Faecalibacterium, Bacteroides and Romboutsia, which appear to be depleted in cancerogenic mucosa as well as in adenomatous polyps, thus representing novel microbial biomarkers associated with early tumor formation. Furthermore, an absolute quantification of Fusubacterium nucleatum in polyps further compounded the important role of this microorganism as a valuable putative microbial biomarker for early diagnosis of colorectal cancer.The human intestine retains a complex microbial ecosystem, which performs crucial functions that impact on host health. Several studies have indicated that intestinal dysbiosis may impact on the establishment of life-threatening intestinal diseases such as colorectal cancer. An adenomatous polyp is the result of abnormal tissue growth, which is benign but is considered to be associated with a high risk of developing colorectal cancer, based on its grade of dysplasia. Development of diagnostic tools that are based on surveying the gut microbiota and are aimed at early detection of colorectal cancer represent highly desirable target. For this purpose, we performed a pilot study in which we applied a metataxonomic analysis based on 16S rRNA gene sequencing approach to unveil the composition of microbial communities of intestinal polyps. Moreover, we performed a meta-analysis involving the reconstructed microbiota composition of adenomatous polyps and publicly available metagenomics datasets of colorectal cancer. These analyses allowed the identification of microbial taxa such as  Colorectal cancer (CRC) is one of the most commonly diagnosed cancers worldwide and consequently one of the major causes of death in developed countries3. It is known that adenomatous polyps in some cases may evolve to colorectal cancer, even though no currently available scientific evidence unequivocally demonstrate in how many years and what are the precise causes of degeneration. So far there is a growing number of studies that highlight a direct correlation between polyp size, histology and progression of this pathology to CRC4. Furthermore, the molecular causes promoting such malignant transformation of polyps are essentially unknown5. Several factors have been investigated for polyp involvement in cancer development, such as genetics, epigenetics, diet, life style, obesity, alcohol intake and smoking6.Adenomatous polyps or adenomas have long been recognized as precursors of colorectal cancer10, phenolic compound, and indole production11, as well as conversion of dietary factors into carcinogens and tumor promoters12, and induction of proinflammatory and procarcinogenic pathways in host epithelial cells15. These proposed mechanisms have an impact in altering the metabolic environment of the host, which may directly or indirectly influence mutagenesis rates and thus carcinogenesis16. Various studies have explored the gut microbiota of individuals with CRC, resulting in the identification of a range of different bacterial groups being associated with carcinogenesis, including Helicobacter pylori, Bacteroides fragilis, Fusobacterium nucleatum, various pathogenic Escherichia coli, Streptococcus gallolyticus and members of the genus Clostridium26. Although screening and lifestyle play important roles in early detection and prevention of colorectal cancer, the identification of new diagnostic tools in particular for the early detection of tumors is important. Previous studies have compared the gut microbiota composition of stool samples from healthy people versus patient with polyps28. However, the fecal microbiota is only partially representing the autochthonous microbiota that is in direct contact with the intestinal mucosa30. Thus, in order to evaluate the role of the indigenous gut microbiota of subjects undergoing colic mucosectomies of polypoid lesions, we performed a pilot study collecting gut biopsies from healthy mucosa and polyps from the same subjects. On these samples, we performed a meta-analysis involving the here reconstructed microbiota composition of adenomatous polyps and publicly available metagenomic datasets of colorectal cancer based on 16S rRNA microbial profiling data33 in order to search for the occurrence of common microorganisms between polyps and colon cancer. Altogether, these analyses revealed potential novel microbial biomarkers associated with early malignant transformation.In recent decades, it has become evident that gut bacteria and their metabolites may participate in triggering or progression of colorectal cancer through various proposed mechanisms, including the production of reactive oxygen radicals and other genotoxinsTM Environmental RNA/DNA Isolation Kit Sample (Qiagen) following the manufacturer’s instructions . This study protocol was approved by Ethics Committee of Parma Province and all experiments were performed in accordance with relevant guidelines and regulations. Each patient was characterized by demographic data, level of disease indicators and therapy.At the Gastroenterology and Digestive Endoscopy Unit, University Hospital of Parma, Italy, 12 patients were enrolled, after obtaining informed consent Table . Exclusi34. Illumina adapter overhang nucleotide sequences were then added to the partial 16S rRNA gene-specific amplicons, which in turn were further processed by employing the 16S Metagenomic Sequencing Library Preparation Protocol . Amplifications were carried out using a Verity Thermocycler (Applied Biosystems). The integrity of the PCR amplicons was analysed by electrophoresis on a 2200 TapeStation Instrument .Partial 16S rRNA gene sequences were amplified from extracted DNA using primer pair Probio_Uni (5′-CCTACGGGRSGCAGCAG-3′)/Probio_Rev (5′-ATTACCGCGGCTGCT-3′), which targets the V3 region of the 16S rRNA gene sequencePCR products obtained following the amplification of a section of the 16S rRNA gene were purified by a magnetic purification step involving Agencourt AMPure XP DNA purification beads  in order to remove primer dimers. The DNA concentration of the amplified sequence library was estimated employing a fluorimetric Qubit quantification system (Life Technologies). Amplicons were diluted to 4 nM and 5 µl of each diluted DNA amplicon sample was mixed to prepare the pooled final library. Paired-end sequencing (250 bp × 2) was performed using an Illumina MiSeq sequencer with MiSeq Reagent Kit v3 chemicals-600 cycles .35 as previously described34. Paired-end reads were merged, and quality control implementation allowed the retention of sequences with a length between 140 and 400 bp, mean sequence quality score >25 and with truncation of a sequence at the first base if a low quality sequence within a rolling 10 bp window was found. Sequences with mismatched forward and/or reverse primers were omitted. 16S rRNA Operational Taxonomic Units (OTUs) were defined at ≥97% sequence homology using uclust36 and OTUs with less than 10 sequences were filtered. The Biological Observation Matrix (BIOM) obtained was analysed by summarize_taxa.py script in order to get the relative abundance of each taxonomic group for all samples. All reads were classified to the lowest possible taxonomic rank using QIIME35 and a reference dataset from the SILVA database37. The microbial richness of the samples  was evaluated with the Chao1 index through the alpha_rarefaction.py script included in the Qiime software suite using default parameters. Similarities between samples (beta-diversity) were calculated by unweighted uniFrac38. Hierarchical clusterings were performed with TMeV 4.8.1 (http://www.tm4.org) using Pearson correlation.The fastq files were processed using QIIMEwww.ibm.com/software/it/analytics/spss/) were used to complete statistical analysis. All data were presented as means ± SD. Furthermore, differential abundance of bacteria taxa and alpha-diversity were tested by one-way analysis of variance (ANOVA).SPSS software (Fusobacterium nucleatum targeting a previously described prostaglandin transporter-encoding gene39. The genome copy-number and the deduced cell number (since the genes targeted were in single copy per genome) was evaluated by comparing the cycle threshold (Ct) values obtained with those from a standard curve. Standard curves were calculated from serial dilutions of a culture with a known cell number (as determined by viable count assessment) for the bacterial strain versus Ct produced for each target gene. The primer sequences were as follows: forward primer 5′-CAACCATTACTTTAACTCTACCATGTTCA-3′ and reverse primer 5′-GTTGACTTTACAGAAGGAGATTATGTAAAAATC-3′. qPCR was performed using the CFX96 system . Each PCR reaction mix contained the following: 12.5 μl 2x SYBR SuperMix Green , 1 μl of DNA dilution, each of the forward and reverse primers at 0.5 μM and nuclease-free water was added to obtain a final volume of 20 μl. PCR products were detected with SYBR Green fluorescent dye and amplified according to the following protocol: one cycle of 95 °C for 2 minutes, followed by 42 cycles of 95 °C for 5 s and 60 °C for 30 s. Melting curve: 65 °C to 95 °C with increments of 0.5 °C/s. In each run, negative controls for each primer set were included. Cycle thresholding was calculated using the automated settings for Biorad CFX Manager 3.1 software (BioRad). The entire qPCR experiment was performed a second time using the same samples and methods as outlined above, for the purpose of replication, and very similar results were obtained.qPCR experiments were based on species-specific primers of The 16S rRNA profiling data sequenced in this study were deposited in SRA database under accession number PRJNA415554.A total of twelve patients with polyps were included in this pilot study. From those, histological analyses revealed that four corresponded to adenomatous polyps and eight to hyperplastic polyps. All adenomatous polyps were found in males, while the remaining hyperplastic polyps were collected from three males and five females. Two biopsies were collected from each patient, one corresponding to the polyp and one to healthy mucosa. Patient characteristics and details of endoscopic treatment performed are outlined in Table 34.Twelve Colonic Mucosa with Polyp (CMP) and corresponding Healthy Marginal Tissue (HMT) samples , retrieved from twelve hospitalized subjects, were used to evaluate the microbiota taxonomic profile by means of 16S rRNA gene sequencing analysis as described previouslyNext generation sequencing of these samples produced a total of 2,059,497 sequencing reads with an average of 85,812 reads per sample Table . SubsequRating of rarefaction curves based on the Chao1 biodiversity indexes calculated for the two subgroups of sequenced read pools showed that both curves tend to reach a plateau confirming the high accuracy of the performed 16S rRNA