Rectal Samples Research Articles

A non-invasive method to monitor farmland bird exposure to triazole fungicides

The treatment of seeds with pesticides is an extended practice in current agriculture. There is a high risk of exposure in granivorous birds, such as the red-legged partridge (Alectoris rufa), that can consume those seeds remaining on the surface during sowing. Fungicide exposure could in turn affect bird reproductive capacity. To better understand to what extent triazole fungicides are a threat to granivorous birds, we need an easy and reliable method to quantify field exposure. In this study, we tested a novel non-invasive method to detect the presence of triazole fungicide residues in farmland bird faeces. We experimentally exposed captive red-legged partridges to validate the method, and then applied it in a real scenario to assess exposure of wild partridges. We exposed adult partridges to seeds treated with two formulations containing triazole fungicides as active ingredients: Vincit®Minima (flutriafol 2.5%) and Raxil®Plus (prothioconazole 25% and tebuconazole 15%). We collected two types of faeces (caecal and rectal samples) immediately after exposure and 7 days later and quantified the concentrations of the three triazoles and their common metabolite (1,2,4-triazole). The three active ingredients and 1,2,4-triazole were only detected in faeces collected immediately after exposure. Triazole fungicide detection rates in rectal stool were 28.6%, 73.3% and 80% for flutriafol, prothioconazole and tebuconazole, respectively. In caecal samples, detection rates were 40%, 93.3% and 33.3%, respectively. 1,2,4-triazole was detected in 53% of rectal samples. For an applied use of the method in the field, we collected 43 faecal samples from wild red-legged partridges during autumn cereal seed sowing and found detectable levels of tebuconazole in 18.6% of the analysed wild partridges. The results of the experiment were then used to estimate actual exposure levels from this prevalence value found in wild birds. Our study shows that faecal analysis can be a useful tool to assess farmland bird exposure to triazole fungicides, when samples are fresh and the method has been validated for the detection of target molecules.

Associated factors for bacterial colonization in patients admitted to the intensive care unit of the Clinical Hospital of Infectious Diseases.

This study aimed to identify isolates from colonization and assess the risk factors for bacterial colonization and the risk of death in patients admitted to the intensive care unit (ICU) of the Constanţa County Infectious Diseases Hospital between September 2017 and September 2019. This was a retrospective case-control study in a single center that included all patients admitted to the ICU in Constanţa, Romania, who underwent bacteriological screening upon admission and 7 days after admission, between September 2017 and September 2019. In total, 253 patients were included in this study. The nasal exudate, pharyngeal exudate, and rectal swab samples were screened. In this study, 253 patients were screened bacteriologically, of which 53 had bacterial colonization and 200 did not. Among the bacterial strains, Klebsiella spp. (43.39%) was the most frequently isolated. The predominant resistance mechanism detected in the bacterial isolates was extended-spectrum β-lactamase (ESBL). Multivariate analysis identified a Carmeli score of 3 as an independent risk factor for acquiring bacterial colonization in the ICU. The mortality rate of patients with bacterial colonization was 11.32% and 6% for the patients without colonization (p>0.05). Our study revealed an increased prevalence of Enterobacterales colonization in the ICU. Risk factors for acquiring bacterial colonization differed depending on the type of bacterial colonization, such as ESBL, carbapenemases, methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant enterococci (VRE). An independent risk factor for acquiring bacterial colonization was the Carmeli score of 3.

ESBL-Positive Enterobacteriaceae from Dogs of Santiago and Boa Vista Islands, Cape Verde: A Public Health Concern.

Antimicrobial resistance is a public health threat with an increasing expression in low- and middle-income countries such as Cape Verde. In this country, there is an overpopulation of dogs, which may facilitate the spread of resistant bacteria, including extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae. To clarify the role of dogs as reservoirs for the dissemination of this bacterial group, 100 rectal swab samples were collected from confined (n = 50) and non-confined (n = 50) dogs in Santiago and Boa Vista Islands, Cape Verde. These were analyzed using conventional bacteriological techniques for the detection of ESBL-producing Enterobacteriaceae and characterization of their pathogenic and resistance profiles. Twenty-nine samples displayed ESBL-positive bacteria, from which 48 ESBL-producing isolates were obtained and mostly identified as Escherichia coli. Multiple antimicrobial resistance indexes ranged from 0.18 to 0.70 and half of the isolates were classified as multidrug-resistant. Isolates were capable of producing relevant virulence factors, including biofilm, showing virulence indexes between 0.29 and 0.71. As such, dogs in Cape Verde may act as reservoirs of resistant bacteria, including pathogenic and zoonotic species, representing a public health concern. Although further investigation is needed, this study proposes the periodical analysis of dogs' fecal samples to monitor resistance dissemination in the country, in a One-Health perspective.

