Mucosal Swabs Research Articles

Validated HPLC method for simultaneous determination of azelastine hydrochloride fluticasone propionate and oxymetazoline in nasal mucosa and nasopharyngeal swabs from real human samples

A combination of three co-administrated drugs, such as azelastine hydrochloride (AZT), fluticasone propionate (FP), and oxymetazoline (OXY), is more effective than single therapy for the treatment of seasonal allergy and COVID-19. We established an efficient methodology for the determination of those analytes in spiked nasal mucosa and nasopharyngeal swabs from real human samples. A simple and quick protein precipitation method was used for sample extraction, using acetonitrile. RP-HPLC/DAD method was performed using an Exsil 100 ODS C18 (250 × 4.6 mm, 5 μm) column with an acetonitrile: water (70:30 v/v) solvent system at a flow rate of 0.7 mL/min. A photodiode array detector was applied at 240 nm. A good separation of the three proposed analytes with a short run time of 10 min was noted. Our method was validated according to FDA guidelines for bioanalytical validation methods. Calibration curves were linear in nasal mucosa samples at concentration ranges of 8–125, 10–100, and 10–125 µg/mL, with average recoveries ± SD of 101.56%±0.39, 102.45%±0.86, and 104.61%±4.52 for AZT, FP, and OXY; respectively. The results of precision and accuracy are within acceptable limits. According to stability assays, the three analytes under investigation were stable throughout sample preparation, storage, and injection. Our method was applied to real nasopharyngeal swabs. It shows that the results of the swabs were not affected by gender or age. Good recoveries with low % RSD were observed: 99.03% ± 0.75, 100.02% ± 0.94, and 100.94% ± 1.98 for both genders, and 100.45% ± 0.96, 100.69% ± 1.08, and 100.32% ± 1.53 for different ages for AZT, FP, and OXY; respectively. Moreover, the amount of those drugs in the nasal mucosa was observed for seven hours, and a constant concentration with a low% RSD was noted for the first four hours. Therefore, this method can be applied to monitor the therapeutic dose in the nasal mucosa for the determination of those analytes.

The Effectiveness of Salivary Sampling for the Detection and Quantification of Aggregatibacter actinomycetemcomitans in Periodontitis Patients

The objective was to evaluate using unstimulated saliva in detecting Aggregatibacter actinomycetemcomitans and to compare the saliva and subgingival and mucosa membrane occurrence of this periodontal pathogen in patients diagnosed with advanced periodontitis. Patients with advanced forms of periodontitis (n = 220; mean age: 54.03 ± 03 years) at stage III/IV were sampled. Unstimulated saliva, buccal cheek mucosa, and pooled subgingival plaque samples were collected. The identification of A. actinomycetemcomitans was performed using qPCR. A descriptive analysis and Wilcoxon test and analysis of variance were performed. A. actinomycetemcomitans was isolated from 28.18% of the subjects. A total of 660 samples were obtained, 220 from unstimulated saliva, 220 from buccal cheek mucosa surfaces, and 220 from pooled subgingival plaque samples. A. actinomycetemcomitans was isolated from 21.80% of unstimulated saliva, 19.50% of buccal cheek swabs, and 17.70% of subgingival samples. There was a statistically significant difference between the presence of A. actinomycetemcomitans in the unstimulated saliva samples and in the buccal cheek mucosa swab samples and pooled subgingival plaque samples (p < 0.001). These results suggest that in advanced periodontitis, unstimulated saliva is representative of pooled subgingival plaque/buccal cheek mucosa samples and its use is adequate in the oral detection of A. actinomycetemcomitans in a cohort of patients with stage III and IV periodontitis.

Application of machine learning algorithms to identify serological predictors of COVID-19 severity and outcomes

BackgroundCritically ill hospitalized patients with COVID-19 have greater antibody titers than those with mild to moderate illness, but their association with recovery or death from COVID-19 has not been characterized.MethodsIn a cohort study of 178 COVID-19 patients, 73 non-hospitalized and 105 hospitalized patients, mucosal swabs and plasma samples were collected at hospital enrollment and up to 3 months post-enrollment (MPE) to measure virus RNA, cytokines/chemokines, binding antibodies, ACE2 binding inhibition, and Fc effector antibody responses against SARS-CoV-2. The association of demographic variables and more than 20 serological antibody measures with intubation or death due to COVID-19 was determined using machine learning algorithms.ResultsPredictive models reveal that IgG binding and ACE2 binding inhibition responses at 1 MPE are positively and anti-Spike antibody-mediated complement activation at enrollment is negatively associated with an increased probability of intubation or death from COVID-19 within 3 MPE.ConclusionsAt enrollment, serological antibody measures are more predictive than demographic variables of subsequent intubation or death among hospitalized COVID-19 patients.