profiling analysis. Furthermore, average rarefaction curves revealed a different trend of bacterial richness of the gut microbiota of healthy samples compared to that found in the polyp sample group, i.e. higher level of HMT vs CMP samples Fig. . HoweverInspection of predicted taxonomic profiles at phylum level for all samples showed that Firmicutes represented the dominant phylum (average of 57.59% ± 10.48%) of the colonic mucosa samples, out-numbering the Bacteroidetes (average of 9.24% ± 8.01%) and Proteobacteria (average of 10.26% ± 8.19%) phyla.Nevertheless, no major shifts were observed in bacterial populations between healthy mucosa and Polyp-associated mucosa, although we did observe a moderate reduction of Actinobacteria (relative percentage difference of −13.43%) and Bacteroides (relative percentage difference of −1.02%) in CMP and a concomitant modest increase of Firmicutes (relative percentage difference of 2.84%) compared to HMT Fig. .Figure 140, which may include specific species important for the maintenance of an efficiently functioning gut ecosystem41  to 641% (Romboutsia), or a decrease ranging from −16% (Ruminococcaceae UCG-002) to −74% (Ruminococcus I) in HMT datasets compared to those obtained from CMP samples  in mucosa associated with polyps may represent a potential microbial indicator of a disease condition50.In order to further identify differences in microbiota composition between CMP and HMT patients, we focused on 30 taxa, which were shown to be present as the absolute change of >±0.1% or that displayed a significant variation in terms of relative abundance . In this context, these 30 taxa varied in terms of relative abundance, encompassing an increase ranging from 10% , Unclassified member (U. m.) of Peptostreptococcaceae family occurs with an increased relative abundance of 337.30% in HMT vs. CMP samples, while Peptoclostridium is totally absent in all analyzed CMP samples.In contrast, the higher presence of Romboutsia displayed the same trend in at least 80% of HMT samples, while all taxa trend of CMP were <75%. These results reinforce the notion that Romboutsia may play a key role in maintaining the health status of the host and render this taxon a very valuable candidate as biomarker of intestinal dysbiosis.In order to integrate and confirm the observed differences in relative abundance between HMT and CMP samples, we defined the trend (increase or decrease) for each bacterial taxa in healthy subjects versus polyp condition. Considering all patiens, we identified that only the genus 28. In this context, it is worth mentioning that previous studies on healthy patients or children with rectal bleeding indicated that fecal and mucosal samples belonging to the same individual displayed a very dissimilar microbial composition30. It has been reported that, based on different statistical analyses, fecal and mucosal samples cluster separately, at family and species taxonomic level30. To circumvent the issue of absence of data from polyp-associated biopsies, we decided to use human CRC datasets. In fact, as discussed above, CRC has been considered to represent a later stage of adenomas54. Consequently, we retrieved 293 samples from the four publically available CRC BioProjects33  and CRC-polyps Tissue (CpT) samples highlighted 38 genera with a p-value < 0.05 calculated thought Analysis of variance (ANOVA)  Fig. . FurtherCampylobacter  and Fusobacterium  compared to healthy samples  and Gemella 60. In contrast, HT samples showed higher abundance of ‘beneficial’ bacterial genera, as well as U. m. of Lachnospiraceae family and Faecalibacterium , Bacteroides , and Romboutsia  compared to CpT.Association of ANOVA and trend percentage analyses indicated that CpT samples had a higher abundance of genera belonging to Proteobacteria and Fusobacteria phyla, specifically les Fig. . InteresLachnospiraceae family, Lachnoclostridium, Faecalibacterium, Ruminiclostridium and Subdoligranulum, while disease-associated CpT samples showed high abundance of Campylobacter. These results reflect the assumption that genera belonging to Lachnospiraceae and Ruminococcaceae families may protect healthy subjects from intestinal disease61, such as through the production of short-chain fatty acids (SCFAs)62. In contrast, the higher abundance of bacteria belonging to Campylobacter genus may confirm its correlation with the development of gastrointestinal diseases64.Furthermore, focusing on genera with a taxonomic trend with a prevalence of 100% and a significant p-value (<0.05) calculated between HT and CpT Fig. , it was Overall, we found a correspondence between the trend that we retrieved in our HMT vs CMP samples, and that of the HT vs CRC samples, revealing a small number of microbial taxa that could be considered as bacterial biomarkers for early detection of tumor or, conversely, reflecting a healthy condition.Fusobacterium nucleatum with a formation and progression of colorectal cancer67 the relative abundances of this species were measured by qPCR in all 24 samples (HTM and CMP biopsies of 12 patients). Notably qPCR analyses displayed that this bacterium has been found in six patients  with a genome copy number ranging from 2.24E1 to 1.25E3 for the HTM samples, and a genome copy number range of 2.08E2 to 3.26E3 for CMP biopsies  higher for samples 5, 6, 7 and 9 in the CMP vs. HMT mucosa for each patient. Interestingly, sample 13, showed a total load of Fusobacterium nucleatum higher respect all other samples, i.e., a mean value of 1.02E3 for the HMT as compared to a mean value of 2.37E3 for the CMP  (Fig. 66, but may also serve as a putative valuable microbial biomarker for early diagnosis of malignant displacement of polyps.Since several studies associated the presence of 68.Recent findings have shown that intestinal diseases are correlated with microbiota alterations such as in the case of ulcerative colitis67, but previous in vivo murine based-analyses highlight the potential role of many individual bacteria to promote carcinogenesis69.The contribution of gastrointestinal microbiota in the progression and development of colorectal cancer is complex and not fully understoodRomboutsia genus is more abundant in healthy compared to polyp-associated tissue. Remarkably, the relative abundance of Romboutsia in CRC tissue was found to be drastically reduced. Thus, we may argue that the absence of this microbial genus is a first indicator of an alteration of the mucosa. Remarkably, this microbial taxon has already been observed for other gut-associated diseases51.In the current pilot study, we compared bacterial population in mucosa samples of patients with polyps or CRC, versus healthy mucosa, showing that the Peptoclostridium genus was undetectable in all CMP sample tested, while analysis of the HMT samples revealed a relatively high number of U. m. of Peptostreptococcaceae family.Interestingly, the 33. Our metagenomic analysis revealed that CpT samples had a higher abundance of the Fusobacteria phylum compared to healthy samples. Interestingly, the Fusobacterium genus is often involved in tumor progression70.Our findings were in line with the findings of a meta-analysis in which we compared the CMP samples with CRC samples from publically available metagenomic datasetsLachnospiraceae family, Lachnoclostridium, Faecalibacterium, Ruminiclostridium and Subdoligranulum, while CpT samples showed high abundance of Campylobacter. The higher abundance of bacteria belonging to the Campylobacter genus in CRC mucosa, as well as polyp-associated samples reinforces its correlation with the development of intestinal pathologies64. On the other hand, genera belonging to Lachnospiraceae and Ruminococcaceae families appear to be correlated to beneficial effects on the host as described previously61.At genus level, we identified five taxa characteristic of HT and one taxon typical of CpT subjects. In detail, healthy samples were shown to contain a high abundance of U. m. of 72. The identification of microbial players that are distinctly altered in their abundance in CRC-associated samples may allow early diagnosis of malignant transformation as well as the identification of protective bacteria missing in polyps or CRC tissue.Gut microbiota include a very complex array of microorganisms and its compositional changes have been shown to impact on human health statusIn conclusion, the data obtained in this pilot study have allowed us to identify possible microbial biomarkers associated with polyps and/or CRC. However, such findings need further validation by analysis of a larger number of samples, in order to further substantiate and fully characterize such potential microbial biomarkers of CRC development, as well as beneficial bacteria, and to identify their functions and interactions with the host.Supplementary filesTable S3


Background: Previous studies showed that confocal laser endomicroscopy (CLE) images of brain tumors acquired by a first-generation (Gen1) CLE system using fluorescein sodium (FNa) contrast yielded a diagnostic accuracy similar to frozen surgical sections and histologic analysis. We investigated performance improvements of a second-generation (Gen2) CLE system designed specifically for neurosurgical use.Methods: Rodent glioma models were used for in vivo and rapid ex vivo CLE imaging. FNa and 5-aminolevulinic acid were used as contrast agents. Gen1 and Gen2 CLE images were compared to distinguish cytoarchitectural features of tumor mass and margin and surrounding and normal brain regions. We assessed imaging parameters  and evaluated optimal values for better neurosurgical imaging performance with Gen2.Results: Efficacy of Gen1 and Gen2 was similar in identifying normal brain tissue, vasculature, and tumor cells in masses or at margins. Gen2 had smaller field of view, but higher image resolution, and sharper, clearer images. Other advantages of the Gen2 were auto-brightness correction, user interface, image metadata handling, and image transfer. CLE imaging with FNa allowed identification of nuclear and cytoplasmic contours in tumor cells. Injection of higher dosages of FNa (20 and 40 mg/kg vs. 0.1–8 mg/kg) resulted in better image clarity and structural identification. When used with 5-aminolevulinic acid, CLE was not able to detect individual glioma cells labeled with protoporphyrin IX, but overall fluorescence intensity was higher (p < 0.01) than in the normal hemisphere. Gen2 Z-stack imaging allowed a unique 3D image volume presentation through the focal depth.Conclusion: Compared with Gen1, advantages of Gen2 CLE included a more responsive and intuitive user interface, collection of metadata with each image, automatic Z-stack imaging, sharper images, and a sterile sheath. Shortcomings of Gen2 