Molecular surveillance reveals widespread colonisation by carbapenemase and extended spectrum beta-lactamase producing organisms in neonatal units in Kenya and Nigeria

ObjectivesNeonatal sepsis, a major cause of death amongst infants in sub-Saharan Africa, is often gut derived. Gut colonisation by Enterobacteriaceae producing extended spectrum beta-lactamase (ESBL) or carbapenemase enzymes can lead to antimicrobial-resistant (AMR) or untreatable infections. We sought to explore the rates of colonisation by ESBL or carbapenemase producers in two neonatal units (NNUs) in West and East Africa.MethodsStool and rectal swab samples were taken at multiple timepoints from newborns admitted to the NNUs at the University College Hospital, Ibadan, Nigeria and the Jaramogi Oginga Odinga Teaching and Referral Hospital, Kisumu, western Kenya. Samples were tested for ESBL and carbapenemase genes using a previously validated qPCR assay. Kaplan–Meier survival analysis was used to examine colonisation rates at both sites.ResultsIn total 119 stool and rectal swab samples were taken from 42 infants admitted to the two NNUs. Colonisation with ESBL (37 infants, 89%) was more common than with carbapenemase producers (26, 62.4%; P = 0.093). Median survival time before colonisation with ESBL organisms was 7 days and with carbapenemase producers 16 days (P = 0.035). The majority of ESBL genes detected belonged to the CTX-M-1 (36/38; 95%), and CTX-M-9 (2/36; 5%) groups, and the most prevalent carbapenemase was blaNDM (27/29, 93%).ConclusionsGut colonisation of neonates by AMR organisms was common and occurred rapidly in NNUs in Kenya and Nigeria. Active surveillance of colonisation will improve the understanding of AMR in these settings and guide infection control and antibiotic prescribing practice to improve clinical outcomes.

Molecular detection and genotype analysis of Kudoa septempunctata from food poisoning outbreaks in Korea.

Concerns about foodborne illnesses caused by Kudoa septempunctata are steadily growing, but reports of K. septempunctata in clinical and food specimens related to food poisoning in Korea are limited. This study aimed to genetically identify K. septempunctata in patients with acute diarrhea and in clinical and food samples related to food poisoning caused by sashimi consumption. Both real-time and nested polymerase chain reaction assays were performed to detect K. septempunctata 18S and 28S rDNA genes in the stools of 348 patients with acute diarrhea, 11 samples (6 stool and 5 rectal swab samples) from patients with food poisoning, and 2 raw Paralichthys olivaceus samples collected from a restaurant where a food poisoning incident occurred. K. septempunctata was identified in 5 clinical specimens (4 stools and 1 rectal swab) and 1 P. olivaceus sashimi sample. All detected K. septempunctata were of genotype ST3. This is the first study to identify K. septempunctata in both patients and food samples with epidemiological relevance in Korea, providing evidence that it is a pathogen that causes food poisoning. Also, this is the first study to confirm the presence of K. septempunctata genes in rectal swabs. Despite continuing suspected occurrences of Kudoa foodborne outbreaks, the rate of identification of K. septempunctata is very low. One reason for this is the limitation in obtaining stool and vomit samples for the diagnosis of Kudoa infection. We strongly suggest the inclusion of rectal swabs among the diagnostic specimens for Kudoa food poisoning.

Evaluation of Non-Invasive Sampling Methods for Detection of Hepatitis E Virus Infected Pigs in Pens

Pigs are a reservoir of hepatitis E virus (HEV), which causes hepatitis in humans. To study the epidemiology of HEV in pig farms, sampling methods are currently used that cause discomfort to pigs, such as rectal sampling. In line with the 3Rs principle, we aimed to evaluate non-invasive methods to detect pens with HEV-shedding pigs. Twenty-eight pens of one farm were sampled cross-sectionally. Individual rectal swabs (IRS) were collected to determine prevalence within pens. Four pen-level samples were compared: a pool of IRS per pen (P), boot socks (BS), oral fluid (OF) and pooled faecal droppings (FD). Each sample was tested by RT-PCR and the sensitivity and specificity of each method was determined by Bayesian latent class analysis. According to IRS, 19/28 pens were HEV positive. BS had a sensitivity of 95% and detected HEV in pens with 10% of pigs shedding; however, specificity was below 30%. FD were comparably accurate to P, with a sensitivity and specificity of 94% and 86%, respectively. BS sampling is thus advised to detect early shedding of HEV or pen contamination, and FD to determine the duration of shedding. This study demonstrates that non-invasive sampling can replace rectal swabs in research on HEV in pigs.

