Mucosal Swab Samples Research Articles

Preliminary analysis of the buccal mucosal fungal microbiome in oral lichen planus patients.

To determine the structure and co-occurrence patterns of mucosal fungal community in oral lichen planus (OLP) patients. Mucosal swab samples from 20 OLP patients and 10 healthy controls (HCs) were collected and the mucosal mycobiomes were sequenced. The abundance, frequency, and diversity of fungi were analyzed, as well as the inter-genera interactions. The associations between fungal genera and OLP severity were further identified. At the genus level, the relative abundance of unclassified_Trichocomaceae was significantly decreased in the reticular and erosive OLP groups compared to HCs. Meanwhile, significantly lower levels of Pseudozyma were observed in the reticular OLP group compared to HCs. The negative:positive cohesiveness ratio was significantly lower in the OLP group than HCs, indicating a relatively unstable fungal ecological system in the OLP group. In the OLP group, the abundance of unclassified_Nectriaceae was significantly correlated with the reticulation/erythema/ulceration (REU) score. Compared to HCs, the decreased stability of fungal communities and the decreased abundances of two genera (unclassified_Trichocomaceae and Pseudozyma) on buccal mucosa were identified in OLP patients.

A Screening for Virus Infections among Wild Eurasian Tundra Reindeer (Rangifer tarandus tarandus) in Iceland, 2017-2019.

A winter population of around 4000-5000 wild Eurasian tundra reindeer (Rangifer t. tarandus) in the eastern part of Iceland represents descendants from 35 semi-domesticated reindeer imported to Iceland from Finnmark county, Norway, in 1787. While previous studies have indicated that they host fewer parasite species as compared to reindeer in Fennoscandia, little information exists on their exposure to reindeer viral pathogens. The aim of this study was to investigate blood from hunted reindeer for antibodies against alphaherpesvirus and gammaherpesviruses (malignant catarrhal fever viruses, MCFV), pestivirus, bluetongue virus, and Schmallenberg virus, and to investigate nasal and oral mucosal membrane swab samples for the presence of parapoxvirus-specific DNA. Blood samples collected during the hunting seasons in 2017 (n = 40), 2018 (n = 103), and 2019 (n = 138) were tested for viral antibodies using enzyme-linked immunosorbent assays (ELISA). Screening for parapoxvirus DNA was conducted on swab samples from 181 reindeer by polymerase chain reaction (PCR), targeting the B2L and GIF genes. Antibodies against pestivirus were detected in two animals from 2017, and antibodies against MCFV were detected in two reindeer from 2018. No antibodies were detected against the other viruses tested. Parapoxvirus-specific DNA was detected in nasal swab samples from two animals sampled in 2019. This study suggests that the investigated viral infections are either not present or present at a low prevalence only, probably not representing a major health threat to this reindeer population. The lack of exposure to alphaherpesvirus, an enzootic pathogen in most investigated Rangifer populations, was unexpected.

Effect of a direct-fed microbial (10-G Armor) on feedlot performance, carcass characteristics, and prevalence of Salmonella in fed-beef heifers.