were a slightly slower maximal imaging speed and smaller field of view. Optimal Gen2 imaging parameters to visualize brain tumor cytoarchitecture with FNa as a fluorescent contrast were defined to aid further neurosurgical clinical in vivo and rapid ex vivo use. Further validation of the Gen2 CLE for microscopic visualization and diagnosis of brain tumors is ongoing. Although this technique is considered to be the “gold standard” for establishing a histopathologic diagnosis, it entails a number of significant limitations. These limitations include the time required for transferring, processing, and interpreting the tissue ; the prePrevious studies using a CLE system originally designed for nonneurosurgical use  showed that a blinded review of CLE imaging of brain gliomas and meningiomas by a neuropathologist yielded an accuracy rate of 92.9%, similar to those previously reported with frozen-section analysis , 7–9. InVarious CLE devices have been developed; the proximal scanning fiber optic CLE , 20 and in vivo investigation of distal scanning CLE in human and animal model brain tumors were conducted using the Gen1 device, which had several performance characteristics that limited its use in neurosurgery. These included lack of a sterile attachment cover sheath for the imaging probe, a different design and function of the handheld probe that was not optimal for the neurosurgeon's usual hand position, and nonoptimal imaging processing and display , the primary fluorophore with which they were designed to work, and with 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence. Although the fluorescence signal from PpIX is not within the optimal detection range for these CLE systems, 5-ALA is also of interest in brain tumor surgery, especially for use in invasive gliomas, and our preliminary studies suggested that the PpIX signal may be faintly detected with the CLE systems. This study aimed to expand on the current literature related to CLE for brain tumor imaging, including further investigating the relationship between FNa dosage and image quality, the ability to differentiate cellular structures with CLE and FNa, and the utility of both generations of CLE with 5-ALA for the detection of tumor tissue.Guide for the Care and Use of Laboratory Animals and with approval from the Institutional Animal Care and Use Committee of the Barrow Neurological Institute and St. Joseph's Hospital and Medical Center, Phoenix, Arizona. Animals were maintained under approved veterinary care in the vivarium of St. Joseph's Hospital and Medical Center.All animal investigations were performed according to the guidelines outlined by the National Institutes of Health ex vivo CLE analysis. The same samples were then sent for routine histologic processing and review by a neuropathologist. All patients gave voluntary informed consent as a part of a study protocol approved by the Institutional Review Board of the Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, Phoenix, Arizona. A flowchart describing the study protocol is presented in Patient tissues used for this project were acquired from a prospective ongoing brain tumor clinical study. Patients with preoperative diagnoses of brain masses requiring surgical removal and for whom the decision was to use the assistance of fluorescence surgical guidance with FNa were prospectively enrolled. Extra biopsies from tumor tissue that would have to be safely removed during the normal course of the surgery were used. De-identified samples were placed on a wet telfa and submitted for immediate c−2J/J mice  weighing 20 g (mean) were anesthetized and placed in a small-animal stereotactic head frame. Glioma models were created according to the previously described protocol  were infused 2 mm lateral and 2 mm posterior to bregma at a depth of 2.5 mm from the brain surface through a bur hole at the targeted brain location after the syringe needle was withdrawn 0.5 mm to a total depth of 2 mm below the surface of the brain to create a 0.5 mm pocket for the cells. The 2-μL cell suspension was infused using a controllable microinjector (0.67 μL/min × 3 min with the needle in place for 2 min afterward) -Tyrprotocol . Brieflyterward) .in vivo imaging system (PerkinElmer). Bioluminescence images were examined and quantified in Living Image 4.3 software (PerkinElmer) , anesthetized using isoflurane in a 37°C chamber, and glioma growth was confirmed using bioluminescence detection in the IVIS Spectrum inElmer) .n = 4), 1 (n = 7), 2 (n = 2), 5 (n = 7), 8 (n = 5), 20 (n = 6), and 40 (n = 6) mg/kg. These doses approximate the doses of FNa that can be used in humans, as we have previously used 5 mg/kg IV in clinical study. The 5-ALA  was injected intraperitoneally 2 h before surgery at a dose of 5 mg dissolved in 200 μL of normal saline (n = 5). Chosen dosages are similar to those previously used in comparable animal studies with 5-ALA  was used to visualize the exposed surfaces of both cerebral hemispheres. Brain tumors were identified macroscopically in the right hemisphere of all mice, and the CLE scanning was performed. After imaging was performed, the anesthetized animals were euthanized according to protocol guidelines. Brains were extracted, sliced fresh coronally (1 mm in thickness) through the center of the tumor in a mouse brain slicer matrix , and imaged rapidly n = 37), 5-ALA (n = 5), or no dye injection (n = 4 control). This comparison was completed initially using the Gen1 CLE in a designated area. Once adequate images were acquired, the Gen2 CLE was used to take further images at the same area. All scanning was performed in vivo and then ex vivo within the 2-h interval alternating Gen1 and Gen2 confocal microscopes targeting multiple regions of interest. Further testing included evaluation of Gen2 imaging with different available filters (green bandpass or green longpass and red longpass) and with various gain, laser intensity, brightness, acquisition speed, and zoom settings controlling for image quality and noise levels. In 2 animals, topical acridine orange was used for nuclear staining as described elsewhere  and Gen2 CLE  were compared in terms of intraoperative fluorescence visualization using either FNa .The benchtop laser scanning microscopy (LSM) imaging  was performed with a C-Apochromat 40×/1.20 W Korr M27 objective. Sliced fresh brain samples were imaged on 35-mm glass-bottom Petri dishes . For PpIX visualization, the excitation and detection wavelengths used were 405 and 635–750 nm, respectively. Additionally, topical staining was performed with acridine orange (AO), acriflavine (AF), Hoechst 33342 , and 4′6-diamidino-2-phenylindole (DAPI). The excitation wavelength was 488 nm for AO, AF, or FNa, and 405 nm for DAPI and Hoechst; and the detection wavelength was 493–625 nm for AO, AF, or FNa, and 410–585 nm for DAPI and Hoechst. Examples of imaging modalities are shown in t-test, Mann-Whitney U test, and Kruskal-Wallis analysis of variance (ANOVA) with significance set at p < 0.05 were used to establish whether differences were statistically significant, and the Spearman R-value was used to assess correlations.Imaging analysis and cell and nuclei size measurements were performed in FIJI software (open source software) . StatistTo produce clear images, several parameters were controlled in both systems: gain, brightness level, laser power, scanning speed, imaging depth, and Z-stack thickness (Gen2 only). Because many imaging parameters were similar between Gen1 and Gen2 CLE, salient results are presented of observations for Gen2, and are specifically mentioned when differences were observed with Gen1. Gen2 imaging parameters are summarized in The field of view (FOV) of the Gen2  was half the size of the Gen1 image . Gen2 images were essentially twice the resolution. Gen2 images appear sharper, but upon magnification, they have a grainier appearance compared with Gen1 images. This graininess is due to the raw image TIFF format of Gen2 images, compared to compressed and smoothing JPG formatting of Gen1 images.Specimens with grossly average fluorescence brightness produced clear CLE images on Gen2 gain setting of 2,400 . When th× 512-pixel image and 1.2 s per frame for a 1,024 × 1,024-pixel image. The scanning speed for Gen2 ranged from 1.3 s per frame for the highest quality imaging to 0.27 s per frame for the lowest quality imaging. Despite the difference in scanning speed, all images were recorded and stored as 1,920 × 1,080-pixel red, green, blue (RGB) TIFF files.Scanning speed was critically important for time series imaging. Comparison of imaging speed recorded for both generations of CLEs is presented in Optimal imaging depth with FNa was within the first few microns in depth from the surface of the imaged tissue. The CLE Z-stack function (Gen2 only) resulted in a series of images, the first of which was a central image from the current position on the Z axis. A series of images were acquired starting from the surface and ending at the deepest position, with the Z-step image thickness selection to be between 3 and 20 μm. The interval of the scanning depth could be chosen by selecting two offset positions. The maximal range was obtained at the offsets from −47 to +50, which resulted in a 97-μm thick Z-stack. The number of images per Z-stack was limited by the size of the Z-step (3 μm minimum), which for the 97-μm Z-stack resulted in 35 images. Higher ranges are not practically necessary, so the available CLE Z-stack range is sufficient for use in human brain tissue. Previous experiments have shown that the maximal depth of brain tumor tissue (using FNa) for interrogation through Z-stack imaging is 36 μm in animal and 28 μm in human brain tissue .Probe movements during image capturing produced similar artifacts in both Gen1 and Gen2 CLEs. Although free-hand probe positioning yielded informative images, remaining in a steady position for an optimal Z-stack was difficult for image series acquisition. A semiflexible surgical probe holder was used to decrease movement artifacts.in vivo imaging, which is not possible on fixed tissues. In contrast to the static hematoxylin and eosin (H&E) slide, in vivo CLE revealed histologic features of the living tumor cells and their behavior. In animal gliomas, many tumor cells were actually moving independently, squeezing and pushing each other, captivated by the stream of oozing blood. Such tumor cells were flowing independent of the tumor core, especially when they were at the border of the brain slice. When imaged in vivo, GL261 glioma cells were actually not tightly connected to one another, as they appear on H&E slides, although they grow as relatively solid tumors. CLE imaging revealed pericellular spaces that were filled with FNa, which