Development of a new multiplex real-time PCR assay for rapid screening of hospital-acquired infection agents

AimsA new multiplex real-time PCR (qPCR) assay was developed to detect antibiotic-resistant hospital-acquired infectious agents in nasal and rectal swab samples in 1.5 h without the need for nucleic acid extraction. MethodsSpiked negative clinical specimens were used for the analytical performance evaluation. Double-blind samples were collected from 1788 patients to assess the relative clinical performance of the qPCR assay to the conventional culture-based methods. Bio-Speedy® Fast Lysis Buffer (FLB) and 2× qPCR-Mix for hydrolysis probes (Bioeksen R&D Technologies, Istanbul, Turkey) and LightCycler® 96 Instrument (Roche Inc., Branchburg, NJ, USA) were used for all molecular analyses. The samples were transferred into 400 L FLB, homogenized and immediately used in qPCRs. The target DNA regions are vanA and vanB genes for vancomycin-resistant Enterococcus (VRE); blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-23, blaOXA-48, blaOXA-58 genes for carbapenem-resistant Enterobacteriaceae (CRE); and mecA, mecC and spa for methicillin-resistant Staphylococcus aureus (MRSA). ResultsNo qPCR tests produced positive results for the samples spiked with the potential cross-reacting organisms. The limit of detection (LOD) of the assay for all targets was 100 colony-forming unit (cfu)/swab-sample. Results of the repeatability studies in two different centers were in 96%–100% (69/72–72/72) agreement. The relative specificity and sensitivity of the qPCR assay were respectively 96.8% and 98.8% for VRE; 94.9% and 95.1% for CRE; 99.9% and 97.1% for MRSA. ConclusionsThe developed qPCR assay can screen antibiotic-resistant hospital-acquired infectious agents in infected/colonized patients with an equal clinical performance to the culture-based methods.

Pharmacokinetics, the Immunological Impact, and the Effect on HIV Ex-Vivo Infectivity of Maraviroc, Raltegravir, and Lopinavir in Men Who Have Sex with Men Using Postexposure Prophylaxis.

Most of the studies using the colorectal tissue explants challenge model have been conducted after one single dose and before reaching a steady state. We consider that longer exposure as in 28-day postexposure prophylaxis (PEP) course and in an at-risk setting, such as after a sexual risk exposure to HIV could give us valuable information about these drugs. In a substudy we assessed pharmacokinetics, changes on immune system and ex-vivo rectal mucosal susceptibility to HIV-1 infection after taking maraviroc (MVC), raltegravir (RAL), and ritonavir-boosted lopinavir (LPV/r) PEP-based regimens in 30 men who have sex with men. Participants received 28 days of twice-daily MVC (n = 11), RAL (n = 10) or LPV/r (n = 9) all with tenofovir/emtricitabine (TDF/FTC) backbone. Blood, rectal fluid, and rectal tissue samples were collected at days 7, 28, and 90 after starting PEP. The samples obtained at day 90 were considered baseline. All studied antiretrovirals were quantifiable at 7 and 28 days in all tissues. Activation markers were increased in CD4 mucosal mononuclear cells (MMCs) after 28 days of MVC: CD38 + 68.5 versus 85.1, p = .008 and CD38+DR +16.1 versus 26.7, p = .008. Exposure to MVC at both endpoints (7 and 28 days) was associated with significant suppression of HIV-1BAL (p = .005 and p = .028), but we did not observe this effect with RAL or LPV/r. Merging together changes in MMC in all arms, we found a positive correlation in the CD8 T cell lineage between the infectivity at day 7 and activation (CD38+ r = 0.43, p = .025, DR + r = 0.547, p = .003 and 38+DR+ r = 0.526, p = .05), senescence (CD57+CD28- r = 0.479, p = .012), naive cells (RA+CCR7+ r = 0.484, p = .01), and CCR5 expression (r = 0.593, p = .001). We conclude that MVC in combination with TDF/FTC was associated with viral suppression in rectal explants and that overall ex-vivo HIV infectivity correlated with activation and senescence in CD8 MMCs.

Rectal cancer–specific subtyping using transcriptomics datasets derived from treatment-naive patients to assess novel prognostic signatures and key differences to CMS-based subtyping.