Fatal borealpox in an immunosuppressed patient treated with antivirals and vaccinia immunoglobulin - Alaska, 2023.

Borealpox virus (BRPV, formerly known as Alaskapox virus) is a zoonotic member of the Orthopoxvirus genus first identified in a person in 2015. In the six patients with infection previously observed BRPV involved mild, self-limiting illness. We report the first fatal BRPV infection in an immunosuppressed patient. A man aged 69 years from Alaska's Kenai Peninsula was receiving anti-CD20 therapy for chronic lymphocytic leukemia. He presented to care for a tender, red papule in his right axilla with increasing induration and pain. The patient failed to respond to multiple prescribed antibiotic regimens and was hospitalized 65 days postsymptom onset for progression of presumed infectious cellulitis. BRPV was eventually detected through orthopoxvirus real-time polymerase chain reaction testing of mucosal swabs. He received combination antiviral therapy, including 21 days of intravenous tecovirimat, intravenous vaccinia immunoglobulin, and oral brincidofovir. Serial serology was conducted on specimens obtained posttreatment initiation. The patient's condition initially improved with plaque recession, reduced erythema, and epithelization around the axillary lesion beginning one-week post-therapy. He later exhibited delayed wound healing, malnutrition, acute renal failure, and respiratory failure. He died 138 days postsymptom onset. Serologic testing revealed no evidence the patient generated a humoral immune response. No secondary cases were detected. This report demonstrates that BRPV can cause overwhelming disseminated infection in certain immunocompromised patients. Based on the patient's initial response, early BRPV identification and antiviral therapies might have been beneficial. These therapies, in combination with optimized immune function, should be considered for patients at risk for manifestations of BRPV.

Analysis Of The Result Of DNA (Deoxyribonucleic Acid) Electrophoresis On Vaginal Mucosal Swabs With Cervical Cancer Patients

Background: Cervical cancer is one of the second-highest sexually transmitted diseases in the world, accounting for 13% of the total 22% of deaths caused by non-communicable diseases. Today there have been many developments about cervical cancer diagnosis methods especially in early detection aimed at recognizing cervical cancer at an earlier stage, including molecular biology detection using PCR tools and electrophoresis. Objective: To identify the results of DNA electrophoresis in vaginal mucosal swabs with cervical cancer. Method : The research method used is experimental. Results: Based on the results of a study using a documentary gel, 2 samples of HPV DNA tape were detected, it was tested positive because there was 450 bp DNA tape and 4 samples of negative results were obtained. Conclusion: The frequency distribution of respondents from 6 samples by using electrophoresis and focumerary gel as readers detected 2 positive samples. A positive result is expressed if a result in the documentary gel is found to be a DNA band. The size of the DNA ribbon produced with the DNA marker markers is at 450 bp, according to the sample target. This shows that the DNA bands in the sample are about 450 bp in size.

Untangling the role of environmental and host-related determinants for on-farm transmission of verotoxin-producing Escherichia coli O157

ABSTRACT Background: Cattle colonised by the zoonotic pathogen verotoxin-producing Escherichia coli of serotype O157 (VTEC O157) can shed high levels of the pathogen in their faeces. A suggested key for controlling VTEC O157 is preventing colonisation of individuals. Aim: In this study the role of individual super-shedders and factors related to susceptibility and environmental exposure in the transmission of VTEC O157 among dairy calves are explored. Methods: The association between sex, age, pen hygiene, pen type and stocking density and colonisation of individual calves, established by recto-anal mucosal swabs, on farms where pathogenic VTEC O157 had been confirmed was investigated. In a follow-up sampling, the consistency of previously identified risk factors and the role of shedding pen mates was assessed by studying the risk of new/re-colonisation. Results: The results suggest an important role of stocking density that decreases with age, possibly due to increased resistance to colonisation following exposure. However, previous colonisation did not influence the risk of being colonised in the second sampling. Super-shedders (shedding >103 colony forming units/g faeces) significantly increased the risk of colonisation in peers (OR = 10, CI 4.2–52). In addition, environmental factors associated with survival of the bacteria, affected risk. Conclusion: The results confirm the suggested importance of super-shedders but also emphasises the importance of considering the combined exposure from peers and the environment.