Crossbred beef heifers (N = 1,394; initial shrunk body weight [BW] 291 ± 9.9 kg) were used to investigate the efficacy of 10-G Armor (Life Products, Inc., Norfolk, NE; 10-G) upon feedlot performance, carcass characteristics, and fecal and subiliac lymph nodes Salmonella prevalence. Heifers were blocked by day of arrival and allocated to 1 of 20 pens (N = 70 heifers/pen) and assigned one of two treatments (10 pens/treatment): no direct-fed microbial (CON) or 2 g/heifer/d of Lactobacillus acidophilus, Enterococcus faecium, Pediococcus pentosaceus, Lactobacillus brevis and Lactobacillus plantarum, respectively (Life Products, Inc., Norfolk, NE; 10-G). Twenty-four animals were randomly selected from each pen for Salmonella sampling. Recto-anal mucosal swab samples (RAMS) were obtained at initial processing and harvest; subiliac lymph nodes were collected at harvest. In addition, pen surface fecal pats were collected and composited by pen (10 pats per composite, 5 composites per pen) on days 0, 52, 120, and 192. Data were analyzed as a generalized complete block design, and pen served as the experimental unit. No differences were observed in live growth performance metrics (P ≥ 0.55). Yield grade distributions did not differ between treatments (P ≥ 0.62); however, cattle fed 10-G tended (P = 0.06; 14.6% vs. 18.9%) to have fewer USDA Select carcasses and more (P = 0.09; 73.6% vs. 78.0%) USDA Choice carcasses. Cattle fed 10-G tended (P = 0.10; 9.2% vs. 12.3%) to have fewer liver abscesses and had fewer (P = 0.04; 5.3% vs. 8.5%) severe liver abscesses. Salmonella prevalence of RAMS did not differ between treatments at initial processing (P = 0.97; CON = 11.6%, 10-G = 11.5%) or at harvest (P = 0.91; CON = 99.0%, 10-G = 98.6%); however, RAMS differed (P < 0.01) in Salmonella prevalence between the two collection times. Cattle fed 10-G had a lower frequency of Salmonella positive lymph nodes (P = 0.01; CON = 15.8%, 10-G = 7.4%) than CON. However, Salmonella log (mpn/g) of lymph nodes did not differ between treatments at harvest (P = 0.34; CON = 0.73, 10-G = 0.34). These data indicate that cattle fed 10-G have decreased rates of severe liver abscesses without altering live animal performance or carcass characteristics. Supplementation of 10-G significantly reduced the prevalence rate of Salmonella recovered from the subiliac lymph nodes. The factors responsible for the observed difference in the effects of 10-G on Salmonella warrant further investigation

COVID-19 cynomolgus macaque model reflecting human COVID-19 pathological conditions

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global threat to human health and life. A useful pathological animal model accurately reflecting human pathology is needed to overcome the COVID-19 crisis. In the present study, COVID-19 cynomolgus monkey models including monkeys with underlying diseases causing severe pathogenicity such as metabolic disease and elderly monkeys were examined. Cynomolgus macaques with various clinical conditions were intranasally and/or intratracheally inoculated with SARS-CoV-2. Infection with SARS-CoV-2 was found in mucosal swab samples, and a higher level and longer period of viral RNA was detected in elderly monkeys than in young monkeys. Pneumonia was confirmed in all of the monkeys by computed tomography images. When monkeys were readministrated SARS-CoV-2 at 56 d or later after initial infection all of the animals showed inflammatory responses without virus detection in swab samples. Surprisingly, in elderly monkeys reinfection showed transient severe pneumonia with increased levels of various serum cytokines and chemokines compared with those in primary infection. The results of this study indicated that the COVID-19 cynomolgus monkey model reflects the pathophysiology of humans and would be useful for elucidating the pathophysiology and developing therapeutic agents and vaccines.

Pathogen surveillance in Southern Ocean pinnipeds

Knowledge of the health status and potential effect of disease outbreaks among Southern Ocean fauna may be decisive for its conservation. We assessed the exposure and infection of Antarctic fur seals (Arctocephalus gazella, AFS) and Southern elephant seals (Mirounga leonine, SES) to parapoxvirus, Phocid alphaherpesvirus-1 (PhHV-1), smooth Brucella spp. and Toxoplasma gondii. AFS (n = 65) serum and swab samples, and SES (n = 13) serum samples from the sub--Antarctic island of Bouvetøya (54°25’S, 03°22’E) were collected during two austral summers (2014/15, 2017/18). Three polymerase chain reaction (PCR) tests amplifying the DNA polymerase, B2L and GIF parapoxvirus genomic regions were performed, investigating DNA from mucosal swab samples. The glycoprotein B gene was targeted to detect PhHV-1 viral DNA. Sera were assayed for T. gondii and smooth Brucella spp. antibodies with indirect enzyme-linked immunosorbent assays. Parapoxvirus PCR amplicons of the expected size were generated in two of the 29 AFS pups (nasal swabs, 2014/15), targeting the B2L (n = 2) and DNA polymerase (n = 1) genes, whereas the GIF PCR did not amplify target sequences. The PCR amplicons were sequenced and blasted in GenBank, revealing highest similarity with a seal parapoxvirus, confirming the presence of the virus in AFS for the first time. No PhHV-1 amplicons were generated, and antibodies against T. gondii or smooth Brucella spp. were not detected. Our data indicate that these seals are host for parapoxvirus but are neither exposed to smooth Brucella spp. nor T. gondii. Evidence of PhHV-1 shedding was not detected.