235 Background: Molecular subtyping in colorectal cancer provided novel insights into tumour heterogeneity and delivered new prognostic signatures. Existing methods (such as CMS-based subtyping) were generated without much consideration of differences in expression profiles between colon and rectal cancer tissues. Moreover, stage 2 and 3 rectal cancer patients often receive neo-adjuvant radio-chemotherapy which may impact on gene expression when determined from resection samples. Methods: We investigated possible differences between treatment-naive colon and rectal cancers at the molecular level. We collected mRNA expression profiles for rectal and colon cancer samples available from Gene Expression Omnibus (GEO) database (n=2139) and applied batch-effect correction using ComBat. Results: To explore whether CMS subtypes changed after neoadjuvant chemoradiation in rectal cancers, we analysed rectal tumour samples from 40 patients where both pre-operative biopsies and resections after chemoradiotherapy were available. More than half of samples shifted from CMS1-3 to CMS4 after patients received chemoradiotherapy. We therefore focused our further analysis on treatment-naive rectal cancers. We observed that CMS-based subtyping showed different prognostic potential in treatment-naïve colon vs. rectal cancers. Rectal cancers had significantly better disease-free survival (DFS) when assigned to CMS 2 or 4 compared to colon cancers. This suggested differences in disease biology that were not captured by CMS subtyping. We next aimed to identify molecular subtypes in rectal cancers, and identified three prevalent rectal-specific subtypes (RSS) by applying hierarchical clustering in 182 untreated rectal cancer samples. We used gene co-expression network analysis to define functional gene modules to identify these subtypes. Based on the gene modules’ signatures, we built a classifier to predict our new subtypes in different transcriptomics datasets (n=692). We observed that RSS1 had better disease-free survival (DFS), whereas RSS2 exhibited worse short-term DFS (less than 3 years) and RSS3 showed worse long-term DFS (3+ years). We discovered the deactivation of EGFR, MAPK and WNT pathways on RSS3 as well as high CD8+ infiltration and low CD4+ counts in these tumours. RSS2 exhibits low plasma cell and regulatory T-cell abundance and high activations of TGF-β, NF-κB, and TNF-α. We also observed high EMT, angiogenesis and inflammatory response in this subtype. Lastly, RSS1 showed high expression on MYC target and low activity on angiogenesis genes. Moreover, the immune and inflammatory responses were also lower than in the other subtypes. Conclusions: Rectal-specific subtypes are of prognostic importance and provide new insight into molecular disease pathways and potential targets.

Performance of two commercial multiplex polymerase chain reaction assays for the etiological diagnosis of sexually transmitted infections among men who have sex with men.

This study aimed to investigate the etiologies of sexually transmitted infections (STIs) among men who have sex with men (MSM) in Taiwan. Two commercial assays, the BD MAX Chlamydia trachomatis (CT), Neisseria gonorrhoeae (GC), and Trichomonas vaginalis (TV) panel and the Allplex™ STI Essential assay (CT, GC, Mycoplasma genitalium [MG], Mycoplasma hominis [MH], Ureaplasma urealyticum [UU], Ureaplasma parvum [UP], and TV) were evaluated. During the first stage, urine and rectal swab samples from 168 patients were evaluated using the BD MAX assay, and the multiplex RT-PCR Allplex™ STI Essential assay was applied only to the patients with positive results on the BD MAX asay (n=49). During the second stage, urine and rectal swab samples from 90 patients were evaluated using the BD MAX assay and the Allplex™ qPCR. The Allplex qPCR identified all CT, missed one and additionally one TV from the positive samples (n=49) by the BD MAX assay in the first stage. At the second stage, both commercial assays showed similar detection rate of CT, NG or CT/NG coinfection (11.1%, 1.1% and 4.4% by the BD MAX assay; 10.0%, 1.1% and 2.2% by the Allplex qPCR). The positivity rates of MG, MH, and UU by the Allplex qPCR were 4.4%, 2.2%, and 12.2%, respectively, for urine samples and 10%, 13.3%, and 22.2%, respectively, for anal swab samples. High rates of STI-associated etiologies were observed in MSM. The positive rates were higher in rectal swabs than in urine samples.

Intestinal colonization of vancomycin-resistant Enterococcus in children admitted to Mofid children's hospital intensive care unit at admission and at discharge.

