MicroPET Imaging of Bacterial Infection with Specifically Nitroreductase Responsive 18F-labeled Nitrogen Mustard Analogues

Purpose Bacterial infection and antibiotic-resistant are still serious threats to human health. Here, we aim to develop two novel radiotracers 18F-NTRP and 18F-NCRP with specific nitroreductase (NTR) responsive to image the deep-seated bacterial infection with PET for differentiating infection from sterile inflammation. Methods 18F-NTRP and 18F-NCRP were synthesized via a one-step method. All the steps of tracers radiosynthesis were successfully adapted in the All-In-One automated module. After the physiochemical properties of 18F-NTRP and 18F-NCRP were characterized, their specificity and selectivity to NTR were verified in E. coli and S. aureus as well. The ex vivo biodistribution of tracers was evaluated in normal mice. MicroPET-CT imaging was acquired in mouse models of bacterial infection and inflammation after the administration of 18F-NTRP and 18F-NCRP. Results Fully automated radiosynthesis of 18F-NTRP and 18F-NCRP were achieved within 90 - 110 min with overall decay-uncorrected isolated radiochemical yields of 21.24 ± 4.25 % and 11.3 ± 3.78 % respectively. Molar activities of 18F-NTRP and 18F-NCRP were 320 ± 40 GBq/µmol and 275 ± 33 GBq/µmol respectively. In addition, 18F-NTRP and 18F-NCRP exhibited good selectivity and specificity to NTR response. PET-CT imaging in bacterial infected mice models with 18F-NTRP and 18F-NCRP showed significant radioactivity uptakes in both E. coli and S. aureus infected muscles. Especially, the E. coli infected muscle uptakes were up to 2.4 ± 0.2 %ID/g (18F-NTRP) and 4.05 ± 0.49 %ID/g (18F-NCRP), 3-fold to control uptake. Furthermore, both 18F-NTRP and 18F-NCRP showed a 2.6-fold higher uptake in bacterial infection compared to inflammation counterparts, indicating that they can distinguish infection from inflammation effectively. Conclusion Based on the promising results of this preliminary study, 18F-NTRP and 18F-NCRP are worth further study to verify their potentials for bacterial infection imaging via NTR responsive.

Drug interaction evaluation of [99mTc]Tc-ketoconazole uptake with ketoconazole administration in Candidiasis mice model

The use of radiopharmaceuticals for infection detection has gained increasing attention for their application in nuclear medicine. The administration of [99mTc]Tc-ketoconazole may altere pharmacological aspects including interaction data with some antifungals especially ketoconazole as a common drug for candidiasis treatment. The current study investigated the ex vivo biodistribution and pharmacokinetic interaction of [99mTc]Tc-ketoconazole after ketoconazole administration in BALB/c mice. In this research,The [99mTc]Tc-ketoconazole was prepared with radiochemical purity 94.59% (n=3). The ex vivo biodistribution uptake in infected muscle as a target organ showed 0.16±0.13%ID/g for control, 0.17±0.12 for 1 h, and 0.05±0.04 for 3 h after ketoconazole administration. The Target/Non-Target (T/NT) ratio between infected muscle compared with normal muscle showed 1.19±0.13 for the control group, 2.56±1.71 for 1 h and 0.86±0.67 for 3 h. The ex vivo biodistribution also showed high radioactivity uptake on the liver, lung, spleen and kidney. Pharmacokinetics analysis using PKSolver showed [99mTc]Tc-ketoconazole half-life elimination (t1/2 beta) for the therapy group showed shorter elimination time 13.78±4.77 h compared with the control group 44.77±2.74 h after ketoconazole administration. Pharmacokinetics parameter change also occurred on the area under the curve of therapy group (AUC 0-inf) that is 26.10±18.97 %ID/g*h and maximum concentration (Cmax) 13.05±9.48 %ID/g which was related with radiopharmaceutical absorption rate. This study proves that based on biodistribution and pharmacokinetics evaluation, the [99mTc]Tc-ketoconazole application was recommended at 1 h post ketoconazole administration.

