,46. For invasive pulmonary aspergillosis, anesthetized mice were hung by their upper incisors and secured to a support. A superficial 1 cm incision was made in the midline of the neck to visualize and successfully cannulate the trachea47. The intubation was confirmed upon connection to a ventilator, and then 40 pL suspension of fungal conidia [7xl06A. fumigatus (ATCC 1022) conidia / mouse] was used for the infection followed by two days of incubation prior to imaging. Rhinosinual infection was carried out by a slow intranasal instillation of 15 pL of the fungal conidia suspension [IxlO8A. fumigatus (ATCC 1022), Rhizopus oryzae or Mucor circinelloides conidia / mouse) under anesthesia with two days of incubation prior to imaging. For invasive cerebral aspergillosis, anesthetized mice were infected with 3 pL suspension of fungal3843844.601_P18516-01conidia [Ix lO6A. fumigatus (ATCC 1022) conidia / mouse] via a burr hole using a Hamilton syringe (Hamilton, 88,000) and stereotaxic instrument (David KOPF instrument, model 900) with the following coordinates: 2 mm dorsal to bregma, 2 mm lateral to middle line and 2 mm deep. After the inoculation, the hole was sealed with bone wax and the scalp was closed using a surgical staple and the infection incubated for one day prior to imaging. The model of sterile pneumonia was developed using an intratracheal instillation of 30 pL of LPS solution (5 pg / g). For systemic inflammation mice were intraperitoneally administered 50 pL of LPS solution four days prior (2 pg / g) and the day of imaging (6 pg / g)48,49.

[0156] Imaging: Animals were injected via tail vain with a bolus of 8.89 MBq ± 1.43 of18F-FDG or 8.54 ± 1.56 MBq of18F-FDS. A 15 min PET was acquired 45 min after injection of18F-FDG, and 120 minutes after injection of18F-FDS, using a nanoScan PET / CT (Mediso). A CT scan was obtained for anatomical reference. MRI was performed using a 7T preclinical MRI (MR Solutions) and mouse 'H body coils with 1 mm slices and fat saturation. The T1W and T2W sequences utilized a repetition time of 1000 ms and an echo time of 11 and 45 ms respectively.

[0157] Ex vivo Biodistribution: These studies were performed immediately after the completion of the imaging studies. Animals were sacrificed, and the tissues were perfused by infusing 6 mL of PBS per animal through the left ventricle. The organs were extracted thereafter and the activity in each organ was measured immediately using an automated gamma counter (PerkinElmer) and presented as the percentage injected dose per gram of tissue (%ID / g).

[0158] Histopathology: Slides were prepared from tissues fixed in 4% paraformaldehyde from our current mold infection animal models. Multiple random slides were obtained from each tissue sample, stained with GMS, digitally scanned and analyzed as described above. Fixed tissue samples from mice with E. coli myositis with a target implantation of 7 logio CFU units were obtained from our prior studies21. Multiple random slides were obtained from each tissue sample, Gram stained, digitally scanned and analyzed as described above.

[0159] Image analysis

[0160] Human images were analyzed using Mirada XD™ 3.6.8 (Mirada Medical) and PMOD version 3.402 (PMOD Technologies LLC) while the animal images were analyzed using VivoQuant 2020 (Invicro)21,22,50. Three-dimensional VOIs were drawn manually using the CT as a reference and the PET activity was measured. The human PET data are presented as TNT which is the ratio of the PET signal at the sites of pathology to the unaffected sites of the same tissue.3943844.601_P18516-01The animal PET data are presented as SUVr which is the ratio of the PET signal at the sites of pathology to the same tissue from uninfected animals. All data were visualized using AMIRA 5.2.1 (Visage Imaging, Inc.) or AMIDE 1.0.6 (Andreas Loening).

[0161] Statistical analysis

[0162] Data were analyzed using Prism 10.0 (GraphPad Software Inc). All data are represented as median ± IQR and comparisons were made using a two-tailed Mann- Whitney U test. P values < 0.05 were considered statistically significant.REFERENCES

[0163] All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents form part of the common general knowledge in the art.

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[0214] Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.4443844.601_P18516-01

Claims

THAT WHICH IS CLAIMED:

1. A method for detecting a clinical mold infection in a subject, the method comprising administering18F-fluorodeoxysorbitol (18F-FDS) to the subject and imaging the18F- fluorodeoxy sorbitol (18F-FDS) in the subject to detect uptake of the18F-fluorodeoxysorbitol (18F- FDS) by mold thereby indicating the presence or absence a clinical mold infection.

2. The method of claim 1, wherein the subject is suspected of having a clinical mold infection or being at risk of a clinical mold infection.

3. The method of claim 1 or claim 2, wherein the absence of uptake of18F-FDS in the image is indicative of the absence of a mold infection by more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more species of mold.

4. The method of claim 1 or claim 2, further comprising the step of invasively sampling the mold infection after first detecting the presence of uptake of18F-FDS by mold in the subject.

5. A method for monitoring a response of a subject to a treatment for a mold infection comprising: administering a treatment for a mold infection to the subject; administering18F-fluorodeoxysorbitol (18F-FDS) to the subject; and imaging the18F-fluorodeoxy sorbitol (18F-FDS) in the subject to detect uptake of the18F- fluorodeoxy sorbitol (18F-FDS) by mold thereby providing an indication of the response of the subject to the treatment.

6. The method of claim 5, wherein the steps of administering18F-fluorodeoxysorbitol (18F-FDS) to the subject and imaging the18F-fluorodeoxysorbitol (18F-FDS) to detect uptake of the18F-fluorodeoxysorbitol (18F-FDS) by mold in the subject are repeated two or more times to provide two or more images.