Assessing tissue transcription biomarkers of chronic rhinosinusitis: a comparison of sampling methodologies.

Chronic rhinosinusitis (CRS) is a spectrum of complex inflammatory conditions of the sinonasal mucosa. Identification of biomarkers that enable classification and improved delineation among CRS endotypes is of increasing interest. However, the extent to which less invasive sampling methods identify genuine tissue inflammatory patterns is not well understood. The aim of this study was to investigate mucosal swab and cytobrush sampling as less invasive proxies for tissue transcription levels of putative biomarkers of CRS. Expression levels of 21 biomarkers of interest were assessed via custom TaqMan array cards from mucosal biopsy, cytobrush, and swab samples, in 32 patients with CRS. Reported expression levels were compared between each of the 3 sample types within each patient. Reported transcription levels from swab samples for IL33, MUC5AC, IL1RN, CXCL8 (IL-8), TNF, IFNG, IL5, OSM, IL1A, and IL17C, and cytobrush levels for IL33, MUC5AC, IL5RA, IL1RN, CXCL8 (IL-8), and IL5 were significantly different to tissue levels from matched biopsy samples. Reported expression via swab and cytobrush sampling differed from patterns observed in matched tissue for 10 of 21 and 6 of 21 markers, respectively. Non-biopsy-based studies for these particular markers may therefore not adequately represent tissue inflammatory processes and should be interpreted with caution. Cytobrush samples largely tracked tissue patterns for the remaining target biomarkers. In these cases, cytobrush sampling appears to adequately reflect tissue patterns for several putative biomarkers of CRS, supporting their use in clinical and research settings as a less-invasive proxy for the assessment of mucosal tissue inflammatory transcription patterns.

Recto-Anal Junction (RAJ) and Fecal Microbiomes of Cattle Experimentally Challenged With Escherichia coli O157:H7.

Cattle are the asymptomatic reservoirs of Escherichia coli O157:H7 (O157) that preferentially colonizes the bovine recto-anal junction (RAJ). Understanding the influence of O157 on the diversity of the RAJ microbiota could give insights into its persistence at the RAJ in cattle. Hence, we compared changes in bovine RAJ and fecal microbiota following O157 challenge under experimental conditions. Cattle were either orally challenged (n = 4) with1010 CFU of a streptomycin-resistant O157 strain 86–24, or mock-challenged (n = 4) with phosphate buffered saline. Rectoanal mucosal swab (RAMS) and fecal samples were collected at different time points for analysis. Alpha diversity measures (Chao1 species richness and Shannon diversity index) were found to be significantly different between RAMS and fecal samples but not influenced by O157 challenge. The Firmicutes to Bacteroidetes (F: B) ratio was higher in RAMS samples from O157 colonized animals and this may have influenced the consistent yet decreased O157 colonization at the RAJ. Specific bacterial genera that were present in relative low abundance in fecal and RAMS microbiota did not affect overall microbial diversity but were associated with O157 colonization. Differential abundance analysis (DAA) of genera in samples from O157 shedding cattle indicated significantly higher relative abundance of Paenibacillus and Fusobacterium in RAMS, and Tyzzerella in fecal samples. Mock-challenged cattle showed higher relative abundance of Intestinimonas and Citrobacter in RAMS samples, and Succinivibrio, and Prevotella 1 in fecal samples. These results suggest that O157 challenge exerts transient influence on the intestinal microbial community which in turn might promote O157 colonization in a site-specific manner.

Comparative Assessment of DNA Targets and Amplification Methods for Leishmania (Viannia) Detection in Human Samples.

.Multiple polymerase chain reaction (PCR)-based approaches have been developed for Leishmania detection in clinical and laboratory samples, and this diversity limits inter-study comparisons, meta-analyses, and generalization of findings. Towards harmonization of a molecular tool for detection of Leishmania (Viannia) for research purposes, we evaluated the concordance of 18SrDNA quantitative polymerase chain reaction (qPCR) and minicircle kinetoplastid DNA (mkDNA) PCR followed by Southern blot (PCR-SB) in in vitro infection systems and in lesion and mucosal swab samples from Colombian patients with cutaneous leishmaniasis caused by L. (Viannia). The lower limit of parasite detection of 18SrDNA qPCR and mkDNA PCR-SB was 10−1 promastigotes and one intracellular amastigote per reaction. From cutaneous lesions (n = 63), an almost perfect concordance was found between the methods (κ = 0.92, 95% CI: 0.82–1.00). Despite equal limits of detection, mkDNA PCR-SB was more efficient for parasite detection in mucosal samples than 18SrDNA qPCR or 18SrDNA digital droplet PCR. The high concordance, sensitivity, scaling potential, and feasibility of implementation of the 18SrDNA qPCR, support its selection as the L. (Viannia) in research laboratories, as a first step towards harmonization of research protocols in the region.