SARS-CoV-2 infection is associated with intestinal permeability, systemic inflammation, and microbial dysbiosis in hospitalized patients.

Coronavirus disease 2019 (COVID-19) and its associated severity have been linked to uncontrolled inflammation and may be associated with changes in the microbiome of mucosal sites including the gastrointestinal tract and oral cavity. These sites play an important role in host-microbe homeostasis, and disruption of epithelial barrier integrity during COVID-19 may potentially lead to exacerbated inflammation and immune dysfunction. Outcomes in COVID-19 are highly disparate, ranging from asymptomatic to fatal, and the impact of microbial dysbiosis on disease severity is unclear. Here, we obtained plasma, rectal swabs, oropharyngeal swabs, and nasal swabs from 86 patients hospitalized with COVID-19 and 12 healthy volunteers. We performed 16S rRNA sequencing to characterize the microbial communities in the mucosal swabs and measured concentrations of circulating cytokines, markers of gut barrier integrity, and fatty acids in the plasma samples. We compared these plasma concentrations and microbiomes between healthy volunteers and COVID-19 patients, some of whom had unfortunately died by the end of the study enrollment, and performed a correlation analysis between plasma variables and bacterial abundances. Rectal swabs of COVID-19 patients had reduced abundances of several commensal bacteria including Faecalibacterium prausnitzii and an increased abundance of the opportunistic pathogens Eggerthella lenta and Hungatella hathewayi. Furthermore, the oral pathogen Scardovia wiggsiae was more abundant in the oropharyngeal swabs of COVID-19 patients who died. The abundance of both H. hathewayi and S. wiggsiae correlated with circulating inflammatory markers including IL-6, highlighting the possible role of the microbiome in COVID-19 severity and providing potential therapeutic targets for managing COVID-19.IMPORTANCEOutcomes in coronavirus disease 2019 (COVID-19) are highly disparate and are associated with uncontrolled inflammation; however, the individual factors that lead to this uncontrolled inflammation are not fully understood. Here, we report that severe COVID-19 is associated with systemic inflammation, microbial translocation, and microbial dysbiosis. The rectal and oropharyngeal microbiomes of COVID-19 patients were characterized by a decreased abundance of commensal bacteria and an increased abundance of opportunistic pathogens, which positively correlated with markers of inflammation and microbial translocation. These microbial perturbations may, therefore, contribute to disease severity in COVID-19 and highlight the potential for microbiome-based interventions in improving COVID-19 outcomes.

Intergenerational continuation of parent-child separation and 1-year telomere length attrition among mother-offspring dyads in rural China: The moderating effects of resilience

BackgroundAlthough stressor exposure early in life was known risk factor for telomere length (TL) attrition, limited literature explored it across generations. Furthermore, the effects of resilience have rarely been examined. Here, we examined whether the effects of intergenerational parent-child separation on offspring 1-year TL attrition vary by the levels of resilience. MethodIn a sample of 342 mother-child dyads living in rural China, the intergenerational continuation of parent-child separation was defined as the two generations both experiencing parent-child separation from both parents for >6 months a year early in life assessed by the parent-reported questionnaire, whereas intergenerational discontinuity refers to parent-child separation exposed in one generation only. TL was measured at baseline (from June to November 2021) and 1-year later with children's buccal mucosa swabs, with resilience polygenic risk scores (PRS) evaluated based on 4 single-nucleotide variations in 4 resilience-related genes (OXTR, FKBP5, NPY, and TNF-α). ResultsAmong 342 mother-offspring dyads, 35 (10.2 %) experienced intergenerational continuation of parent-child separation, and 139 (40.6 %) were identified as discontinuous. Remarkably, a 0.12-point reduction in TL attrition was only associated with intergenerational continuation of parent-child separation (95 % CI: 0.04, 0.21, P < 0.01) but not discontinuity. Importantly, the association between intergenerational continuation of parent-child separation with accelerated TL attrition disappeared in offspring with high resilience PRS (β = 0.07, 95%CI: −0.06, 0.21). ConclusionOur findings highlight the importance of breaking the intergenerational cycle of parent-child separation and the moderating effects of resilience on TL attrition for children exposed to adversity.