were not previously visualized well on H&E stained slides.Time series imaging (in both Gen1 and Gen2) allowed observation of blood cell movements inside and outside the vessels . Tumor cIn no case was the tumor location uncertain based on CLE images after successful intravenous FNa injection. When imaging normal brain without tumor using CLE, FNa could be seen inside blood vessels, and occasionally leaking from vessels because of damage from surgery. Red blood cells could be visualized in these vessels as dark moving objects. No FNa was seen in other areas of the normal brain, other than intravascularly, except for sparse autofluorescent cells. Ultimately, the area of the brain away from the vessels appeared similar to the brain when FNa had not been injected, with both generally lacking fluorescence, except for rare autofluorescent cells and with limited fluorescence . After FAbnormal tumor vasculature was seen in several tumor biopsy spots . A gradip = 0.10) from those measured on FNa-contrasted CLE brain tumor images (11.6 ± 2.5 μm), suggesting validity of identification of nuclei on the FNa-based confocal images. The average nuclei size was significantly smaller (p < 0.01) than the cell sizes (20.8 ± 3.9 μm)  and Hoechst  showed images similar to CLE grayscale images and provided additional confirmation regarding the dimensions of nuclei and cells observed with CLE (Average nuclei sizes measured on AO-stained tumor images (11.9 ± 2.5 μm) were not different ( 3.9 μm) . Benchtowith CLE .in vivo and rapidly counterstained ex vivo with Hoechst suggested that FNa signal “followed” invasions of the tumor cells in the normal brain. It confirmed that many small cell contours visible with FNa are actually anuclear red blood cells. A gradient of FNa distribution from the tumor into the normal brain was also visible with the higher FOV confocal microscopy.The tumor margin was visible in the GL261 model with both CLEs. The tumor border region appeared as a nonuniform delineation between the area with low FNa signal that did not contain atypical cellular features  and an area with high FNa signal containing silhouettes of the abnormal tumor cells . BenchtoWhen normal brain tissue was injured, leaking FNa signal was visible. Brain regions of injury without tumor could be differentiated from regions of brain with tumor on CLE images by small and nonvariable red blood cells being present, rather than large, numerous, and variable tumor cells .R = 0.5, p < 0.05)  . The bes < 0.05) ; 8 mg/kg < 0.05) ; 7.7 mg/ < 0.05) ]. Based p < 0.01)  .Imaging using green bandpass or green longpass filters was compared on both systems to visualize FNa in mouse glioma models. Images acquired with the longpass filter appeared brighter. At the same time, quantitative analysis showed that the bandpass filter resulted in significantly better-contrasted images . With auWhen FNa was used, images were acquired mainly with a gain at the mid-position . Very bright fluorescent specimens (10–40 mg/kg FNa) required gain adjustments on only rare occasions, as control was usually possible for oversaturated pixels and image quality by lowering the laser power and brightness .For FNa imaging, the laser power was usually at 50% (500 μm). In locations that produced dark images (low FNa or bleached areas), increasing the laser intensity improved image quality and brightness to some extent, but further increases in laser power did not result in quality improvement. The functioning of laser power control was not different between Gen1 and Gen2 systems. Overall, grossly bright fluorescent samples resulted in excellent contrast and quality at 50% laser power and gain position in the midpoint range  .The autobrightness function auto-adjusts the brightness of the image on the fly during continuous imaging based on the brightness histogram of the precedent image, independently from the gain and laser power settings. With one exception, the autobrightness function of the Gen2 was advantageous for rapid image and optimal brightness display, contrasting with the Gen1 that lacked this function and required frequent manual adjustments. The exception occurred when the image had abnormally bright artifacts. In such situations, the autobrightness function decreased the overall image brightness, with the FNa signal in the tumor interstitium becoming low and barely detectable—this was observed both qualitatively in real time and quantitatively during post-processing. In such situations, manual brightness setup was used to obtain better overall image quality.When the autobrightness function was on, the increase in gain resulted in a proportional decrease in automatic brightness level. Brightness values can be indicative of noise level. Brightness levels below 80% were the most optimal with less black noise, compared to a brightness level of 80% and above. The brightness values were maintained below 80% to keep the noise levels low .Background noise levels were further studied to assess the effects of Gen2 imaging parameters on image quality. Imaging of normal brain with various manually set brightness levels revealed that mean image intensity and standard deviation of the individual pixels had a positive correlation with brightness level . At a brMost images were acquired at a speed setting of 1080 pixels (~1.26 seconds per frame), which resulted in the best image quality . Faster The Gen2 CLE system is compatible with the specific demands of neurosurgical use. With the disposable sterile sheath that includes a coverslip, the Gen2 CLE can now be safely used on the human brain during surgery without the need for a complex sterilization procedure after surgery, as was the case with the Gen1 system . The ergMultiple imaging parameters can be adjusted in both Gen1 and Gen2 systems making them flexible and capable of detecting a single-channel fluorescent signal over a broad range of intensity values. Gen2 is more adjustable, as it allows for changing most parameters rapidly during scanning, while with the Gen1, the user is required to create a new imaging session using the operating software interface to change the gain or filter. Another convenient advantage is that, unlike the Gen1, the Gen2 imaging parameters are recorded as part of the file properties. Having access to these parameters is important for quantitative analysis and comparison among various images, especially to evaluate fluorophore signal intensity in the image, which correlates with the concentration of the fluorophore in the sample. For FNa-guided optical biopsy in brain tumors, such information may be used to assess the degree of blood-brain barrier disruption, to eliminate imaging artifacts, to identify potential causes of suboptimal images, or to improve images in post-processing.While continuous imaging occurs at a chosen speed in both CLE systems, the recording options are different. Both systems have the option to record a single still image and time series, while the Gen2 can also automatically record Z-stacks. Still image recordings were used when the sample quality was perfect and when there was no concern about having only a few selected imaging frames recorded while interrogating the tissue. However, in most cases, continuous recording was preferred and advantageous, which is discussed later. Finding the exact same position again is nearly impossible with a CLE system due to the minute dimensions on the order of fractions of a millimeter , contact probe design, and pliable nature of unfixed brain tissue. Thus, there was always a concern about not being able to return the CLE to the previous imaging plane. Therefore, still recordings were used less frequently than Z-stacks or time series. Incorporation and registration of the probe into an image-guided surgery navigation system would allow improvement in reassessing tissue imaging location.A Z-stack is a series of images taken in rapid succession at different depths and a constant position . The “Z-It is also worth mentioning that the size of objects becomes larger when they are imaged out of focus and deeper within the tissue. This could potentially make small red blood cells look larger than they are, thus imposing a minor potential for misdiagnosing small cells as tumor cells.Time series (or “cine”-series) recordings were used extensively for post-processing and creation of video loops. Continuous nonstop recording during Gen1 or Gen2 CLE interrogation of the sample was helpful compared with “on-demand” snapshot recording because with the latter technique, some locations were bleached out before the image was recorded. Gradual photobleaching was observed, but with practice, minimizing the scan time avoided this problem. We did not investigate whether laser interrogation results in tissue damage. Although tissue damage is unlikely, we are conducting an ongoing investigation of the potential phototoxicity of CLE scanning. Additionally, minute tissue shifts changed the observed image before taking a picture, and the probe often could not be returned to the desired location because of the pliable nature of unfixed brain tissue. Z-position, brightness, and laser power can be adjusted during the time series imaging to record optimal quality images.In vivo CLE demonstrated dynamics of the glioma environment for the first time, something that is not possible in fixed brain tissue.Notably, time series imaging revealed a “movement” feature of the tissue and supplemented visual information gained from still images. This feature is useful in several aspects for tissue histologic characteristics and CLE image interpretation. Primarily, differentiation of freely moving red blood cells from other moving or stationary cells becomes much easier and thus fosters the diagnostic accuracy of image and tissue interpretation. Additionally, it becomes easier to differentiate real signal from noise. By viewing time series, differentiation is possible between what is meaningful and what is merely stochastic noise. Such judgment is usually difficult on still images because of the nature of FNa. FNa is a nonspecific dye that is not bound to any structure, but rather provides a bright background  on which to observe, contrast, and differentiate structures. Noise is an inherent limitation of FNa. in vivo tracking of blood flow in vessels. Tracking of individual blood cells is not always possible because of fast movements that require high-speed scanning confocal imaging systems, such as spinning disk confocal microscopes  is not optimally tuned for excitation of PpIX, and CLE images did not show specific information. CLE images did not reproduce findings when LSM with the appropriate excitation laser was used to image PpIX in the same mouse glioma model .While it was