This study aimed to investigate the frequency of intestinal colonization by vancomycin-resistant Enterococcus (VRE) carrying vanA and vanB genes in patients at ICU admission and at discharge from ICU in Mofid children's Hospital, Tehran, Iran. Sampling was performed using rectal swabs and vancomycin susceptibility testing for Enterococcus spp. was carried out using a minimum inhibitory concentration (MIC) assay on Muller Hinton Agar (MHA) medium using an E-test kit. The molecular detection of VRE isolates was performed by the PCR method using the vanA and vanB resistance genes. A total of 234 and 186 non-duplicate rectal swab samples were collected from patients at ICU admission and at discharge from ICU, respectively. Enterococcus spp. was detected in 34.6% (n = 81/234) of rectal swab samples collected from patients at ICU admission, of which 44.4% (n = 36/81) were VRE isolates. In contrast, the prevalence of Enterococcus spp. and VRE isolates among patients at discharge from ICU was 17.7% (n = 33/186) and 57.6% (n = 19/33), respectively. Out of 19 VRE isolated from patients at ICU admission, 4 (21%) and 1 (5.3%) contained vanA and vanB genes, respectively. In contrast, out of 36 VRE isolated from patients at discharge from ICU, 11 (30.5%) were positive for the vanA gene. Results revealed that the prevalence of Enterococcus spp. among patients at ICU admission was high. However, VRE was frequently isolated from patients who were hospitalized for several days in ICUs. The implementation of proper infection control strategies and the use of suitable protocols to guide the appropriate prescribing of antibiotics are necessary.

DOP29 Single cell analysis of Crohn’s disease fistula; comparison of different locations

Abstract Background A common complication of Crohn’s disease(CD) are perianal fistulae, highly impacting the quality of life in these patients. The pathogenesis of these fistulas is incompletely understood. Treatment is often given based on knowledge of disease processes in the inflamed intestine itself, but whether the immune activity in the fistula tract is similar remains unknown. Full characterization of the intestinal mucosa versus the fistula tract itself may reveal immunological differences between the two, and generate leads for both development of new interventional options as well as allow better stratification of patients. Methods Matched biopsies were obtained from the rectal mucosa, the internal opening of the fistula and the tract itself. A total of 30 patients were included (20 CD; 10 non-IBD patients). Tissues were processed to cell suspensions and selected for live CD66b- cells. Single cell RNASeq analysis was performed using 10x Chromium. In total, 111.955 immune cells were annotated and used for analysis. Results T cells comprised the largest cluster of immune cells, which was subclustered into 19 clusters. Both the internal opening and the fistula tracts contained elevated proportions of effector memory and cycling T cells. Conversely, the rectum contained more central and tissue resident memory cells. T cells expressing IL-22, which were previously described in fistula were detected in considerable number in the rectum and internal opening, but were scarce in the fistula tract itself. These cells were clearly distinct from the IL17A producing subset, and IL17F expressing cells and mainly found in the fistula tracts. Only the tract contained a clear subset of CXCL13 expressing T cells, suggesting follicular helper T cells. Presence of these cells was strongly correlated to the presence of lymphoid inducer cells as well as IgG producing B cells rather than the IgA subset found in the rectum. Clusters of myeloid cells were largely derived from the fistula tracts. Subclustering into 10 clusters indicated a shift from anti-inflammatory to pro-inflammatory macrophages in the tracts compared to the rectal mucosa. Finally, a striking difference in NK cells subsets was observed, with significantly more CD56bright NK cells present in the rectal samples while the internal opening and the tracts were comparable. Conclusion Cellular composition and activity of the immune cell compartment differs considerably between the rectum, the internal opening of the fistula and the tract. Subsequently, therapy targeted at processes involved in mucosal inflammation may not actually target the fistula itself, which may explain the low success rate in fistula resolution. Careful analysis of the fistula may help to identify targets useful in the treatment.

P001 Obefazimod upregulates miR-124 and downregulates the expression of some cytokines in blood and rectal biopsies of patients with moderate-to-severe ulcerative colitis.