Genetic Diversity and Zoonotic Potential of Shiga Toxin-Producing E. coli (STEC) in Cattle and Buffaloes from Islamabad, Pakistan

Shiga toxin-producing E. coli (STEC) are considered important zoonotic pathogens of great economic significance, associated with diarrhea, hemolytic uremic syndrome (HUS), hemorrhagic colitis (HC), and death in humans. This study aimed to investigate the distribution of various STEC virulence gene markers and antimicrobial susceptibility (AST) profiles associated within E. coli isolates from the recto-anal mucosal swabs (RAMSs) of slaughtered cattle and buffaloes in Islamabad, Pakistan. The RAMSs (n = 200) were analyzed using multiplex PCR for the presence of stx1, stx2, eae, and ehxA genes. Samples that were positive for one or more of the virulence genes were inoculated with Sorbitol MacConkey agar (SMAC) for isolation of STEC. The isolates were further analyzed for the presence of virulence genes using multiplex PCR. Of the 200 RAMS, 118 (59%) were positive for one or more virulence genes. E. coli isolates (n = 18) with one or more virulence genes were recovered from the 118 positive samples. The DNA of the isolates positive for one or more virulent genes was extracted and subjected to whole genome sequencing using Illumina. Analysis of the WGS data indicated that the E. coli isolates could be differentiated into 11 serotypes. Most E. coli isolates (13/18; 72.2%) carried five genes (stx1, stx2, Iha, iss, and IpfA) in various combinations. In addition to these five genes, other virulence genes identified in these isolates were espI, ireA, espP, exhA, epeA, mcmA, mch, ast, celB, eilA, katP, and capU. The AST was performed using the Kirby–Bauer disk diffusion test. The study indicated that all the isolates were resistant to rifampicin and a significant proportion of the isolates were MDR. A wide range of antimicrobial resistance genes (ARGs) were detected among the isolates, reflecting the complex nature of resistance mechanisms. The study results indicate that cattle and buffaloes slaughtered in Islamabad might be the carriers of antimicrobial resistant STEC of zoonotic significance, thus representing a source of human infection.

Genetic Influences of Proinflammatory Cytokines on Pain Severity in Patients with Temporomandibular Disorders.

This case-control study investigated single nucleotide polymorphism (SNP) genotypes (CXC motif chemokine ligand 8 (CXCL8): rs2227306 and rs2227307 and tumor necrosis factor (TNF): rs1800629) in 85 patients with pain-related temporomandibular disorders (TMDp) and 85 controls to explore their associations with TMDp presence, pain intensity (low/high), and the presence of chronic arthralgia/myalgia. TMDp was diagnosed using a validated protocol, and polymorphisms were genotyped from buccal mucosa swabs using TaqMan assays. High pain intensity individuals had an increased risk for carrying minor allele "G" (rs2227307) and "T" (rs2227306) compared to controls (76% vs. 55.3%, p = 0.012; 72% vs. 54.1%, p = 0.030, respectively). Carriers of the minor allele "G" (rs2227307) were more prevalent in TMDp patients with arthralgia compared to controls (70.30% vs. 55.30%, p = 0.037). According to logistic regression, the most important predictors for high pain intensity were minor allele "G" of rs2227307 (OR 2.435, 95% CI 1.123-5.282), increasing age (OR 1.038, 95% CI 1.002-1.075), and female sex (OR 4.592, 95% CI 1.289-16.361). The explored gene polymorphisms were not significant risk factors for TMDp presence. These findings highlight the importance of genetic variations, particularly rs2227307, in understanding the diverse clinical manifestations of temporomandibular disorders.

Potential role of salivary lactic acid bacteria in pathogenesis of oral lichen planus

BackgroundEmerging evidence emphasized the role of oral microbiome in oral lichen planus (OLP). To date, no dominant pathogenic bacteria have been identified consistently. It is noteworthy that a decreased abundance of Streptococcus, a member of lactic acid bacteria (LAB) in OLP patients has been commonly reported, indicating its possible effect on OLP. This study aims to investigate the composition of LAB genera in OLP patients by high-throughput sequencing, and to explore the possible relationship between them.MethodsWe collected saliva samples from patients with OLP (n = 21) and healthy controls (n = 22) and performed 16 S rRNA gene high-throughput sequencing. In addition, the abundance of LAB genera was comprehensively analyzed and compared between OLP and HC group. To verify the expression of Lactococcus lactis, real time PCR was conducted in buccal mucosa swab from another 14 patients with OLP and 10 HC. Furthermore, the correlation was conducted between clinical severity of OLP and LAB.ResultsOLP and HC groups showed similar community richness and diversity. The members of LAB, Lactococcus and Lactococcus lactis significantly decreased in saliva of OLP cases and negatively associated with OLP severity. In addition, Lactococcus and Lactococcus lactis showed negative relationship with Fusobacterium and Aggregatibacter, which were considered as potential pathogens of OLP. Similarly, compared with healthy controls, the amount of Lactococcus lactis in mucosa lesion of OLP patients was significantly decreased.ConclusionsA lower amount of Lactococcus at genus level, Lactococcus lactis at species level was observed in OLP cases and associated with disease severity. Further studies to verify the relationship between LAB and OLP, as well as to explore the precise mechanism is needed.