Potential association between Fusobacterium nucleatum enrichment on oral mucosal surface and oral lichen planus.

We determined the bacterial community structure of the buccal mucosa in patients with oral lichen planus (OLP) and evaluated the potential association of Fusobacterium nucleatum with OLP. We collected buccal mucosal swab samples of patients with OLP (n=20) and healthy controls (n=10) and performed 16S rRNA gene sequencing and real-time PCR to determine potentially different bacteria. Damaged and adjacent non-damaged mucosal swab samples of 25 OLP patients were used to detect the amount of F.nucleatum by real-time PCR. Compared with healthy controls, enrichment of Fusobacterium and Granulicatella was more abundant in patients with OLP (p=.0146 and 0.0034). The abundance of Fusobacterium and F.nucleatum was significantly enriched on buccal mucosa of patients with OLP compared with healthy controls (p=.0043 and 0.0235). Compared with adjacent non-damaged buccal mucosa of OLP patients, the amount of F.nucleatum in the damaged mucosa was significantly increased (p=.001). We examined third-level KEGG pathways for bacteria on mucosal surface and found that genes controlling sporulation and ether lipid metabolism were enriched in patients with OLP. A high amount of F.nucleatum may be associated with OLP. Further studies are required to investigate the precise association of F.nucleatum with OLP.

Viral nucleic acid analysis with PCR in lacrimal tissue and nasal swab samples of primary acquired nasolacrimal duct obstruction cases.

Purpose:To evaluate the role of viral infections in the pathogenesis of primary acquired nasolacrimal duct obstruction.Methods:The study included 48 patients diagnosed with primary acquired nasolacrimal duct obstruction undergoing dacryocystorhinostomy surgery. Prior to dacryocystorhinostomy surgery, nasal swab sample was taken from the inferior meatus at the same side. During dacryocystorhinostomy, tissue biopsy sample (2 × 2 mm) was taken from the junction area of the lacrimal sac and nasolacrimal duct. Following nucleic acid extraction, polymerase chain reaction was performed.Results:The patients consisted of 9 (18.8%) men and 39 (81.2%) women with a mean age of 51.0 ± 14.3 years. Qualitative polymerase chain reaction showed viral genome in the nasal swabs of 10 (20.8%) patients, including coronavirus 229E (three cases), coronavirus HKU1 (two cases), respiratory syncytial virus (two cases), coronavirus OC43 (one case), coronavirus NL63 (one case), and adenovirus (one case). In the dacryocystorhinostomy samples, viral genomes were detected in four (8.3%) cases, including respiratory syncytial virus (two cases), coronavirus HKU1 (one case), and adenovirus (one case). There was a statistically significant agreement between nasal mucosal swab and dacryocystorhinostomy biopsy samples in terms of respiratory syncytial virus positivity (kappa = 1.000, p = 0.001).Conclusion:Although the viral genome was detected in the samples, a direct relationship between viruses and pathogenesis of primary acquired nasolacrimal duct obstruction could not be revealed because of the low number of positive results. However, considering the profibrotic characteristics of specific viruses such as respiratory syncytial virus and adenovirus, viral infections may be one of the many predisposing factors of primary acquired nasolacrimal duct obstruction.