Prevalence of bovine sarcocystosis in slaughtered cattle in Duhok abattoir, Iraq

Background: Sarcocystosis or infection with Sarcocystis species is a parasitic zoonotic disease caused by cyst forming coccidian intracellular protozoa that caused by different species of Sarcocystis. The cyst forming parasite has obligatory two hosts life cycle including carnivorous as definitive host and omnivorous or herbivorous as intermediate host. The parasitic infestation causes serious health problems and economical loses because of abortion in pregnant animals, carcass condemnation after slaughtering due to severe emaciation and pathological lesions, low quality of meat, milk and wool, as well as restriction on animal importation by authorities.Methods: The present work was conducted to study the prevalence of sarcocysts infection in slaughtered cattle at Duhok abattoir, Iraq. Muscle samples from different organs comprising esophagus, diaphragm and heart were collected from 150 cattle aged from one to two years old. Different techniques were used for detection of macroscopic and microscopic types of sarcocysts. The techniques including inspection by naked eye, peptic digestion method, muscle mincing and squash preparation and staining with giemsa stain, as well as histopathological examination.Results: The overall prevalence of infected muscle samples was 76%. The infection rate of microscopic type of sarcocyst was 41.3% in heart, 92% in diaphragm and 94% in oesophagus. The histopathological examination of infected muscle tissues revealed mild infiltration of inflammatory cells and slight degeneration of muscle fibers. Significant difference (p≤0.05) was recorded between the prevalence rate of macrocysts and microcysts of sarcocyst but there was no significant difference (p≥0.05) in the prevalence rate of sarcocystis infection among different organs.Conclusions: The results of the current study indicated a high prevalence of sarcocystis infection among slaughtered cattle in Duhok province, Iraq, that could be due existence of large numbers of dogs and cats around the slaughter house which are involved in life cycle of the parasite and spread of the infection.

Radiolabeled Cationic Peptides for Targeted Imaging of Infection

Molecular probes targeting bacteria provide opportunities to target bacterial infections in vivo for both imaging and therapy. In the current study, we report the development of positron emission tomography (PET) probes for imaging of live bacterial infection based on the small molecules HLys-DOTA, a polycationic peptide synthesized as the D-isomer (RYWVAWRNRG) conjugated to 1, 4, 7, 10-tetraazacyclododecane-N′,N″,N‴,N-tetraacetic acid (DOTA) and AB1-HLys-DOTA, which includes an unnatural amino acid AB1 that preferentially binds to bacteria membrane lipids with amine groups via formation of iminoboronates. HLys-DOTA and AB1-HLys-DOTA peptides were radiolabeled with 64Cu and investigated as PET imaging agents to track bacterial infection in vitro and in intramuscularly infected (IM) mice models. Cell uptake studies at 37°C in Staphylococcus aureus (SA) show higher uptake of 64Cu-AB1-HLys-DOTA; 98.47 ± 3.54% vs 64Cu-HLys-DOTA; 39.12 ± 3.27% at 24 h. Standard uptake values (SUV) analysis of the PET images resulted in mean SUV of 0.70 ± 0.08, 0.49 ± 0.04, and 0.31 ± 0.01 for 64Cu-AB1-HLys-DOTA and 0.17 ± 0.06, 0.16 ± 0.02, and 0.13 ± 0.01 for 64Cu-HLys-DOTA at 1, 4, and 24 h post injection, respectively, in the infected muscles. Similarly, in the biodistribution studies, dose uptake in the infected muscles was 4 times higher in the targeted 64Cu-AB1-HLys-DOTA group than in the 64Cu-HLys-DOTA group and 2‐3 times higher than in the PBS control group at 1, 4, and 24 h post injection. 64Cu-AB1-HLys-DOTA was able to distinguish between SA-infected muscle and Pseudomonas aeruginosa (PA) infected muscle with lower mean SUV of 0.28 ± 0.10 at 1 h post injection. This illustrates the utility of the AB1 covalently targeting group in synergy with the HLys peptide, which noncovalently binds to bacterial membranes. These results suggest that 64Cu-labeled AB1-HLys-DOTA peptide could be used as an imaging probe for detection of bacterial infection in vivo with specificity for Gram-positive bacteria.