Abstract Background The oral small molecule obefazimod (ABX464) has demonstrated efficacy and safety in patients with moderate-to-severe ulcerative colitis (UC) in a randomized, placebo-controlled, 8-week phase 2b induction study1. We examined the effects of obefazimod on miR-124 and cytokines levels in blood (baseline, weeks 4 and 8) and rectal biopsies (baseline, week 8) of these patients. Methods Absolute quantification (QuantaSoft Pro) of the miR-124 copy number was performed by using droplet digital PCR technology on 644 rectal biopsy samples and 1104 whole blood samples from patients receiving obefazimod (25mg, 50mg or 100mg once daily) or placebo. A total of 15 cytokines (IL1b, IL13, IL17, IL6, IL10, IL23, MCP-1, IL34, CXCL-12, IL22, LIF, TGFb, GM-CSF, IFNg and TNFa) were examined by immunohistochemistry (IHC) on 208 rectal biopsies. For each cytokine, antibodies were tested for the chromogenic IHC detection on paraffin tissue sections; repeatability and reproducibility tests were conducted to validate protocols with robust signal. IHC signal was digitized and quantified with Halo® image analysis software. IL17 was also measured in blood samples using an ELLA immunoassay (Bio-Techne). Results The three tested doses of obefazimod (25mg, 50mg or 100mg once daily) significantly upregulated miR-124 in blood at week 1 (A) and 8 (B), and in rectal tissue at week 8 (C) (from p<0.001 to p<0.0001 vs. placeb The three doses of obefazimod significantly reduced IL17 levels in blood of patients at week 1, 4 and 8 (from p<0.01 to p<0.0001 vs. placebo). IHC protocols in rectal tissue were validated for 4 out of 15 cytokines: IL17, IL23, CXCL-12, INFg. Compared with baseline levels, an 8-week obefazimod treatment significantly down-regulated the expression of IL17 (at all doses, (from p<0.05 to p<0.001), IL23 (p<0.01 at 50mg, p<0.001 at 100 mg), and INFg (p<0.001 at 50 mg). Within obefazimod treated subjects, clinical responders showed higher reduction of IL-17A and IL-23 expression in rectal tissue than non-clinical responders at week 8 (reduction in MMS ≥2 points and ≥30% from baseline + decrease in rectal bleeding subscore ≥1 or absolute rectal bleeding subscore ≤1 point). No significant change was observed with CXCL-12. Conclusion The 8-week efficacy of obefazimod in patients with moderate-to-severe UC is associated with an increase in miR-124 expression in blood and tissue and with a decrease of IL17, IL23 and INFg in the colorectal tissue.

DOP65 T-cell derived TGFβ1 remodels the rectum mesenchyme in Crohn’s Disease patients with perianal fistulizing disease

Abstract Background Perianal fistulizing disease (PFD) is a complication occurring in about 20% of Crohn’s Disease (CD) patients. It consists of the formation of new epithelised tracks connecting the intestine with the outer perianal region, which dramatically worsens a patient’s quality of life. The mechanisms driving fistula formation have not been elucidated and therefore, there are currently no specific treatments for PFD. Methods We collected rectum biopsies of CD patients with and without PFD in the absence of rectal inflammation (CD and CD+PFD), and with active rectal inflammation (CDinf and CD+PFDinf) (Fig.1). Rectal tissue samples were processed for RT-qPCR, or histologic analysis, and single-cell RNA sequencing (scRNAseq) for each patient. Results Differential expression gene (DEG) analysis of CD4+ and CD8+ T cells comparing CD vs CD+PFD and CDinf vs CD+PFDinf patients, respectively, revealed increased expression of TGFB1 in patients with fistulae. To investigate the main cellular subsets TGFB1 was interacting with, we performed ligand-receptor interaction analysis using CellphoneDB, which indicated that T-cell derived TGFB1 was primary interacting with the stromal compartment (Fig.2). 365 DEG were found in lamina propria (LP) fibroblasts of CD+PFD compared to CD, including TGFβ1-inducible genes such as TFPI2, SERPINE2 and MMPs 1 and 3. These changes were validated by bulk RT-qPCR, underscoring their potential use as PFD biomarkers (Fig.3). Moreover, we also identified a signature of elastin fibre assembly genes dysregulated in CD+PFD fibroblasts, suggesting molecular changes in the formation of elastin fibres in these patients. To determine whether the differentiation of fibroblasts was altered in the context of PFD, we conducted trajectory analysis using RNA velocity. Whilst fibroblasts in CD patients preferentially differentiate towards myofibroblasts, patients with PFD are biased towards LP fibroblasts, indicating an overall structural rearrangement of the mesenchyme in the rectum of patients with PFD, regardless of their inflammatory status (Fig.4). Importantly, analysis of an independent scRNAseq dataset of CD and ulcerative colitis (UC) patients showed higher expression of TGFB1 in T cells of active CD compared to active UC patients, suggesting that these alterations are already present in CD patients before the onset of a fistulizing phenotype and might potentially contribute to explain the absence of PFD in UC patients. Conclusion Based on these results, we suggest that the rectum of patients suffering from PFD present cellular and transcriptomic differences compared to CD patients without PFD. These changes are inflammation independent and point towards the TGFβ pathway as the main driver of this complication.