Comparison of DNA extraction methods for genotyping equine histidine-rich glycoprotein insertion/deletion polymorphisms using oral mucosa swabs and feces

Previously, we demonstrated unique insertion/deletion polymorphisms of equine histidine-rich glycoprotein (eHRG) with five genotypes composed of 45-bp or 90-bp deletions in the histidine-rich region of eHRG in Thoroughbred horses. Although leukocytes are typically used to collect DNA for genotyping, blood sampling from animals is sometimes difficult and invasive. Moreover, the method for extracting DNA from blood leukocytes involves complicated steps and must be performed soon after blood sampling for sensitive gene analysis. In the present study, we performed eHRG genotyping using DNA, isolated from oral mucosa swabs collected by rubbing the mucosa on the underside of the upper lip of horses and 100 mg of freshly excreted feces obtained by scraping their surface. In the present study, we performed eHRG genotyping using DNA isolated from oral mucosa swabs and feces of horses (18 Thoroughbreds, 17 mixed breeds, 2 warm bloods), and compared the accuracy of this method with that of the method using DNA from leukocytes. The DNA derived from oral mucosa swabs was sufficient in quantity and quality for eHRG genotyping. However, DNA derived from fecal samples requires a more sensitive detection system because of contamination with non-horse DNA, and the test quality is low. Collection of oral mucosa swabs is less invasive than blood sampling; further, oral swabs can be stored for a longer period in a specified high-quality solution. Therefore, collecting DNA samples from oral mucosa swabs is recommended for the genetic analysis of not only horses but also other animals that are not accustomed to humans.

Genetic influence on treatment outcomes in patients with pain-related temporomandibular disorders.

Single nucleotide polymorphisms (SNPs) may influence pain susceptibility and impact treatment response in pain-related temporomandibular disorders (TMDp). Explore the role of COMT (rs4646310, rs6269, rs4818, rs4680) and OPRM1 (rs1799971) genotypes in regulating treatment response. Sixty TMDp patients (55 females and 5 males), diagnosed with the Diagnostic Criteria for TMD (DC/TMD), underwent standardised treatment (information and education, home physical therapy, occlusal splint) for 6 months. Treatment outcomes included: pain intensity, pain-free mouth opening, jaw functional limitation, depression, and anxiety. Genotyping for COMT and OPRM1 SNPs was performed using DNA from buccal mucosa swabs and TaqMan assays. Statistical analysis was carried out to compare the changes in treatment outcomes and the influence of genotypes on treatment response. Significantly less pain reduction was observed in minor allele carriers of rs4646310, and rs4680 compared to dominant homozygous (p < .025). Minor allele carriers of rs1799971 and rs4646310 demonstrated worsening in pain-free mouth opening while dominant homozygous exhibited improvement (p < .025). Significantly less anxiety reduction was observed in minor allele carriers of rs4646310 compared to dominant homozygous (p = .003). Of the all variables assessed in the regression model, carrying a minor allele of rs1799971 predicted a poorer treatment response considering pain-free mouth opening while carrying a minor allele of rs4646310 predicted less pain and less anxiety reduction. Our findings indicate that certain SNP variants of the COMT and OPRM1 genes were associated with poorer treatment response and may therefore play a significant role in the classification of TMDp patients. Also, assessment of patient genotype could potentially aid in predicting treatment response.

