Peptide nucleic acid probe-assisted paper-based electrochemical biosensor for multiplexed detection of respiratory viruses.

The similar transmission patterns and early symptoms of respiratory viral infections, particularly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza (H1N1), and respiratory syncytial virus (RSV), pose substantial challenges in the diagnosis, therapeutic management, and handling of these infectious diseases. Multiplexed point-of-care testing for detection is urgently needed for prompt and efficient disease management. Here, we introduce an electrochemical paper-based analytical device (ePAD) platform for multiplexed and label-free detection of SARS-CoV-2, H1N1, and RSV infection using immobilized pyrrolidinyl peptide nucleic acid probes. Hybridization between the probes and viral nucleic acid targets causes changes in the electrochemical response. The resulting sensor offers high sensitivity and low detection limits of 0.12, 0.35, and 0.36 pM for SARS-CoV-2 (N gene), H1N1, and RSV, respectively, without showing any cross-reactivities. The amplification-free detection of extracted RNA from 42 nasopharyngeal swab samples was successfully demonstrated and validated against reverse-transcription polymerase chain reaction (range of cycle threshold values: 17.43-25.89). The proposed platform showed excellent clinical sensitivity (100 %) and specificity (≥97 %) to achieve excellent agreement (κ ≥ 0.914) with the standard assay, thereby demonstrating its applicability for the screening and diagnosis of these respiratory diseases.

Extraction-Free Testing for SARS-CoV-2 in Nasal Swab and Saliva Samples on a Single High-Throughput Platform.

The COVID-19 pandemic introduced an urgent need for rapid and high-throughput testing for SARS-CoV-2. RNA extraction is a major bottleneck for RT-qPCR. We describe a semi-automated, extraction-free RT-qPCR assay for detection of SARS-CoV-2 in nasal swab and saliva samples on a single platform. With a limit of detection of 4 copies/mL, this laboratory developed test performed equivalently to established methods requiring nucleic acid extraction. Five technologists staffing two shifts per day (80 person-hours) processed more than 400,000 samples over 10 months. Patients opted to provide nasal swab samples (83.6%) more frequently than saliva (16.4%), creating the added challenge of producing swab collection kits. Real-world testing data indicated a higher frequency of SARS-CoV-2 detection in saliva (10.1%) compared to nasal swab (7.7%). This cost-effective and quickly scalable approach is suitable for pandemic preparedness planning related to surveillance and diagnostic testing.

Unsupervised Machine Learning Reveals a Vulvodynia-Predominant Subtype in Bladder Pain Syndrome/Interstitial Cystitis.

Background Bladder pain syndrome/interstitial cystitis (BPS/IC) is a chronic condition characterized by pelvic pain and urinary symptoms. Despite its significant impact on patients' quality of life, the heterogeneity of BPS/IC symptoms and the presence of comorbidities such as vulvodynia may not be adequately captured by validated questionnaires. Identifying vulvodynia in BPS/IC patients is crucial for providing appropriate treatment options. This study aimed to identify subtypes of BPS/IC patients using unsupervised machine learning and to investigate the prevalence of vulvodynia in each subtype. Methods We conducted a prospective cross-sectional study of 123 BPS/IC patients and 64 age-matched controls. Hierarchical clustering was performed using data from validated questionnaires, including the Numerical Rating Scale-11, Interstitial Cystitis Symptom Index (ICSI), Interstitial Cystitis Problem Index (ICPI), Pelvic Pain and Urgency/Frequency scores, Overactive Bladder Questionnaire Short Form (OABq SF), Overactive Bladder Symptom Score (OABSS), and Pelvic Floor Distress Inventory-20. The optimal number of clusters was determined using the elbow method, and the characteristics of each cluster were analyzed. All participants underwent a vulvodynia swab test to assess vulvodynia symptoms. Results Unsupervised machine learning revealed three distinct clusters of BPS/IC patients. Clusters 0 and 2 differed significantly, with Cluster 2 characterized by significantly higher vulvodynia scores compared to other clusters (P < 0.001). In contrast, Cluster 2 had lower bladder pain scores (ICSI and ICPI) and overactive bladder symptom scores (OABq SF and OABSS) compared to other clusters. Clusters 0 and 1 were characterized by a predominance of bladder pain and urinary frequency symptoms, with Cluster 0 exhibiting more severe symptoms. Conclusions Our study identified distinct subtypes of BPS/IC patients using unsupervised machine learning, with Cluster 2 representing a vulvodynia-predominant subtype. This finding, along with the potential of targeted therapies such as non-ablative erbium YAG laser for vulvodynia, underscores the importance of assessing extravesical symptoms, particularly vulvodynia, for the diagnosis and treatment of BPS/IC. A tailored approach, including laser therapy for vulvodynia-predominant patients, may be necessary for optimal management of BPS/IC. The vulvodynia swab test plays a crucial role in assessing vulvodynia symptoms, underlining the limitations of validated questionnaires in capturing the full spectrum of BPS/IC symptoms. A comprehensive evaluation of patients, including the vulvodynia swab test, is essential for accurate subtyping and management of BPS/IC. Further research with larger sample sizes and investigation of the relationship between identified subtypes and other clinical data is warranted to advance our understanding and management of BPS/IC.

Evaluating diagnostic accuracy of an RT-PCR test for the detection of SARS-CoV-2 in saliva.