Molecular characterisation of oomycetes from fish farm located in Mymensingh sadar during summer

Oomycetes, also known as water molds, can cause serious infection to plants and animals especially at lower temperature although they dwell in freshwater and moist ecosystem throughout the year. The aim of this research was to investigate the diversity of oomycetes inhabiting in small water bodies during summer. Three types of samples i.e. water, fish mucus and apparently infected muscle samples of fish were collected from a large fish farm consisting of over 100 medium to large ponds in Mymensingh during summer (March to June) in 2015. A total number of 385 samples (284 of water, 79 of mucus and 22 of apparently infected muscle samples) were collected in 15 ml sterile falcon tubes with baits in each. Eleven of the isolates were isolated in Potato Dextrose Agar (PDA) plates and were identified using molecular methods that included DNA extraction, PCR amplification and subsequent sequencing of the ITS region of the genomic DNA of the samples. BLAST analysis to GenBank revealed that two of the isolates were 99% similar to Pythium sp. (HQ643814), three of the isolates were 98-99% similar to Pythium sp. (KT247392), and each of the remaining four isolates was similar up to 99% to Pythium sp. (KF836354), 99% to Pythium sp. (EU544193), 99% to Pythium rhizo-oryzae (HQ643757) and 100% to Pythium catenulatum (KP862946). Two of the eleven isolates were not assessed due to sequencing error. Phylogenetic analysis revealed that six of the isolates are of clade B1 and three of the isolates are of clade B2 in the Pythium phylogeny. The results partially suggest that plant pathogenic oomycetes are more common in summer than animal or fish pathogenic isolates in the sampled farm however; intensive sampling with a broad range of freshwater ecosystems during summer can give a clearer view on oomycete diversity in Bangladesh.Asian J. Med. Biol. Res. June 2016, 2(2): 236-246

Studies on the epidemiology and histopathology of Euclinostomum heterostomum (Trematoda; Digenea) infection in Channa punctata from North India

A survey on the occurrence and epidemiology of the encysted progenetic metacercariae of Euclinostomum heterostomum infection in Channa punctata in the Aligarh region of North India revealed a mean prevalence, intensity, and abundance of 18.61, 1.52, and 0.38%, respectively, during the period from April 2011 to March 2012. Liver, kidney, peritoneum, muscle, and ovary were found to be infected with this parasite, and the later three are reported for the first time in this fish species. The histopathology of the infected tissues indicated the following at the host-parasite interface: tissue damage, infiltration of immune cells into the cyst wall, chronic inflammatory responses, and granulomatous lesions. The infected liver showed degeneration of hepatocytes, cytoplasmic vacuolation, nuclear alterations, mallory body formation, fibrosis, and necrosis. The pathology of the infected kidney included distortion and dilation of renal tubules, vacuolar degeneration, hypertrophy and hyperplasia of tubular epithelial cells, occlusion of tubules, fibrosis, hemorrhage, and congestion of glomeruli. The infected muscle demonstrated comparatively fewer pathological changes confined only to the circumference of the cyst wall. The ovary displayed the least changes. The conclusions drawn from the study are that the large metacercarial cysts formed by E. heterostomum in the vital organs of the economically important fish C. punctata could result in the impairment of fish physiology and health, thereby affecting their productivity and quality for human consumption.

Synthesis,68Ga-Radiolabeling, and PreliminaryIn VivoAssessment of a Depsipeptide-Derived Compound as a Potential PET/CT Infection Imaging Agent

Noninvasive imaging is a powerful tool for early diagnosis and monitoring of various disease processes, such as infections. An alarming shortage of infection-selective radiopharmaceuticals exists for overcoming the diagnostic limitations with unspecific tracers such as67/68Ga-citrate or18F-FDG. We report here TBIA101, an antimicrobial peptide derivative that was conjugated to DOTA and radiolabeled with68Ga for a subsequentin vitroassessment andin vivoinfection imaging usingEscherichia coli-bearing mice by targeting bacterial lipopolysaccharides with PET/CT. Following DOTA-conjugation, the compound was verified for its cytotoxic and bacterial binding behaviour and compound stability, followed by68Gallium-radiolabeling.µPET/CT using68Ga-DOTA-TBIA101 was employed to detect muscularE. coli-infection in BALB/c mice, as warranted by thein vitroresults.68Ga-DOTA-TBIA101-PET detectedE. coli-infected muscle tissue (SUV = 1.3–2.4) > noninfected thighs(P=0.322)> forearm muscles(P=0.092)> background(P=0.021)in the same animal. Normalization of the infected thigh muscle to reference tissue showed a ratio of 3.0 ± 0.8 and a ratio of 2.3 ± 0.6 compared to the identical healthy tissue. The majority of the activity was cleared by renal excretion. The latter findings warrant further preclinical imaging studies of greater depth, as the DOTA-conjugation did not compromise the TBIA101’s capacity as targeting vector.