possible to differentiate glioma from normal brain tissue by comparing overall average image intensity, this charcteristic was not deemed useful. Similar “fluorescence intensity” sampling has been shown previously using spectrophotometric methods –43. Of nThe first parameter to consider during CLE imaging is the filter. Between the two types of green filters , the longpass filter produced brighter images. Although images with such longpass filters may look more pleasing, they include nonspecific light from the background that attenuates FNa-induced contrast. Ultimately, the bandpass filter produced images with higher contrast and was preferable for obtaining better quality and interpretable images.Unlike in the benchtop confocal microscope, where the gain is fine-tuned during the constant image capturing, with the Gen2 CLE the gain control is located in a separate window and is not available for rapid adjustments on the fly. However, adjustments of brightness and laser power were enough to obtain quality images in most cases without the need to adjust the gain. Only in a few very bright or very dim specimens was there a need to adjust the gain for optimal image quality.Laser power at mid-range (50% or 500 μm) was generally optimal for imaging, with increasing intensity providing some improvements in image quality. Lack of significant improvement in image quality with increasing laser power is mainly explained by the nature of FNa, which, after extravasation, is diffused through almost all tissue thickness. Therefore, increased excitation results in increased emission in all image regions, including background and cells. Moreover, autofluorescence and noise begin to be visible at higher laser settings. This association of laser power and gain with contrast and autofluorescence is shown schematically in The brightness is the built-in adjustable proprietary setting parameter used during CLE imaging. It has a range of settings from 0 to 100% and, unlike the gain and laser power settings, could be adjusted during the imaging. Brightness was a primary setting of both CLE systems. Autobrightness is a new Gen2 function that saved time and made imaging process faster. Images acquired in autobrightness mode had good quality, comparable with the quality of images acquired at manually set brightness levels. Additionally, an optimal brightness level, or even pseudocoloring, can be applied later during post-processing .The speed of image acquisition was inversely correlated with image quality. This relationship illustrates the tradeoff of the convenience of using high-speed imaging to locate informative structures quickly within a large area with little detail versus taking more time to look at higher resolution images in greater detail. For areas of high contrast, such as intact vessels with FNa within the normal brain, or pronounced hypercellular tumor areas with bright FNa nearby, high-speed imaging was able to resolve structures well. However, in most cases, fast scanning was not able to resolve red blood cells and fine cellular details.Although the FOV of Gen2 images is about half the size of Gen1 images, it was sufficient to obtain similar image interpretation for corresponding histological structures. Subjective image quality appraisal between the systems is dependent on the display size. The Gen2 is capable of increasing optical zoom by a factor of two, but this results in a corresponding decrease in FOV size, which is not particularly helpful for the identification of cell or tissue architecture using FNa imaging. However, a zoom function might be advantageous for dyes such as AO or AF . Unlike Because of the inherent cellular composition of different tumor types, including cellular and regional tissue architectural heterogeneity, CLE system functionality may benefit the surgeon or pathologist by use of a lower resolution and larger FOV image assessment. This situation would allow a closer match to the lower power magnification of a conventional pathology microscope, which would allow the surgeon a larger FOV for scanning the tissue surface . HoweverFNa doses in animals are higher than human doses used in brain tumor surgery either with epifluorescence guidance using the operating microscope with a dedicated filter [2–20 mg/kg ] or prevThe current study was performed using a mouse GL261 glioma model. Histologic patterns and the feasibility of FNa imaging with CLE in other experimental tumor models were not addressed in this project. However, clinical experience  and the in vivo) and human tumor samples (ex vivo) were employed to discern and describe optimal imaging parameters for microscopic histologic visualization with a clinical-grade CLE system and intravenous FNa as a contrast fluorophore. Visualized histologic patterns and pixel intensity values were of comparable quality on Gen1 and Gen2 systems, if not improved with the Gen2 system. The Gen2 had a limitation of a smaller FOV, while having higher resolution, clearer images, and a more responsive user interface, including an autobrightness function and volumetric Z-stack image acquisition that enrich diagnostic possibilities and imaging functionality.Functionality and parameters were assessed for a CLE system (Gen2) designed specifically to perform to the demands and progress of neurosurgery to potentially assist in managing invasive brain tumors in a preclinical setting. Experimental brain tumor models . Although CLE has been used routinely in other medical and surgical specialties, such as for gastrointestinal diagnosis, neurosurgery is in its infancy with respect to CLE use. CLE technology does appear to be responding to the demands of neurosurgeons and neuropathologists for practical introduction into the operating room as an aid in surgery for invasive brain tumors after nearly a decade of developmental exploration and testing.Concepts for CLE use that must be considered are those that will make the system meaningful in a surgical setting. CLE usefulness to neurosurgery must be proven by being shown to provide a measurable ability to discriminate and guide removal of tumor invasion, thereby extending or optimizing the resection. As much as CLE may provide precise information on where to biopsy or remove tissue, it may be just as crucially used to inform the neurosurgeon where to stop the resection. Further clinical investigation of the diagnostic value of the Gen2 CLE system in fluorescence-guided brain tumor surgery is ongoing.All patients gave voluntary informed consent as a part of a study protocol approved by the Institutional Review Board of the Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, Phoenix, Arizona.EB, PN, and MP conception and design. EB, EM, AC, AP, and CC acquisition of data, experiments. EB, EM, NM, VB, JE, PN, and MP analysis and interpretation of data. MP, DH, and AS resources, animal model development. MP, PN, and ML supervision. EB, EM, and CC drafting the article. EB, AC, AP, DH, NM, VB, AS, ML, JE, PN, and MP revising article critically for important intellectual content. All authors final approval of the manuscript.Carl Zeiss AG had no influence on project design, image acquisition, data interpretation and analysis, or manuscript preparation, and has no agreements for marketing or financial incentive with the Barrow Neurological Institute for the technology in this study. PN has a consultant agreement with Carl Zeiss AG. EB and JE have received travel support from Carl Zeiss AG for attendance at a scientific conference. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Salmonella in foods owing to its simplicity, rapidity, and robustness. This multi-laboratory validation (MLV) study aimed to validate a Salmonella LAMP-based method against the United States Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 5 Salmonella reference method in a representative animal food matrix (dry dog food). Fourteen independent collaborators from seven laboratories in the United States and Canada participated in the study. Each collaborator received two sets of 24 blind-coded dry dog food samples  and initiated the testing on the same day. The MLV study used an unpaired design where different test portions were analyzed by the LAMP and BAM methods using different preenrichment protocols (buffered peptone water for LAMP and lactose broth for BAM). All LAMP samples were confirmed by culture using the BAM method. BAM samples were also tested by LAMP following lactose broth preenrichment (paired samples). Statistical analysis was carried out by the probability of detection (POD) per AOAC guidelines and by a random intercept logistic regression model. Overall, no significant differences in POD between the Salmonella LAMP and BAM methods were observed with either unpaired or paired samples, indicating the methods were comparable. LAMP testing following preenrichment in buffered peptone water or lactose broth also resulted in insignificant POD differences (P > 0.05). The MLV study strongly supports the utility and applicability of this rapid and reliable LAMP method in routine regulatory screening of Salmonella in animal food.Loop-mediated isothermal amplification (LAMP) has gained wide popularity in the detection of  Salmonella is a ubiquitous human and animal pathogen, with human outbreak-related illnesses broadly attributed to multiple food categories of plant and animal origins  is also well documented  tested and was capable of detecting <1 CFU/25 g in animal food   against the FDA BAM Chapter 5 Salmonella (reference method) in a representative animal food matrix (dry dog food) for future incorporation into the FDA’s compendium of analytical laboratory methods for food and feed safety. MLV participants included 14 independent collaborators from seven FDA, state, and academic laboratories in the United States and Canada. The MLV study also compared the effects of two preenrichment buffers used in LAMP and BAM on Salmonella detection in animal food.This MLV collaborative study aimed to validate the Fourteen independent collaborators (or independent teams), two each from seven FDA, state, and academic laboratories participated in the MLV. The FDA laboratories were from the Office of Regulatory Science at FDA’s Center for Food Safety and Applied Nutrition, and Northeast Food and Feed Laboratory, Pacific Southwest Food and Feed Laboratory, and San Francisco Laboratory at FDA’s Office of Regulatory Affairs. Other participants were the Food and Drug Laboratory Branch at California Department of Public Health, Office of Indiana State Chemist, and Animal Health Laboratory at University of Guelph .Salmonella by the BAM Chapter 5 reference method  to confirm negative results.Inoculated samples were prepared by Q Laboratories . Briefly, bulk