IRAP-PCR As A Tool For Screening HERV Polymorphisms In Nasal Mucosal Swabs

Objective: Inter-retrotransposon polymorphism Polymerase Chain Reaction (IRAP-PCR) technique allows for detecting insertional polymorphisms via amplification of the DNA fragment between two retrotransposons in plant genomes. However, this method has not been reported to be used for analyzing human samples to date. Recently, Human Endogenous Retrovirus (HERV) polymorphisms gained interest due to their potential effect on pathophysiology of certain diseases. Nevertheless, the association between HERV polymorphisms and the risk for developing nasal polyposis (NP) has not been studied. In this study, we aimed to investigate whether or not IRAP-PCR could be performed in nasal swab samples for comparing HERV polymorphisms in different nasal mucosal samples. Methods: Nasal swab samples from 16 patients were used for DNA isolation. These DNA samples were used as templates for IRAP PCR of HERV-K6, HERV-K11, HERV-L1 and HERV-L2 and PCR products were analyzed by agarose gel electrophoresis. Results: Nasal swab samples yielded enough DNA material for successfully performing IRAP-PCR. We obtained specific banding patterns the three out of four HERV sequences tested in this study. No polymorphisms was detected between samples from different patients. Similarly, polymorphic bands was not detected between the polyps or nasal mucosal swab samples obtained from the same patient. Conclusion: We have, for the first time, shown that IRAPPCR can be performed in nasal swabs. Our findings suggest that this technique can serve as an inexpensive and effective screening tool for investigating links between nasal mucosal diseases and HERV polymorphisms such as nasal polyposis.

The sinonasal mycobiota in chronic rhinosinusitis and control patients.

While bacterial associations with chronic rhinosinusitis (CRS) are increasingly well described, fewer studies have examined the fungal component of the sinonasal microbiota. Here we present a study of the sinonasal mycobiota in a cohort of 144 patients (106 patients with CRS and 38 controls). Fungal communities were characterised by analysis of mucosal swab samples of the left and right middle meatuses via ITS2 marker amplicon sequencing on the Illumina MiSeq platform. Fungal associations with previously published bacterial community and inflammatory cytokine and cell data for this cohort (collected at the same intra-operative time point) were also investigated. Malassezia spp. were ubiquitous and often highly predominant. Season of sampling explained more of the variability in the data than any of the clinical parameters. The predominant Malassezia sp. was distinct in patients with cystic fibrosis compared to those without. However, distinctions in the mycobiota were not evident between any other patient groupings assessed, and few fungal-bacterial or fungal-inflammatory associations were observed. This study confirms the prominent place of Malassezia spp. within the upper respiratory tract. Overall, few distinctions between patient groups were evident, and these data lend further support to the hypothesis that fungal community types may have no direct causative association with idiopathic CRS. Additional studies incorporating a broader array of inflammatory markers are required to assess whether these ubiquitous fungi nonetheless play an exacerbating role in some sensitive individuals.

An investigation of shedding and super-shedding of Shiga toxigenic Escherichia coli O157 and E.coli O26 in cattle presented for slaughter in the Republic of Ireland.

Shiga toxigenic Escherichia coli (STEC) are an important group of pathogens and can be transmitted to humans from direct or indirect contact with cattle faeces. This study investigated the shedding of E.coli O157 and O26 in cattle at the time of slaughter and factors associated with super-shedding (SS) animals. Rectoanal mucosal swab (RAMS) samples were collected from cattle (n=1,317) at three large Irish commercial beef abattoirs over an 18month period, and metadata were collected at the time of sampling regarding farm of origin, animal age, breed and gender. RAMS swabs were examined for the presence and numbers of E.coli O157 and O26 using a previously developed quantitative real-time PCR protocol. Samples positive by PCR were culturally examined and isolates analysed for the presence of stx subtypes, eae and phylogroup. Any samples with counts >104 CFU/swab of STEC O157 or O26 were deemed to be super-shedders. Overall, 4.18% (55/1,317) of RAMS samples were positive for STEC O157, and 2.13% (28/1,317) were classified as STEC O157 SS. For STEC O26, 0.76% (10/1,317) of cattle were positive for STEC O26, and 0.23% (3/1,317) were classified as super-shedders. Fewer STEC shedders and SS were noted among older animals (>37months). There was a seasonal trend observed for STEC O157, with the highest prevalence of shedding and SS events in the autumn (August to October). The majority of E.coli O157 (50/55) isolates had stx2 and were eae positive, with no significant difference between SS and low shedders (LS). Interestingly, all STEC O26 (n=10) were eae negative and had varied stx profiles. This study demonstrates that, while the overall shedding rates are relatively low in cattle at slaughter, among positive animals there is a high level of SS, which may pose a higher risk of cross-contamination during slaughter.

Impact of systemic antimicrobial therapy on mucosal staphylococci in a population of dogs in Northwest England.