Differentially expressed glycoproteins in pre- and post-digital rectal examination urine samples for detecting aggressive prostate cancer.

Urinary glycoproteins associated with aggressive prostate cancer (AG-PCa) were previously reported using post-digital rectal examination (DRE) urine specimens. To explore the potential of using pre-DRE urine specimens for detecting AG-PCa, we compared glycoproteins between pre- and post-DRE urine specimens, verified the previously identified post-DRE AG-PCa-associated urinary glycoproteins in pre-DRE urine specimens, and explored potential new glycoproteins for AG-PCa detection in pre-DRE urine specimens. Quantitative glycoproteomic data were acquired for 154 pre-DRE urine specimens from 41 patients with no cancer at biopsy, 48 patients with non-AG-PCa (Gleason score=6), and 65 patients with AG-PCa (Gleason score 7 or above). Compared to glycopeptides from the post-DRE urine data, humoral immunity-related proteins were enriched in pre-DRE urine samples, whereas cell mediated immune response proteins were enriched in post-DRE urine samples. Analyses of AG-PCa-associated glycoproteins from pre-DRE urine revealed that the three urinary glycoproteins, prostate-specific antigen (PSA), prostatic acid phosphatase (ACPP), and CD97 antigen (CD97) that were previously identified in post-DRE urine samples, were also observed as AG-PCa associated glycoproteins in pre-DRE urine. In addition, we identified three new glycoproteins, fibrillin 1 (FBN1), vitronectin (VTN), and hemicentin 2 (HMCN2), to be potentially associated with AG-PCa in pre-DRE urine specimens. In summary, glycoprotein profiles differ between pre- and post-DRE urine specimens. The identified AG-PCa-associated glycoproteins may be further evaluated in large cohort of pre-DRE urine specimens for detecting clinically significant PCa.

Genomic data reveals the emergence of the co-occurrence of blaKPC-2 and blaCTX-M-15 in an Escherichia coli ST648 strain isolated from rectal swab within the framework of hospital surveillance

The worldwide dissemination of carbapenemase-producing Escherichia coli lineages belonging to high-risk clones poses a challenging public health menace. The aim of this work was to investigate genomic features of a colonizing multidrug-resistant strain of Klebsiella pneumoniae carbapenemase (KPC)-producing E. coli from our institution. Whole-genome sequencing was done by Illumina MiSeq-I, and de novo assembly was achieved using SPAdes. Resistome, mobilome, plasmids, virulome, and integrons were analysed using ResFinder, AMRFinder, ISFinder, PlasmidFinder, MOB-suite, VirulenceFinder, and IntegronFinder. Sequence types (STs) were identified with pubMLST and BIGSdb databases. Conjugation assays were also performed. Escherichia coli HA25pEc was isolated from a rectal swab sample taken within the framework of the hospital epidemiological surveillance protocol for detection of carbapenemase-producing Enterobacterales. Escherichia coli HA25pEc corresponded to the first report of ST648 co-harbouring blaKPC-2 and blaCTX-M-15 in Latin America from a colonized patient. It had 19 antibiotic resistance genes (ARGs), including blaKPC-2, located on a Tn4401a isoform. Conjugation assays revealed that blaKPC-2 was not transferred by conjugation to E. coli J53 under our experimental conditions. Escherichia coli ST648 has been detected previously in companion and farm animals as well as in hospital- and community-acquired infections worldwide. Although scarcely reported as KPC-producers, our finding in a culture surveillance with several acquired ARGs, including blaCTX-M-15, alerts the potential of this clone for worldwide unnoticed spreading of extreme drug resistance to β-lactams. These data reinforce the importance of carrying out molecular surveillance to identify reservoirs and warn about the dissemination of new international clones in carbapenemase-bearing patients.

Five-Hour Detection of Intestinal Colonization with Extended-Spectrum-β-Lactamase-Producing Enterobacteriaceae Using the β-Lacta Phenotypic Test: the BLESSED Study.

Extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-PE) intestinal colonization is of particular concern as it negatively impacts morbidity and is the main source of external cross-contamination in hospitalized patients. Contact isolation strategies may be caught out due to the turnaround time needed by laboratories to report intestinal colonization, during which patients may be inappropriately isolated or not isolated. Here, we developed a protocol combining enrichment by a rapid selective subculture of rectal swab medium and realization of a β-Lacta test on the obtained bacterial pellet (named the BLESSED protocol). The performances of this protocol were validated in vitro on 12 ESBL-PE strains spiked into calibrated sample suspensions and confirmed in clinical settings using 155 rectal swabs, of which 23 (reference method) and 31 (postenrichment broth culture) came from ESBL-PE carriers. In vitro, the protocol detected, with 100% sensitivity, the presence of the 12 ESBL-PE strains from 104 CFU/mL. In the clinical validation cohort, 22 out of the 23 (reference method) and 28 out of the 31 (postenrichment broth culture) ESBL-PE-positive rectal samples were accurately detected. The diagnostic performances for ESBL-PE detection, considering all ESBL-PE carriers, were 90% sensitivity, 98% specificity, an 87% positive predictive value, and a 98% negative predictive value. Our protocol is a rapid and low-cost method that can detect intestinal colonization with ESBL-PE in less than 5 h more accurately than the reference method, opening the field for further studies assessing a rapid and targeted isolation strategy applied only to patients with a positive BLESSED protocol result. IMPORTANCE To both improve the efficiency of contact isolation among ESBL-PE carriers and avoid the unnecessary isolation of noncolonized patients, we should reduce the turnaround time of ESBL screening in laboratories and improve the sensitivity of diagnostic methods. The development of rapid and low-cost methods that satisfy these two goals is a promising approach. In this study, we developed such a technique and report its good diagnostic performance, opening the door for further studies assessing a rapid and targeted isolation strategy applied in a few hours only for patients truly colonized with ESBL-producing bacteria.

Rectal swabs as a viable alternative to faecal sampling for the analysis of gut microbiota functionality and composition

Faecal or biopsy samples are frequently used to analyse the gut microbiota, but issues remain with the provision and collection of such samples. Rectal swabs are widely-utilised in clinical practice and previous data demonstrate their potential role in microbiota analyses; however, studies to date have been heterogenous, and there are a particular lack of data concerning the utility of swabs for the analysis of the microbiota’s functionality and metabolome. We compared paired stool and rectal swab samples from healthy individuals to investigate whether rectal swabs are a reliable proxy for faecal sampling. There were no significant differences in key alpha and beta diversity measures between swab and faecal samples, and inter-subject variability was preserved. Additionally, no significant differences were demonstrated in abundance of major annotated phyla. Inferred gut functionality using Tax4Fun2 showed excellent correlation between the two sampling techniques (Pearson’s coefficient r = 0.9217, P < 0.0001). Proton nuclear magnetic resonance (1H NMR) spectroscopy enabled the detection of 20 metabolites, with overall excellent correlation identified between rectal swab and faecal samples for levels all metabolites collectively, although more variable degrees of association between swab and stool for levels of individual metabolites. These data support the utility of rectal swabs in both compositional and functional analyses of the gut microbiota.

Vertical Transmission of Severe Acute Respiratory Syndrome Coronavirus 2: Current Evidence and Perspectives

Vertical Transmission of Severe Acute Respiratory Syndrome Coronavirus 2: Current Evidence and Perspectives

Monkeypox Outbreak 2022: Clinical and Virological Features of 30 Patients at the Sexually Transmitted Diseases Centre of Sant' Orsola Hospital, Bologna, Northeastern Italy.

Monkeypox infection is a zoonosis first described in humans in 1970 in Congo. While previously manifesting in small, confined outbreaks, the disease is rapidly spreading globally. The aim of this study was to investigate microbiological samples (skin, rectal, and oropharyngeal swab samples and plasma and urine samples) that can help in adequate diagnostic, therapeutic, and prognostic management. We present 30 laboratory-confirmed monkeypox patients with peculiar clinical and virological features admitted to the Sexually Transmitted Diseases Centre of Sant' Orsola Hospital, University of Bologna, in the period between 20 June and 10 August 2022. Demographic, anamnestic, and clinical data were obtained, and microbiological samples were collected and analyzed by real-time PCR to detect the presence of monkeypox virus (MPXV) DNA. All monkeypox patients were adult men who have sex with men (MSM) (mean age, 37.5 years). Nonskin samples were collected from 29 patients during the acute phase of the infection. The detection rates of MPXV DNA in plasma, urine, and oropharyngeal swab samples (82.3%, 64.7%, and 75.0%, respectively) were highest in samples collected 4 to 6 days after symptom onset. The presence of MPXV in plasma and urine samples was analyzed 11 to 38 days after symptom onset to monitor viral shedding duration. Interestingly, MPXV DNA was detected in a urine sample collected on day 21 in one patient. Prolonged positivity in urine after the clinical recovery suggests a potential source of infection by contamination of wastewater and sewage and transmission to possible animal reservoirs and highlights the need for further investigations on nonskin samples to extend and more adequately standardize the patient isolation period.