dry dog food in kibble form was obtained from a local pet store and screened for the presence of e method  and the Salmonella was separated into two sets and inoculated with a lyophilized culture of Salmonella enterica serovar Infantis ATCC 51741 at two target levels: a high level of ca. 2 to 5 CFU/25 g test portion and a low level of ca. 0.2 to 2 CFU/25 g test portion. An uninoculated control set (0 CFU/25 g test portion) was also included. After inoculation, the three sets of bulk samples were homogenized and held at room temperature for 2 weeks for aging to simulate storage. Replicate samples  from the two inoculated sets were evaluated at three time points  by BAM and iQ-Check methods to verify the target levels and homogeneity.Dry dog food confirmed negative for On the day of shipment, a five-tube three-level most probable number (MPN) analysis was performed by evaluating 5 × 50 g replicates, 5 × 25 g replicates, and 5 × 10 g replicates to obtain final inoculation levels in the dry dog food sample sets. The samples were apportioned (25 g each), packaged, labeled , and shipped overnight to the seven participating laboratories. For each laboratory, four sets of eight samples from each of the three inoculation levels were sent, along with two sets of samples from the uninoculated control set reserved for aerobic plate count (APC).All collaborators (or teams) began testing on the same day. APC was performed by the pour plate method according to the FDA BAM Chapter 3  or usingg for 1 min to remove large particles followed by another centrifugation at 16,000 × g for 2 min. The pellets were suspended in 100 μl of PrepMan Ultra reagent, heated at 100°C for 10 min, cooled to room temperature, and centrifuged again at 16,000 × g for 2 min. The supernatants (sample DNA extracts) were stored at -20°C until use.DNA extraction was performed by using the PrepMan Ultra sample preparation reagents . Briefly, aliquots (1 ml) of BPW or LB preenrichment cultures were first centrifuged at 900 × S. enterica Typhimurium ATCC 19585 [LT2] at 1.7 × 104 CFU/reaction) and no template control (molecular grade water) were included in each LAMP run. Briefly, the reagent mixture in a total volume of 25 μl contained 1× isothermal master mix ISO-001 , 1× primer mix , and 2 μl of sample DNA extract. The LAMP reaction was run at 65°C for 30 min followed by an annealing step from 98 to 80°C with 0.05°C decrement per second  were determined when fluorescence ratios reached the maximum value of the amplification rate curve  of LAMP products were obtained in the anneal derivative curve . Additional confirmation tests performed included VITEK 2 Gram-negative biochemical identification method , Bruker MALDI Gram-negative Biotyper method  (Procedures described in the BAM Chapter 5  were fol2017.09) , or real2017.09) .Salmonella isolates were recovered. LAMP samples with the correct mT (approximately 90°C) and maxT values between 5 and 30 min were considered positive. For this MLV, all LAMP testing results were reported as presumptive results (presumptive positive or presumptive negative). BAM and LAMP results for each contamination level (including uninoculated controls) were analyzed by using the probability of detection (POD) statistical model  for the uninoculated dry dog food controls. Salmonella MPNs obtained in the two inoculated sample sets, with a 95% confidence interval, were 0.65 MPN/25 g  for the low level and 3.01 MPN/25 g  for the high level. Two collaborators (9 and 10) mixed up sample bag sets among the four sets of samples received in their laboratory, resulting in uneven number (5–10) of samples tested per inoculation level. Nonetheless, the samples were not compromised as they were individually bagged and blindly coded, and their data were still included in the final statistical analysis for the MLV study. Data from another two collaborators (2 and 4) were excluded due to confirmed positive results among uninoculated controls. In total, there were 288 (12 collaborator × 24 samples/collaborator) data points each for LAMP and BAM in the final comparisons presented below, which include LAMP vs. BAM using unpaired samples, LAMP vs. BAM using paired samples, BPW vs. LB for use as LAMP preenrichment buffers, and BPW vs. LB for use as BAM preenrichment buffers.The average APC was 2.1 × 10Salmonella Infantis ATCC 51741 in 25 g dry dog food test portions by the LAMP alternative method versus the FDA BAM Chapter 5 reference method in an unpaired study design, i.e., different portions were analyzed by LAMP and BAM using different preenrichment buffers. For the uninoculated controls, collaborators 2, 4, and 5 had LAMP presumptive positive results with the rate as high as 75% for both collaborators 2 and 4. The single sample for collaborator 5 did not confirm positive by BAM culturing, but several samples for collaborators 2 and 4 did . Based on these results, data from collaborators 2 and 4 were excluded from the MLV. In addition, collaborator 1 had one LAMP presumptive negative sample confirming positive by BAM and collaborator 4 had one BAM sample testing positive by BAM. Fractional recovery  was obtained for the low inoculation level by all collaborators although 2, 4, 9, and 10 achieved that by only one method (LAMP presumptive or BAM reference). All high-level inoculated samples tested positive, regardless of the method used  produced fractional positive results (25–75%) and three uninoculated control samples were confirmed positive for Salmonella  compared to others (ca. 7 min) (data not shown), indicating the amount of target DNA in the sample DNA extracts was low. This may be attributed to low contamination levels and dead or injured cells in these samples, which failed to reach the detection limit of BAM even after enrichments. The samples were shipped without dry ice since pet food is usually stored and shipped at ambient temperature; however, this may have contributed to some of the variability observed in the study. Another possibility is there was cross-contamination introduced during DNA extraction or assay setup for LAMP. For the uninoculated controls, one false positive (collaborator 5) and one false negative  results were observed. The former had high maxT values (average of 14 min), while the latter was technically true negative  as it was an uninoculated control. Cross-contamination may have occurred when the two collaborators processed the samples for LAMP or BAM. Prior to this MLV, the LAMP assay has been extensively evaluated and high specificity (100% inclusivity and exclusivity) and high sensitivity  have been demonstrated  and no false negative results , we also compared the performance of LAMP and BAM using paired samples, i.e., the same test portions were analyzed by LAMP and BAM following LB preenrichment (LB-LAMP vs. BAM). One false positive (collaborator 13) and one false negative (collaborator 8) results were observed in low-level inoculated samples and one false positive was observed in one uninoculated sample . SimilarSalmonella detection in food  in raw ground beef and wet dog food  in ground beef, beef trim, ground turkey, chicken carcass rinses, bagged mixed lettuce, and fresh spinach . Comparing maxT values generated in this MLV for low- and high-level inoculated samples showed that BPW preenrichment generated maxT values on average 1.9 min and 1.6 min shorter than those using LB preenrichment, suggesting the amount of DNA was higher when BPW was used as the preenrichment buffer. A recent study evaluating the 3M MDA Salmonella and the ANSR (stands for amplified nucleic single temperature reaction) detection system for Salmonella  in egg products also showed that preenrichment in BPW improved the performance of both assays compared to LB . As a result, multiple confirmation methods besides serotyping were performed by participating laboratories, including VITEK 2, Bruker MALDI, and real-time qPCR, extending the time of sample testing by BAM to 2 weeks in contrast to 24 h by LAMP.It is worth noting that the In our single-laboratory validation study , five otSalmonella in animal food. As only LAMP-positive samples should continue with the isolation of Salmonella by the FDA BAM culture method, the LAMP method holds great potential to significantly reduce the time and labor and improve efficiency in animal food testing.In conclusion, the MLV study clearly demonstrated the utility and applicability of this rapid and reliable LAMP method in routine regulatory screening of All datasets generated for this study are included in the manuscript.BG, KD, QRY, TH, and RP contributed to design and coordination of the study. BG, KD, and QRY performed the data analysis and interpretation. SW contributed to statistical analysis. XD, LJH, GZ, YH, XL, KC, JD, KH, SL, RR, DR, AY, PP, CW, TE, DK, JS, DW, LH, LL, CL, QY, DS, and HC contributed to sample testing and coordination of the study in seven participating laboratories. SA and SY assisted with sample analysis in the coordinating laboratory. All authors reviewed the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A full range of near and intermediate vision has not been clinically evaluated for the Symfony extended depth of focus intraocular lens (EDOF IOL).  To evaluate the monocular range of near visual acuity with an EDOF IOL.  Retrospective case series.  Consecutive patients of a single surgeon from January 2017 through March 2018.  Phacoemulsification with implantation of an EDOF IOL.  Uncorrected distance visual acuity (UDVA), uncorrected near visual acuity (UNVA), corrected distance visual acuity (CDVA), distance-corrected near visual acuity (DCNVA), range of DCNVA, and optimal near focal length.  Seventy-six eyes of 48 patients  were included with a mean follow-up of 68 days. Mean values were as follows: logarithm of the minimum angle of resolution (logMAR) UDVA 0.02 ± 0.09, logMAR UNVA 0.12 ± 0.09 at a mean distance of 51 cm, logMAR CDVA −0.05 ± 0.07, logMAR DCNVA 0.08 ± 0.07 at a mean distance of 51 cm, and a spherical equivalent of −0.16 diopters ±0.35. Percentage of eyes achieving DCNVA of 20/30 were 84% at 36 cm, 92% at 41 cm, 99% at 51 cm, 93% at 61 cm, and 74% at 71 cm. DCNVA of 20/40 or better was achieved in nearly 100% of eyes over a range of 35 cm.  