Antimicrobial-resistant bacteria are increasingly isolated from veterinary patients. To determine risk factors for antimicrobial resistance (AMR) among canine mucosal staphylococci following routine antimicrobial treatment with cefalexin (CFX), clavulanate-amoxicillin (AC), cefovecin (CVN), clindamycin (CD) or a fluoroquinolone (FQ). Mucosal swab samples (n = 463) were collected from 127 dogs pre-treatment, immediately, and at one- and three-months post-treatment. Staphylococci were identified phenotypically and biochemically as coagulase negative (CoNS) or coagulase positive (CoPS); CoPS were speciated by nuc gene PCR. Antimicrobial susceptibility was determined using disc diffusion and mecA gene carriage by PCR. Multilevel, multivariable models examined associations between risk factors and presence/absence of CoPS, meticillin resistance (MR), multidrug-resistance (MDR) and fluoroquinolone resistance (FQR). The percentage of samples with CoNS increased and with CoPS (including S. pseudintermedius) decreased immediately post-treatment with CFX, CVN and CD (P ≤ 0.001) and one month post-treatment with CD (P = 0.003). By three months post-treatment, there was no significant difference compared to pre-treatment samples. Immediately post-treatment with FQs there was significantly increased risk of isolating MRS (P = 0.002), MDR (P = 0.002) or FQR (P = 0.013) staphylococci and of MDR following CFX treatment (P = 0.019). The percentage of samples with AMR staphylococci declined from immediately to three months post-treatment and there was no significant difference between resistance prevalence at one or three months post-treatment for most AMR traits and treatment groups. Exceptions include increased MDR following FQ (P = 0.048) or CFX (P = 0.021), at one and three months post-treatment, respectively. Systemic antimicrobials impact on mucosal staphylococci. Immediately after therapy, the mucosa may be a reservoir for AMR staphylococci that are a source of mobile genetic elements carrying AMR genes.

Culture independent analysis using gnd as a target gene to assess Escherichia coli diversity and community structure

Current culture methods to investigate changes in Escherichia coli community structure are often slow and laborious. Genes such as gnd (6-phosphogluconate dehydrogenase) have a highly variable nucleotide sequence and may provide a target for E. coli microbiome analysis using culture-independent methods. Metabarcoded PCR primers were used to generate separate libraries from calf faecal samples for high throughput sequencing. Although a total of 348 separate gnd sequence types (gSTs) were identified, 188 were likely to be due to sequencing errors. Of the remaining 160 gSTs, 92 did not match those in a database of 319 separate gnd sequences. ‘Animal’ was the main determinant of E. coli diversity with limited impact of sample type or DNA extraction method on intra-host E. coli community variation from faeces and recto-anal mucosal swab samples. This culture-independent study has addressed the difficulties of quantifying bacterial intra-species diversity and revealed that, whilst individual animals may harbour >50 separate E. coli strains, communities are dominated by <10 strains alongside a large pool of subdominant strains present at low abundances. This method will be useful for characterising the diversity and population structure of E. coli in experimental studies designed to assess the impact of interventions on the gut microbiome.

Evaluation of Rectoanal Mucosal Swab Sampling for Molecular Detection of Enterohemorrhagic Escherichia coli in Beef Cattle

Cattle are a primary reservoir of enterohemorrhagic Escherichia coli (EHEC), and contaminated beef products are a source of human infections. The U.S. Department of Agriculture Food Safety and Inspection Service declared seven EHEC serogroups (O26, O45, O103, O111, O121, O145, and O157) as adulterants in raw ground beef. Sampling a large number of animals for EHEC surveillance or evaluations of EHEC-focused preharvest interventions requires a convenient and robust sampling method. We evaluated the diagnostic performance of rectoanal mucosal swab (RAMS) for the detection of the top seven EHEC serogroups. Paired fecal grab (FG) and RAMS samples were collected from 176 beef cattle and tested using the NeoSEEK Shiga toxin-producing E. coli (STEC) confirmation method. The prevalence of virulence-associated genes (stx1, stx2, stx2c, eae, and nleB) was higher in RAMS than in FG samples. The results of the two methods had poor agreement, as indicated by kappa statistics, for the detection of the seven serogroups. When FG and RAMS results were combined for comparison, RAMS was more sensitive than FG for the detection of serogroups O103 (82% versus 39%), O157 (75% versus 67%), and O45 (79% versus 73%) with similar sensitivity for the detection of serogroup O145 (67%). Serogroups O111 and O121 were detected from one and two samples, respectively, by FG and were not detected by RAMS. Serogroup O26 was not detected with either method. RAMS appears to be equivalent or superior to FG sampling for detection of the top seven EHEC serogroups in the feces of beef cattle with the NeoSEEK STEC confirmation test.