7. The method of claim 5, wherein the two or more images are compared to monitor response to the treatment.

8. The method of claim 7, wherein a decrease in18F-fluorodeoxysorbitol (18F-FDS) uptake as evidenced by a comparison of the two or more images is evidence of a positive response to the treatment.

9. The method of any one of claims 5 to 8, wherein the treatment is an antibiotic treatment or an antifungal treatment.

10. The method of any one of claims 1 to 9, wherein the imaging comprises positron emission tomography-computed tomography (“PET-CT”) imaging.

11. The method of any one of claims 1 to 10, wherein the subject is a human.

12. The method of any one of claims 1 to 11, wherein the mold infection is selected from an Aspergillus mold infection, a non-Aspergillus (galactomannan-negative) mold infection, and an azole-resistant (Aspergillus calidoustus) mold infection.

13. The method of any one of claims 1 to 12, further comprising testing the subject for galactomannan status before, during or after administration of18F-FDS to the subject.

14. The method of any one of claims 1 to 13, wherein the subject is galactomannan negative and the uptake of18F-FDS is indicative of a non-Aspergillus (galactomannan-negative) mold infection.

15. The method of claim 14, wherein the non-Aspergillus (galactomannan-negative) mold infection is selected from the group consisting of a Rhizopus spp. mold infection, a Mucor spp. mold infection, a Lichtheimia corymbifera mold infection, a Syncephalastrum racemosum mold infection, a Cunninghamella bertholletiae mold infection, and a Cladophialophora bantiana mold infection and a Fusarium solani mold infection, and combinations thereof.

16. The method of claim 14, wherein the non-Aspergillus (galactomannan-ncgativc) mold infection is selected from the group consisting of a Rhizopus spp. mold infection, a Mucor spp. mold infection, and a Fusarium solani mold infection, and combinations thereof.

17. The method of claim 14, wherein the non-Aspergillus (galactomannan-negative) mold infection is a Mucor spp. mold infection.

18. The method of any one of claims 1 to 17, wherein the mold infection is selected from an Aspergillus spp. mold infection, a Rhizopus spp. mold infection, a Mucor spp. mold infection, a Lichtheimia corymbifera mold infection, a Syncephalastrum racemosum mold infection, a Cunninghamella bertholletiae mold infection, a Cladophialophora bantiana mold infection, and a Fusarium solani mold infection, and combinations thereof.

19. The method of claim 18, wherein the subject has two or more of an Aspergillus spp. mold infection, a Rhizopus spp. mold infection, a Mucor spp. mold infection, a Lichtheimia corymbifera mold infection, a Syncephalastrum racemosum mold infection, a Cunninghamella bertholletiae mold infection, a Cladophialophora bantiana mold infection and a Fusarium solani mold infection, and the method allows simultaneous detection of mold infections by two or more different mold species.

20. The method of claim 19, wherein at least one of the mold infections is an Aspergillus spp. mold infection and at least one of the mold infections is a Mucor spp. mold infection.

21. The method of any one of claims 1 to 20, wherein the imaging allows localization of the mold infection to an area of the body and / or determination of the extent of the mold infection in an area of the body.

22. The method of claim 21, wherein the area of the body is selected from the group consisting of the brain, lungs and the nasopharyngeal pathway.

23. The method of any one of claims 1 to 21, wherein the mold infection is selected from an invasive pulmonary mold infection, a cerebral mold infection, and a nasopharyngeal mold infection.

24. The method of any one of claims 1 to 21, wherein the mold infection comprises a rhinosinusal infection due to infection by an Aspergillus ssp., Rhizopus ssp., and / or Mucor ssp.

25. The method of any one of claims 1 to 24, wherein the subject is immunocompromised.

26. The method of any one of claims 1 to 25, wherein the subject has cancer.

27. The method of any one of claims 1 to 26, wherein the imaging comprising taking a whole-body image.

28. The method of any one of claims 1 to 27, wherein the imaging distinguishes a mold infection from a sterile inflammation and / or cancer.

29. The method of any one of claims 1 to 28, wherein the subject has a gram-negative bacterial infection, has been previously diagnosed with a gram-negative bacterial infection, is suspected of having a gram-negative bacterial infection, or being at risk of a gram-negative bacterial infection.

30. The method of any one of claims 1 to 29, wherein the imaging distinguishes a mold infection from an infection with a gram-negative bacteria.

31. The method of any one of claims 1 to 30, further comprising the step of diagnosing or identifying a mold infection when the subject also has an infection by a gram-negative bacteria, has been previously diagnosed with an infection by a gram-negative bacteria, or is suspected of being at risk by infection with a gram-negative bacteria.

32. The method of any one of claims 29 to 31, wherein the gram negative bacteria is a Pseudomonas spp.

33. The method of claim 32, wherein the Pseudomonas spp. is P. aeruginosa.

34. The method of any one of claims 1 to 33, wherein the mold infection is not an infection by Aspergillus fumigatus .

35. The method of any one of claims 1 to 34, further comprising the step of distinguishing a mold infection from a bacterial infection in an infective tissue lesion having a volume in the subject by determining the volume of the infective tissue lesion occupied by an infective biomass by image analysis of the results of the imaging step, wherein a high volume of occupation of the lesion by the biomass is indicative of a mold infection and a low volume of occupation of the lesion by the biomass is indicative a bacterial infection.

36. The method of claim 35, wherein a high volume of occupation of the lesion is defined by a biomass occupation of the lesion of greater than 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, or 10.0%.

37. The method of claim 35 or claim 36, wherein the image analysis comprises automated segmentation of the image.

38. The method of any one of claims 35 to 37, wherein a low volume of occupation of the lesion by the biomass is less than 0.95%, 0.9%, 0.8%, 0.5%, or 0.3%.

Images