The Symfony EDOF IOL achieved excellent distance visual acuity while providing a 35 cm range of near visual acuity at levels useful for many tasks in nearly all patients. Presbyopia can be a significant factor affecting quality of life in the developed and developing world , 2. In 2Currently, the only EDOF IOL available in the United States is the Tecnis Symfony extended depth of focus IOL  , 6, whicA retrospective chart review was conducted for all patients undergoing phacoemulsification by a single surgeon (RTP) with implantation of a Symfony IOL (ZXR00) or Symfony toric IOL  from January 2017 through March 2018. Exclusion criteria included lack of follow-up beyond 21 days postoperatively, concurrent ocular conditions limiting best corrected distance visual acuity to less than or equal to 20/40 Snellen, visually significant posterior capsular opacification (PCO) requiring YAG laser capsulotomy within 4 months of phacoemulsification, or visually significant dysphotopsia requiring IOL exchange. Patients with prior corneal refractive surgery including radial keratotomy were not excluded. An institutional review board approval was obtained by the Houston Methodist Research Institute, Houston, TX.Phacoemulsification was performed using a standard microinvasive cataract surgery technique . Both maVisual acuity was reported at the first postoperative visit after phacoemulsification when eyes were considered stable . Near visual acuity was measured using a card on a phoropter rod with the patient's distance refraction placed in the phoropter.Primary endpoints were uncorrected distance visual acuity (UDVA), uncorrected near visual acuity (UNVA), corrected distance visual acuity (CDVA), distance-corrected near visual acuity (DCNVA), range of DCNVA measured at 36, 41, 51, 61, and 71 cm, UNVA optimal focal length, and DCNVA optimal focal length. These distances were based on the following factors: approximate reading focal lengths seen with traditional multifocal lenses in the same IOL platform used by the surgeon , average focal distance to desktop computer screens, and the maximal length of the phoropter near vision rod (28 inches or 71 cm). Secondary endpoints included incidence of posterior capsular opacification requiring YAG laser capsulotomy and incidence of lens exchange due to patient dissatisfaction.Data analysis was performed using Microsoft Excel 2016 . Snellen visual acuities were converted to logarithm of the minimal angle of resolution (logMAR) for calculation of mean and standard deviation values.A total of 100 eyes of 65 patients were identified that underwent phacoemulsification during the study period with implantation of a Symfony EDOF IOL. Twenty-four eyes of 18 patients were excluded based on the following criteria: lack of adequate follow-up (7 eyes), concurrent ocular conditions limiting DCVA to less than or equal to 20/40 (10 eyes), postoperative PCO requiring YAG capsulotomy (6 eyes), and intractable dysphotopsia requiring IOL exchange (1 eye). The remaining 76 eyes of 48 patients  met inclusion criteria. Sixty-nine (91%) of these eyes underwent FLACS, and 7 (9%) underwent concurrent trabectome surgery along with phacoemulsification. Sixty eyes (79%) were implanted with the Symfony IOL, and the remaining 16 eyes with the Symfony toric IOL. The mean age of patients was 68 years , and the mean follow-up was 68 days . Mean values for UDVA, UNVA, CDVA, DCNVA, spherical equivalent, and optimal focal distance for near vision are reported in Percentage of eyes achieving DCNVA of greater than or equal to 20/20, 20/25, 20/30, or 20/40 at the various near focal lengths tested from 36 to 71 cm are displayed in The range of DCNVA at levels of 20/30 or better and 20/40 or better is displayed in The Symfony extended depth of focus intraocular lens, like many multifocal intraocular lenses, relies on diffractive optics to focus light at different distances from the lens. Benchmark testing of this lens suggests that it can achieve a high level of visual acuity over a range of vision as opposed to providing a distinct near point of focus as with a traditional multifocal lens . To achiWe chose to evaluate monocular visual acuity results only, as we felt that the effect of binocular summation of visual input might lead to falsely elevated visual acuity outcomes. Furthermore, many of our patients are only eligible for implantation of this IOL in one eye since their contralateral eye does not have a visually significant cataract.This study demonstrated that patients achieved an excellent level of DCNVA at intermediate ranges of near vision. Optimal near focal point was at a range most compatible with computer distance and arm's length placement of near reading material, consistent with the clinical trial results of this IOL. Furthermore, this study demonstrated that patients achieved a satisfactory level of distance-corrected near visual acuity over an extended range of near vision, as close as 36 cm and as far as 71 cm in some eyes. At 36 and 71 cm, 34% and 30% of eyes, respectively, had a visual acuity of 20/25 or better. All patients demonstrated 25 cm of range of near vision at 20/40 or better, and 97% of eyes had the same level of acuity over the 35 cm range we tested. The FDA clinical trial results measured near and intermediate visual acuity up to the 20/40 level, beyond that, eyes were grouped in ranges of vision. We feel that 20/40 on a near card represents a size of print that most closely resembles daily reading tasks such as computer and tablet screen print, smartphones, and many magazines and books.This data set included some eyes with ocular comorbidities traditionally considered as relative contraindications for implantation with a MFIOL. We took this approach based on clinical understanding of the optics of this lens resulting in less diffractive loss of light than with traditional MFIOLs, early outcomes in patients with such comorbidities who underwent EDOF IOL implantation, and a thorough informed consent process explaining the risks, benefits, and alternatives to phacoemulsification with implantation of an EDOF IOL. These eyes are included in the data analysis. One patient had moderate primary open-angle glaucoma and a prior Crystalens IOL implanted elsewhere in the contralateral eye; as a result of her dissatisfaction with the contralateral eye and a strong desire for an extended range of vision, she requested an EDOF in her operative eye. Several eyes had prior myopic laser keratorefractive surgery, two of which had significant irregular astigmatism and higher order aberrations on corneal dual Scheimpflug with placido imaging. Limited study data are available on the results of EDOF implantation in postrefractive eyes –27, but Near vision was reported looking at a near card for acuity measurements in ideal lighting conditions, and the range of vision was estimated based on visual acuity measurements at specific numeric distances on the near phoropter rod. This methodology may have affected the study outcomes in two manners. It is possible that the range of DCNVA results might underestimate the true range of vision since measured distances were in steps of 10 cm (4 inches) and stopped at a distance of 71 cm (28 inches). On the other hand, the use of a near acuity card in ideal lighting conditions may overestimate real-world outcomes in patients who are reading text for an extended period, especially if surrounded by poor lighting. This of course would be mitigated in real-world situations through binocular summation in bilaterally implanted patients, which again was not the purpose of this study.Twenty-four percent of eyes during the study period were excluded due to the aforementioned reasons. One of those patients, who had a past ocular history of myopic LASIK, suffered severely from a dysphotopsia at day and night reported since her first postoperative day exam. This dysphotopsia was not described as “bright” or as “dark”, just an “edge of something like a contact lens” appearing constantly in her vision. Despite multiple discussions over multiple visits, the exact nature of her dysphotopsia was still unclear. As a result, her EDOF IOL was exchanged for a three-piece, monofocal, aspheric, silicone IOL placed in the capsular bag with reverse optic capture, which resolved her symptom completely.Of the remaining excluded eyes, seven were lost to follow-up due to lack of geographic proximity to the surgeon. Though follow-up data could not be obtained on these patients, they were very satisfied in their early postoperative period in our clinic. Ten eyes were excluded due to concurrent ocular conditions. Implantation of the EDOF IOL in these patients was planned only after careful and thorough discussion about the potential risks and potential lack of positive near vision outcomes in these patients' eyes. Concurrent conditions included Fuchs corneal dystrophy requiring endothelial keratoplasty, epiretinal membrane, optic atrophy, and high ametropic amblyopia. An additional six eyes were excluded due to early significant posterior capsular opacification affecting visual acuity and requiring YAG laser capsulotomy within four months of their surgery. This represented 6% of the original cohort of patients, all of whom underwent FLACS, as did most of the study patients, and an incidence consistent with other studies of early PCO development .This study was not designed to capture side effects or overall satisfaction rates of patients undergoing phacoemulsification with an EDOF IOL since detailed questionnaires were not administered asking about such symptoms as glare and haloes. It can only be stated that one patient suffered a significant dysphotopsia of uncertain etiology  requiring a lens exchange. Nevertheless, a review of all clinic notes revealed that no patient meeting inclusion criteria was dissatisfied, and though many exhibited early dysphotopsias such as glare, only two patients spontaneously reported persistent moderate symptoms on their final postoperative visit during the study period. The first patient had a history of prior myopic photorefractive keratectomy (PRK) and experienced haloes. The second patient perceived glare indoors and outdoors, which were improving. None of these patients were prevented from driving at night due to their dysphotopsias. On the contrary, an interesting observation was that one study patient had undergone a monofocal lens implant with her cataract surgery in the contralateral eye two weeks prior to the study eye, and due to expected but unsatisfactory lack of near vision, regretted her decision and therefore opted for the EDOF lens in her second eye. She functioned well without glasses for many tasks but did require reading glasses for extended reading and small print.This study is limited by its retrospective nature and the lack of a control group. However, all surgeries were performed by one surgeon and techniques for measuring visual outcomes in the clinic were standardized between visits and between technicians. We therefore feel that the retrospective design does not significantly limit the strength of the visual outcomes reported here. These results are relevant to demonstrating clinical outcomes of phacoemulsification with an EDOF IOL, especially since this data set included eyes that many would consider not ideal candidates for MFIOL implantation due to past ocular history.This study demonstrates that the Symfony EDOF IOL achieves an excellent level of distance visual acuity while providing 35 cm of range of near visual acuity at a level useful for a variety of near visual tasks in nearly all patients.