Characterization of Barmah Forest virus pathogenesis in a mouse model

Alphaviruses including Barmah Forest virus (BFV) and Ross River virus (RRV) cause arthritis, arthralgia and myalgia in humans. The rheumatic symptoms in human BFV infection are very similar to those of RRV. Although RRV disease has been studied extensively, little is known about the pathogenesis of BFV infection. We sought to establish a mouse model for BFV to facilitate our understanding of BFV infectivity, tropism and pathogenesis, and to identify key pathological and immunological mechanisms of BFV infection that may distinguish between infections with BFV and RRV. Here, to the best of our knowledge, we report the first study assessing the virulence and replication of several BFV isolates in a mouse model. We infected newborn Swiss outbred mice with BFV and established that the BFV2193 prototype was the most virulent strain. BFV2193 infection resulted in the highest mortality among all BFV variant isolates, comparable to that of RRV. In comparison with RRV, C57BL/6 mice infected with BFV showed delayed onset, moderate disease scores and early recovery of the disease. BFV replicated poorly in muscle and did not cause the severe myositis seen in RRV-infected mice. The mRNAs for the inflammatory mediators TNF-α, IL-6, CCL2 and arginase-1 were highly upregulated in RRV- but not BFV-infected muscle. To our knowledge, this is the first report of a mouse model of BFV infection, which we have used to demonstrate differences between BFV and RRV infections and to further understand disease pathogenesis. With an increasing number of BFV cases occurring annually, a better understanding of the disease mechanisms is essential for future therapeutic development.

99mTc-prulifloxacin in artificially infected animals

SummaryAim: The radiosynthesis of 99mTc-Prulifloxacin (99mTc-PRN) was assessed in terms of stability, binding with Staphylococcus aureus (S. aureus), biodistribution in rats (RT) and scintigraphic profile in rabbits (RB). Animals, material, methods: 99mTc-PRN was synthesized by mixing 25 μg of stannous fluoride (SnF2) with 18.5 MB of sodium pertechnetate. Thereafter, 0.5 mg of the prufloxacin (PRN) was added to the reaction mixture and the pH was set at 5.1 with 0.01 mol/l HCl. The reaction mixture was incubated at room temperature. The same process was repeated by increasing the concentration of the stannous fluoride from 25 to 250 μg, sodium pertechnetate from 18,5 to 185 MBq and the PRN from 0.5 to 5 mg. The radiochemical stability of the 99mTc-PRN was investigated in higher concentration of the cystein. In-vitro binding investigation was performed using living and heat killed S. aureus to verify specificity of the 99mTc-PRN. Biodistribution was evaluated in artificially infected rats and scintigraphic precision in rabbits at different interval. Results: The 99mTc-RPN prepared by mixing 2 mg of PRN, 74 MBq sodium pertechnetate, 100 μg stannous fluoride at pH 5.4, appeared to be more than 90% stable with a maximum radiochemical yield of 98.15 ± 0.25% at 30 min. The 99mTc-PRN showed higher stability in serum and satisfactory in-vitro binding to living as compared to heat killed S. aureus. 14.25 ± 0.15% of the injected dose was accumulated in the infected muscle of the model RT. Infected to normal muscle ratio was 5.12 and inflamed to normal muscle was 1.2. The biodistribution was validated by the scintigraphic localization of infection in rabbits. Conclusion: This investigation of 99mTc-PRN confirmed its momentous radiochemical immovability in saline, serum, preferential in-vitro binding to living bacteria, higher uptake in the infected muscle of model RT and precise localization in the infected muscle of model RB.