EPIGLOTTIS MICROFLORA OF ADULT PATIENTS WITH ACUTE EPIGLOTTITIS

Introduction. Nowadays acute infectious-inflammatory processes of upper respiratory tract, including acute epiglottitis retain a high proportion among human pathology. In the literature acute epiglottitis is allocated into an independent nosology as severe acute phlegmonous bacterial inflammation of the epiglottis and hypopharynx. There are currently no clear guidelines on how to classify an acute epiglottitis, as well as protocols for patients at various stages of the pathological process. According to common belief, Haemophilus influenzae type -B (Haemophilus influenza type b (Hib)) is the most common cause of epiglottitis. At present, the main etiological role in the genesis of acute epiglottitis in children belongs to haemophilus influenzae. In adults the causes of the disease are beta hemolytic streptococci groups A, B, pneumococcus, Klebsiella, Pseudomonas, Staphylococcus aureus, herpes simplex virus (type 1) and parainfluenza, and others.The aim of this work is to study: the mucosal microflora of the epiglottis in adult patients with acute epiglottitis and to study sensitivity of certain isolates to antimicrobial agents. Material & methods. 86 adult patients with acute epiglottitis were observed: 36 with abscess form of epiglottitis and 50 - with infiltrative. Microbiological analysis of mucosal swab samples taken from hypopharynx were conducted by the conventional technology: for seeding solid or liquid nutrient medium, followed by allocation of isolith and its microscopic, biochemical and serological identification. Microorganisms were classified according to schemes of Bergy. Antimicrobial susceptibility of each strain was determined in accordance with the guidelines. We used discs with antibacterial drugs. The availability of sensitive and resistant strains of microorganisms to antibiotics was assessed. A mucous membrane of the epiglottis was analyzed through microbiological investigation in 86 patients with acute epiglottitis. As a result, 169 strains of microorganisms were sowed from mucous membrane of epiglottis. Results & discussion. In patients with acute epiglottitis Streptococcus progenies dominated in 33 (23.7%) of cases, H. influenza was detected in 27 cases (19.4%), Streptococcus pneumonia strain was in 21 (12.4%) of cases, 4th place -. Staphylococcus aureus -9.3%. There are different types of: staphylococci strains -29, of Enterococcus spp - 11, gram-negative bacilli -41. Noteworthy fact is the high degree of microbial contamination of patients 107-109 CFU. Conclusion. Acute epiglottitis highest sensitivity was observed to the following medications as cephalosporin, namely cefotaxime while it is variable within the same group (1,2,3 cephalosporin’s generation). The isolated strains showed almost 99,4±1,2% sensitivity to fluoroquinolones, especially to levofloxacin and others (S. aureus, S. pyogenes, S. pneumoniae, H. Influenzae). There was a high sensitivity to lincosamides - clindamycin; fluoroquinolones- levofloxacin. In 50% of cases, a resistance to the antibiotic penicillin; and macrolide antibiotic, especially the last generations was observed. According to the obtained findings on the sensitivity of microflora to antibiotics cephalosporin’s II- III generation with moderate and severe degrees of severity in combination with fluoroquinolones (all administered parenterally) are used in acute epiglottitis. Use of fluoroquinolones (levofloxacin, ciprofloxacin, and others) is recommended in case of Cephalosporin intolerance, as well as lincosamides (clindamycin, dalatsin, lincomycin)

Helicobacter pylori in lacrimal secretions.