Abstract 6699: Delineating germline, tumor and extrinsic factors driving mucosal melanoma (MM) risk and response to immune checkpoint inhibitor (ICI) treatment

Abstract Introduction MM is a rare melanoma subtype with distinct biology, low immunogenicity and tumor mutational burden, and subsequently lower response rates to ICIs. The biology of MM is poorly understood. We sought to prospectively, uniformly, and comprehensively profile a cohort of MM patients to determine the interplay of molecular variation, germline predisposition, immunity, gut, mucosal and tumor microbiome and modifiable risk factors on ICI response and resistance in advanced MM. Methods Patients (pts) with MM treated with standard-of-care ICI as any treatment line presenting to MD Anderson are being prospectively enrolled for longitudinal collection for molecular, microbiome, and lifestyle factor profiling. Planned analyses include: whole genome sequencing of normal and tumor tissue, RNA sequencing, and T cell receptor sequencing from fresh tumor samples; germline sequencing and genotype analyses; NanoString Digital Spatial Profiling (DSP) from formalin-fixed paraffin embedded (FFPE) specimens; whole genome shotgun, microbiome profiling and 16S rRNA gene sequencing (16S) of fecal specimens and affected mucosal and tumor sites (swabs); participant lifestyle and patient reported outcomes (PROs) assessments. Fresh tissue is collected and used to develop PDX models and cell lines. Results 98 pts have been enrolled to date; 83 (85%) Caucasians, 8 (8%) Hispanic, 4 (4%) Asians. 67 (68%) females. Median age at MM diagnosis is 64 years (range 32-91 years). MM primary sites include 24 (24%) naso-oral, 29 (30%) urogenital, 26 (27%) anorectal, 7 (7%) conjunctival and 12 (12%) other. Clinical somatic mutation testing was performed in 76 (76%) pts and common mutations were KIT (n = 12, 16%), NRAS (n = 8, 11%) and BRAF V600 or non-V600 (n = 9, 12%). At data cut-off (November 2023), 18 (18%) pts deceased. Median follow-up from first diagnosis to last visit/death was 16 months (range 1-178 months). Sample collections at data cut-off include: blood samples (n = 95; 219 samples); fecal specimens (n = 50; 97 samples); mucosal swabs (tumor/tumor adjacent/oral cavity; n = 38; 101 samples); fresh frozen tumor/normal (n = 38; 55 samples), fresh tumor/normal (n = 28; 36 samples) and FFPE tumor (n = 21; 37 samples) tissue. PROs and dietary assessments have been collected from 47 pts. Fresh tissue for PDX model and cell line development has been collected from 11 pts. Conclusion This is an ongoing prospective study that is expected to drive insights into the tumor/microenvironment/host interactions and factors regulating immunogenicity to predict response and resistance to ICIs in a rare and understudied melanoma subtype. Interrogation of the role of the gut microbiome and its modifiable determinants will lead to the investigation of new therapeutic strategies to modulate the microbiome to improve treatment outcomes in MM. Data will be presented from initial cohort analyses. Citation Format: Florentia Dimitriou, Priyadharsini Nagarajan, Sabitha Prabhakaran, Neus Bota, Mark Knafl, Randy A. Chu, Ashish V. Damania, Pranoti V. Sahasrabhojane, Yasmine Hoballah, Jillian S. Losh, Nadim J. Ajami, Scott E. Woodman, Jennifer A. Wargo, Andrew Futreal, Jennifer L. McQuade. Delineating germline, tumor and extrinsic factors driving mucosal melanoma (MM) risk and response to immune checkpoint inhibitor (ICI) treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6699.

Validation of Salamander Dermal Mucus Swabs as a Novel, Nonlethal Approach for Amphibian Metabolomics and Glutathione Analysis Following Pesticide Exposure.

Evaluating biomarkers of stress in amphibians is critical to conservation, yet current techniques are often destructive and/or time-consuming, which limits ease of use. In the present study, we validate the use of dermal swabs in spotted salamanders (Ambystoma maculatum) for biochemical profiling, as well as glutathione (GSH) stress response following pesticide exposure. Thirty-three purchased spotted salamanders were acclimated to laboratory conditions at Washington College (Chestertown, MD, USA) for 4 weeks. Following acclimation, salamanders were randomly sorted into three groups for an 8-h pesticide exposure on soil: control with no pesticide, 2,4-dichlorophenoxyacetic acid (2,4-D), or chlorpyrifos. Before and after exposure, mucus samples were obtained by gently rubbing a polyester-tipped swab 50 times across the ventral and dorsal surfaces. Salamanders were humanely euthanized and dissected to remove the brain for acetylcholinesterase and liver for GSH and hepatic metabolome analyses, and a whole-body tissue homogenate was used for pesticide quantification. Levels of GSH were present in lower quantities on dermal swabs relative to liver tissues for chlorpyrifos, 2,4-D, and control treatments. However, 2,4-D exposures demonstrated a large effect size increase for GSH levels in livers (Cohen's d = 0.925, p = 0.036). Other GSH increases were statistically insignificant, and effect sizes were characterized as small for 2,4-D mucosal swabs (d = 0.36), medium for chlorpyrifos mucosal swabs (d = 0.713), and negligible for chlorpyrifos liver levels (d = 0.012). The metabolomics analyses indicated that the urea cycle, alanine, and glutamate metabolism biological pathways were perturbed by both sets of pesticide exposures. Obtaining mucus samples through dermal swabbing in amphibians is a viable technique for evaluating health in these imperiled taxa. Environ Toxicol Chem 2024;43:1126-1137. © 2024 SETAC.