Dental pulp stem cells, which are primarily derived from the pulp tissues of human teeth, have rarely been obtained from natal teeth. This study investigated the stem cell and differentiation markers of the dental pulp of natal teeth using immunohistochemistry.The pulp tissue from extracted natal teeth (n = 2) of a 20-day-old healthy male was examined for immunohistochemical expression of stem cell (Oct-4 and SOX 2) and differentiation markers .The pulp tissue of the natal teeth expressed immunopositivity for nestin, CD 44 and SOX2.Natal teeth, if preserved properly, could serve as sources of dental pulp stem cells that are an improvement on deciduous teeth. Natal teeth are present at birth. They are usually removed prophylactically to avoid the complications such as aspiration, ulcerations on the tongue and injuries to the mother during feeding. To study discarded natal teeth as a potential stem cell source, immunohistochemistry was used to check for the expression of stem cells and markers on the dental pulp of the natal teeth. This was compared with the stem cell potential of deciduous teeth. Natal teeth exhibited higher immunohistochemical expression, which suggests that discarded tissues like natal teeth are a potential source for stem cells. The first tooth erupts in the human oral cavity at 6 months of age. However, an occasional entity termed ‘natal teeth’ may be present in some babies at birth. When these teeth erupt within the first month of birth, they are termed neonatal teeth . AlthougDental pulp stem cells (DPSCs) are mainly isolated from the pulp tissue of exfoliated deciduous teeth, deciduous incisors or permanent third molars ,7. ThereTwo natal teeth of a 20-day-old healthy male patient and a deciduous mandibular first molar of an 11-year-old healthy male patient were extracted under local anesthesia based on a protocol approved by the Institutional Review Board and Ethics Committee. Informed consent was obtained from the parents of these patients to extract the teeth and use them for further analysis. The natal teeth were extracted for prophylactic reasons as outlined previously, while the deciduous teeth were extracted due to mobility. The deciduous molar served as the positive control. Following extraction, the teeth were immediately fixed in 10% neutral buffered formalin for 24 h. The dental pulp tissue was removed from the deciduous mandibular first molar by splitting the tooth with a chisel and immediately fixing in 10% neutral buffered formalin for 24 h.After fixation, the natal teeth were demineralized in 10% EDTA (pH = 7.4) for 6 months at room temperature. Subsequently, the samples were rinsed under running water for 4 h followed by dehydration with ascending concentrations of ethanol. Then, the teeth were deparaffinized in xylene, infiltrated, and embedded in paraffin. With the microtome set at 5 μm, longitudinal serial sections were cut and stained with hematoxylin–eosin.Serial sections, 5 μm in thickness, prepared from archived formalin-fixed, paraffin-embedded (FFPE) tissue blocks, were used for immunohistochemical analysis. Sections were deparaffinized and rehydrated following standard methods. Briefly, the sections were deparaffinized three-times with xylene for 5 min and rehydrated in graded ethanol (80–100%) for 5 min. Antigen retrieval was performed by boiling at 98°C for 40 min in 0.01 mol/l sodium citrate buffer (pH 6.0). Details of the primary antibody clones, sources and titers are listed in The endogenous peroxidase activity was blocked and the tissue samples were incubated with OCT4, SOX2, CD44, nestin, osteopontin, desmin and myogenin antibodies for 90 min. Subsequently, the slices were rinsed and incubated with the biotinylated secondary antibody at room temperature for 30 min. The bound antibody complexes were stained for 3–5 min or until appropriate for microscopic examination and then counter stained with hematoxylin (30 s) and mounted.The expression of OCT4, SOX2, CD44, nestin, osteopontin, desmin, myogenin and CD34 on the natal as well as the deciduous tooth (positive control) tissue samples were analyzed. The immunoreactivity of the specimens was interpreted based on the intensity of the staining. Score ranks usually lie in a range from ‘negative’ (mostly marked as ‘-’) to ‘positive’, which was signed with different amount of ‘+’ depending upon the intensity of the stain. The samples were examined with conventional light microscope. For OCT4, SOX2, osteopontin, myogenin nuclear immunohistochemical staining was considered positive whereas for nestin and desmin cytoplasmic immunohistochemical staining was considered positive. Membranous immunohistochemical staining was considered positive for CD34 and CD44. The staining intensity was graded as (+) weak, (++) moderate and (+++) intense according to the overall appearance at different powers of magnification, in other words, 4×, 10× and 40×, respectively and (-) for no staining.Hematoxylin and eosin stained sections of the tissues obtained from the natal and deciduous teeth showed myxomatous connective tissue with blood vessels and inflammatory cells. A clear odontoblastic lining was evident adjacent to the secondary dentin A & 2A.Immunohistochemical examination  demonstrStem cells are an excellent source of generation of a huge number of mature cells through a sequential procedure of proliferation and differentiation, retaining their ability to self renew to maintain the stem cell pool. Stem cell markers are the protein products that identify a multipotent single cell capable of recapitulating. There are four types of cell: embryonic stem cell (ESC), hematopoietic stem cell (HSC), mesenchymal/stromal stem cell (MSC/SSC) and neural stem cell (NSC).in vitro [et al. [et al. [in vitro differentiation of hNDPSCs into induced pluripotent cells that were morphologically similar to pancreatic islet cells.The purpose of this study was to characterize the stem cellness of the dental pulp obtained from natal teeth, which are in general considered waste teeth, using histological and immunohistochemical methods. Only two studies thus far have successfully isolated and characterized human natal DPSCs (hNDPSCs) in vitro ,10. Alth [et al.  demonstrPrior to investigating the multilineage differentiation potential of DPSC, an overview of marker expression in basic pulp tissue is crucial. In the present study, for the first time, we demonstrate the immunohistochemical characterization of stem cell and differentiation markers in FFPE tissue sections of natal teeth. OCT4 and SOX2 serve as reliable markers for identifying the NSC population. OCT4 is an ESC marker, identified as a DNA-binding protein that activates gene transcription via an octamer motif . OCT4 exet al. [Expression of CD44, a positive MSC marker and CD34, a negative MSC marker were also analyzed in this work. CD44, a ubiquitous, multistructural, multifunctional cell surface glycoprotein is involved in cell-to-cell adhesion, cell-matrix interactions and cell migration. It is widely expressed in several cell types. CD44 is expressed at the time of extracellular matrix formation in many sites during embryonic development. CD44, a cell-adhesion factor, is involved in the induction of mineralization in the dental pulp. Interestingly, it was overexpressed in the natal tooth specimen when compared with the control C & 2B. Cet al. .Nestin, a class VI intermediate filament protein, is predominantly expressed in the CNS ,16. Althet al. demonstrated a significant difference between SHED and DPSCs in terms of rates of proliferation, cell population doublings, sphere-like cell cluster formation and osteoinductive capacity, all of which were better in SHED. Our study, which compared natal teeth and deciduous teeth, also found these teeth as an excellent and abundant source of stem cells.Natal teeth provide a unique population of stem cells that can be attributed to their entirely intrauterine development. They are different from deciduous and permanent teeth in their structure as well as function. In their study on exfoliated deciduous teeth, Miura et al. is the lack of expression of myogenic (desmin and myogenin) and osteogenic differentiation markers (osteopontin). This may be attributed to the fact that the above-mentioned study used cell culture techniques, in contrast to our report, which used FFPE tissues. Our study provides evidence that natal teeth have shown very strong positive expression of CD44 (MSC), SOX2 (ESC) and Nestin (NSC) markers. Natal teeth therefore may be a viable source of stem cells to induce repair or regeneration of tissue, wherever applicable and for treatment of degenerative tissues. To the best of our knowledge, this is the first study to demonstrate positive immunoexpression of various stem cell markers in FFPE sections of a usually discarded tooth such as the natal tooth.An interesting finding in the present study that differed to the results of Karaoz Taken together, the results of this study support the use of natal teeth as a potential source of pluripotent stem cells for future cell-based therapies.Since the natal teeth develop during the intrauterine stages, the stem cells of natal teeth originate at much earlier stages of development and exhibit excellent potency to differentiate into various structures. This property could be utilized for treating various clinical conditions. They will also be useful in dentin tissue engineering, teeth replantation and pulp revascularization. However, the stem cells of natal teeth origin have been heretofore underestimated due to the sparse available literature and hence future research in this direction is warranted to use them to their maximal potential.The immunohistochemical expression of OCT4, SOX2, CD34, CD44, nestin, osteopontin, desmin, myogenin and KI67 on the dental pulp of natal as well as the deciduous tooth tissue samples was analyzed.The natal teeth as well as the deciduous tooth specimens expressed immunopositivity for nestin and CD44. However, the natal teeth specimen showed an overexpression of these markers compared with its counterpart.SOX2, a transcription factor considered to be critical for neural stem cell proliferation and differentiation, was expressed only by the natal teeth.Natal teeth, a usually discarded tissue, can be considered as a potential source of pluripotent stem cells for future cell-based therapies.