The aim of this study was to investigate the presence of Helicobacter pylori in human lacrimal and nasal secretions. Eighty patients with complaints of dyspepsia who had undergone endoscopies and gastric antrum biopsies were included in the study. A total of five specimens, including 2 lacrimal secretion samples, 2 nasal mucosal swab samples, and 1 gastric antrum biopsy, were collected from each patient and investigated with polymerase chain reaction (PCR) methods consisting of the urease enzyme coding gene GlmM (UreC) and the H pylori-specific 16S rRNA coding gene. The Reflux Symptom Index and ophthalmologic complaints of the patients were recorded. The detected positivity rates of the H pylori 16S rRNA coding gene in gastric biopsies and nasal mucous and lacrimal secretions were 55, 11.2, and 20%, respectively. The patients were grouped as gastric-antrum-biopsy-negative (Group I [n = 36]) and -positive (Group II [n = 44). In Group II, H pylori positivity in the lacrimal and nasal mucous secretions was 36.3 and 18%, respectively. A comparison between the groups in terms of H pylori presence in nasal mucous and lacrimal secretions yielded statistically significant differences (p = 0.0001, p = 0.003). The simultaneous presence of H pylori in nasal mucous and lacrimal secretions was 13.6% in Group II. H pylori positivity in nasal mucous and lacrimal secretions had a positive moderate correlation (r = 0.40; p = 0.0003). The present study is the first report on the presence of H pylori in lacrimal secretions through nested PCR, which suggested the presence of a number of mechanisms for H pylori transmission to lacrimal secretions.

Nationwide prevalence and risk factors for faecal carriage of Escherichia coli O157 and O26 in very young calves and adult cattle at slaughter in New Zealand.

Nationwide prevalence and risk factors for faecal carriage of Escherichia coli O157 and O26 in cattle were assessed in a 2-year cross-sectional study at four large slaughter plants in New Zealand. Recto-anal mucosal swab samples from a total of 695 young (aged 4-7 days) calves and 895 adult cattle were collected post-slaughter and screened with real-time polymerase chain reaction (PCR) for the presence of E. coli O157 and O26 [Shiga toxin-producing E. coli (STEC) and non-STEC]. Co-infection with either serogroup of E. coli (O157 or O26) was identified as a risk factor in both calves and adult cattle for being tested real-time PCR-positive for E. coli O157 or O26. As confirmed by culture isolation and molecular analysis, the overall prevalence of STEC (STEC O157 and STEC O26 combined) was significantly higher in calves [6·0% (42/695), 95% confidence interval (CI) 4·4-8·1] than in adult cattle [1·8% (16/895), 95% CI 1·1-3·0] (P < 0·001). This study is the first of its kind in New Zealand to assess the relative importance of cattle as a reservoir of STEC O157 and O26 at a national level. Epidemiological data collected will be used in the development of a risk management strategy for STEC in New Zealand.

Parasitological Confirmation and Analysis of Leishmania Diversity in Asymptomatic and Subclinical Infection following Resolution of Cutaneous Leishmaniasis.

BackgroundThe contribution of individuals with subclinical infection to the transmission and endemicity of cutaneous leishmaniasis (CL) is unknown. Immunological evidence of exposure to Leishmania in residents of endemic areas has been the basis for defining the human population with asymptomatic infection. However, parasitological confirmation of subclinical infection is lacking.MethodsWe investigated the presence and viability of Leishmania in blood and non-invasive mucosal tissue samples from individuals with immunological evidence of subclinical infection in endemic areas for CL caused by Leishmania (Viannia) in Colombia. Detection of Leishmania kDNA was conducted by PCR-Southern Blot, and parasite viability was confirmed by amplification of parasite 7SLRNA gene transcripts. A molecular tool for genetic diversity analysis of parasite populations causing persistent subclinical infection based on PCR amplification and sequence analysis of an 82bp region between kDNA conserved blocks 1 and 2 was developed.Principal FindingsPersistent Leishmania infection was demonstrated in 40% (46 of 114) of leishmanin skin test (LST) positive individuals without active disease; parasite viability was established in 59% of these (27 of 46; 24% of total). Parasite burden quantified from circulating blood monocytes, nasal, conjunctival or tonsil mucosal swab samples was comparable, and ranged between 0.2 to 22 parasites per reaction. kDNA sequences were obtained from samples from 2 individuals with asymptomatic infection and from 26 with history of CL, allowing genetic distance analysis that revealed diversity among sequences and clustering within the L. (Viannia) subgenus.ConclusionsOur results provide parasitological confirmation of persistent infection among residents of endemic areas of L. (Viannia) transmission who have experienced asymptomatic infection or recovered from CL, revealing a reservoir of infection that potentially contributes to the endemicity and transmission of disease. kDNA genotyping establishes proof-of-principle of the feasibility of genetic diversity analysis in previously inaccessible and unexplored parasite populations in subclinically infected individuals.