Examination of Oral Candida Infection in Primary Sjögren's Syndrome Patients

Primary Sjögren's syndrome (pSS) is an autoimmune disease characterized by symptoms such as dry mouth, dry eyes, and other systematic symptoms. Due to the hyposalivation experienced by pSS patients, oral dysbacteriosis often occurs. A common complication of pSS is the oral Candida infection. In this article, the authors describe systematic methods that can effectively diagnose oral Candida infection and identify the Candida strains using saliva, oral mucosal swabs, or mouthwash from pSS patients. The Sabouraud's Dextrose Agar (SDA), hyphal formation assay, potassium hydroxide (KOH) smear test, and calcofluor white (CFW) staining assay are used for the diagnosis of oral Candida infection. A Candida diagnostic agar is used for the identification of Candida strains. Finally, antifungal susceptibility testing is used to determine appropriate antifungal drug treatment. This standardized method can enhance the diagnosis, treatment, and future research of pSS-related oral Candida infections. Early diagnosis, using this method, can also prevent any complications arising due to delay in receiving appropriate treatment.

Bacillus amyloliquefaciens Probiotics Mix Supplementation in a Broiler Leaky Gut Model.

The supplementation of antimicrobial growth promoters (AGPs) has been banned in many countries because of the emergence of antimicrobial-resistant pathogens in poultry products and the environment. Probiotics have been broadly studied and demonstrated as a promising AGP substitute. Our study is centred on the effects of a multi-strain Bacillus-based probiotic product on broiler production performance and gut microbial profile in a dexamethasone-induced leaky gut challenge. Two hundred and fifty-six broiler chicks were hatched and randomly assigned into four groups (wheat-soybean meal basal diet (BD) = non-supplemented control (C), BD supplemented with dexamethasone in week 4 (CD), BD containing a probiotic from day one (P), and BD containing a probiotic from day one and supplemented with dexamethasone during challenge week 4 (PD)). The production performance and caecal, gizzard, jejunal lumen and jejunal mucosa swab microbiota were studied by 16S rRNA gene sequencing. The Bacillus probiotic product significantly improved production performance and altered caecal gut microbiota (p ≤ 0.05), but no significant impact on microbiota was observed in other gut sections.

Diagnostic benefits of platelet-to-lymphocyte, neutrophil-to-lymphocyte, and albumin-to-globulin ratios in dogs with nasal cavity diseases

BackgroundA multimodal approach for diagnostic tests under anesthesia is required to diagnose nasal cavity pathology (NP) reliably in dogs. Blood test results may provide clues to the suspected NP.MethodsThis prospective blinded study assessed 72 dogs with chronic nasal discharge due to NPs, and 10 healthy dogs as the control group (CG). NPs were diagnosed using whole-body computed tomography (CT), upper airway endoscopy, examination of nasal mucosal swabs by bacterial and fungal culture, and histopathological examination of nasal mucosa biopsies. The exclusion criteria were the presence of any additional diseases or corticosteroid pre-treatment. In consideration of these exclusion criteria, 55 dogs entered the study. Dogs were classified into benign (benign tumors, idiopathic rhinitis (IR), and others) and malignant (carcinomas and sarcomas) NP groups. Blood count and blood chemistry tests were performed. The neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and albumin-to-globulin ratio (AGR) were calculated and compared.Results25 dogs with malignant NP (13 and 12 with carcinomas and sarcomas, respectively) and 30 dogs with benign NP (seven with benign tumors,13 with IR, and 10 others) were included. In general, in dogs with NP there were only slight abnormalities in complete blood count. However, PLR was significantly higher in dogs with malignant NP (carcinoma and sarcoma) than in those with benign NP and in the CG. Compared with the CG, the NLR was significantly increased in all dogs with NP, and the AGR was mild but significantly lower, except in dogs with sarcomas and benign tumors.ConclusionsIn dogs with nasal disease alone, there are usually no marked abnormalities in blood count. However, while mildly increased NLR and decreased AGR can be observed in almost all NPs, an increased PLR may indicate a malignant NP and can be used as an additional screening tool in dogs with nasal discharge due to nasal cavity pathology.

Oral Health-Related Quality of Life among Complete Denture Stomatitis Patients Treated with Methylene-Blue-Mediated Photodynamic Therapy

Aim: The aim was to assess the effect of antimicrobial photodynamic therapy (a-PDT) on the oral health-related quality of life (OHRQoL) of denture stomatitis patients. Methods: Forty patients were randomly selected to participate. Candidal proliferation was confirmed by using a CHROMagar culture and Gram staining. The denture surface and palatal mucosa were sprayed with a methylene blue photosensitizer prior to the photobiomodulation application. Laser therapy was applied two times a week at 72 h intervals for a period of 8 weeks. The OHIP-EDENT questionnaire was used to analyze the improvement in the OHRQoL. A Wilcoxon test was used to perform the candidal colony-forming unit’s count and comparison. A t-test was applied to evaluate the OHRQoL responses. Results: The overall CFU/mL values were higher in the dentures of the patients compared to a palatal mucosa swab. For instance, the CFU count was reduced from 5.56 ± 2.15 (baseline) to 3.17 ± 2.77 CFU/mL on day 60 on the palates. Similarly, the a-PDT application on the intaglio surface of the denture showed a reduction from 38.83 ± 14.71 to 29.05 ± 15.52 CFU/mL. A significant difference (p &lt; 0.05) was found in function improvement as well as a reduction in physical pain, psychological discomfort, physical disability, and social interaction among the participants after photobiomodulation treatment. Conclusions: The OHRQoL was significantly improved in the DS patients. The Candida albicans abundance was radically reduced after the a-PDT application.

Shiga toxin-producing Escherichia coli O157:H7 among diarrheic patients and their cattle in Amhara National Regional State, Ethiopia.

Shiga toxin-producing Escherichia coli O157:H7 (STEC O157:H7) is a zoonotic pathogen that causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome worldwide. This study aimed to determine the prevalence, antibiotic susceptibility, and associated risk factors of STEC O157:H7 among diarrheic patients and their cattle. A cross-sectional study was conducted among diarrheic patients and their cattle in Amhara National Regional State, Ethiopia from December- 2020 to June- 2022. A total of 1,149 diarrheic patients and 229 cattle were included in the study. STEC O157:H7 detection was done using culture, latex agglutination test, and polymerase chain reaction on diarrheic stool samples and recto-anal mucosal swabs of cattle. Antibiotic susceptibility tests were performed using disk diffusion techniques. Risk factors association were identified using binary and multivariable logistic regression analysis. The overall prevalence of STEC O157:H7 in diarrheic patients and their cattle was 11.1% (128/1149) and 14.4% (33/229) respectively. High percentage of the study subjects were found in under-five children (34.5%). Age less than 5 (AOR: 4.02, 95%CI:1.608-10.058,P = 0.003), and greater than 64 years old (AOR:3.36, 95% CI:1.254-8.986, P = 0.016), presence of diarrheic patient in the house (AOR:2.11, 95%CI:1.309-3.390, P = 0.002), availability of cattle in the house (AOR:2.52, 95%CI:1.261-5.049, P = 0.009), and habit of consuming raw foods (AOR:4.35, 95%CI:2.645-7.148, P = 0.000) were risk factors. Antibiotic resistance was shown in 109(85.2%), and 31(93.9%) isolates from diarrheic patients and their cattle respectively. The highest levels of antibiotic resistance were found to tetracycline (54.7%, 69.7%) in diarrheic patients and their cattle respectively. Multiple drug resistance was also observed among 56(43.8%) and 11(33.3%) isolates in diarrheic patients and their cattle respectively. Our study showed high prevalence of STEC O157:H7 in diarrheic patients and their cattle. Therefore, health education should be given to the community on how to care for animals, proper sanitation, and the impact of raw food consumption.