BACKGROUND AND OBJECTIVE: Saliva has been proposed as a potential more convenient, cost-effective, and easier sample for diagnosing SARS-CoV-2 infections, but there is limited knowledge of the impact of saliva volumes and stages of infection on its sensitivity and specificity. METHODS: In this study, we assessed the performance of SARS-CoV-2 testing in 171 saliva samples from 52 mostly mildly symptomatic patients (aged 18 to 70 years) with a positive reference standard result at screening. The samples were collected at different volumes (50, 100, 300, and 500 µl of saliva) and at different stages of the disease (at enrollment, day 7, 14, and 28 post SARS-CoV-2 diagnosis). Imperfect nasopharyngeal (NP) swab nucleic acid amplification testing was used as a reference. We used a logistic regression with generalized estimating equations to estimate sensitivity, specificity, PPV, and NPV, accounting for the correlation between repeated observations. RESULTS: The sensitivity and specificity values were consistent across saliva volumes. The sensitivity of saliva samples ranged from 70.2% (95% CI, 49.3-85.0%) for 100 µl to 81.0% (95% CI, 51.9-94.4%) for 300 µl of saliva collected. The specificity values ranged between 75.8% (95% CI, 55.0-88.9%) for 50 µl and 78.8% (95% CI, 63.2-88.9%) for 100 µl saliva compared to NP swab samples. The overall percentage of positive results in NP swabs and saliva specimens remained comparable throughout the study visits. We observed no significant difference in cycle number values between saliva and NP swab specimens, irrespective of saliva volume tested. CONCLUSIONS: The saliva collection offers a promising approach for population-based testing.

Omission of alcohol skin cleansing and risk of adverse events in long-term care residents undergoing COVID-19 vaccination: A cohort study.

Despite a lack of clinical data demonstrating the effectiveness of alcohol swab cleansing prior to vaccinations as a prophylactic measure to prevent skin infections, it is recommended for vaccine administration by the Canadian Immunization Guide. The objective of this study was to evaluate the risk of adverse events after omitting alcohol skin cleansing in long-term care (LTC) residents receiving vaccinations during the COVID-19 pandemic. Two medium-sized LTC homes participated in a cohort study, whereby one LTC used alcohol swab cleansing prior to resident vaccinations and the other did not. All residents received two doses of the BNT162b2 COVID-19 vaccine separated by an average (SD) 29.3 (8.5) days. The electronic chart records of participants were reviewed by researchers blinded to group allocation to assess for the presence of adverse events following immunization (AEFI), including reactogenicity, cellulitis, abscess, or systemic reactions. Log-binomial regression was used to compute risk ratios (with 95% confidence intervals) of an AEFI according to alcohol swab status. 189 residents were included, with a total of 56 AEFI between the two doses. The risk of reactogenicity (adjusted RR 0.54, 95% CI 0.17-1.73) or systemic reactions (adjusted RR 0.75, 95% CI 0.26-2.13) did not differ for the residents that received alcohol skin antisepsis compared to those that did not. There were no cases of cellulitis or abscess. This study did not demonstrate an elevated risk of AEFI in LTC residents receiving two doses of the BNT162b2 mRNA COVID vaccine without alcohol skin antisepsis.

Swab Testing to Optimize Pneumonia treatment with empiric Vancomycin (STOP-Vanc): study protocol for a randomized controlled trial.

Background: Vancomycin, an antibiotic with activity against Methicillin-resistant Staphylococcus aureus (MRSA), is frequently included in empiric treatment for community-acquired pneumonia (CAP) despite the fact that MRSA is rarely implicated in CAP. Conducting polymerase chain reaction (PCR) testing on nasal swabs to identify the presence of MRSA colonization has been proposed as an antimicrobial stewardship intervention to reduce the use of vancomycin. Observational studies have shown reductions in vancomycin use after implementation of MRSA colonization testing, and this approach has been adopted by CAP guidelines. However, the ability of this intervention to safely reduce vancomycin use has yet to be tested in a randomized controlled trial. Methods: STOP-Vanc is a pragmatic, prospective, single center, non-blinded randomized trial. Adult patients with suspicion for CAP who are receiving vancomycin and admitted to the Medical Intensive Care Unit at Vanderbilt University Medical Center will be screened for eligibility. Eligible patients will be enrolled and randomized in a 1:1 ratio to either receive MRSA nasal swab PCR testing in addition to usual care (intervention group), or usual care alone (control group). PCR testing results will be transmitted through the electronic health record to the treating clinicians. Primary providers of intervention group patients with negative swab results will also receive a page providing clinical guidance recommending discontinuation of vancomycin. The primary outcome will be vancomycin-free hours alive, defined as the number of hours alive and free of the use of vancomycin within the first seven days following trial enrollment estimated using a proportional odds ratio model. Secondary outcomes include 30-day all-cause mortality and time alive off vancomycin. Discussion: STOP-Vanc will provide the first randomized controlled trial data regarding the use of MRSA nasal swab PCR testing to guide antibiotic de-escalation. This study will provide important information regarding the effect of MRSA PCR testing and antimicrobial stewardship guidance on clinical outcomes in an intensive care unit setting. Trial registration: This trial was registered on ClinicalTrials.gov on February 22, 2024. (ClinicalTrials.gov identifier: NCT06272994).

Lessons Learned from Active Clinical and Laboratory Surveillance during the Sheep Pox Virus Outbreak in Spain, 2022-2023.

In September 2022, more than 50 years after its eradication from Spain, Sheep pox virus was confirmed by laboratory analysis in sheep showing characteristic lesions. This was the start of an outbreak that lasted 9 months and infected 30 farms dispersed over two different areas, Andalusia and Castilla-La Mancha. Early after the initial confirmation, an active surveillance based on clinical inspection with laboratory confirmation of sheep with clinical signs was started in restricted areas. This allowed the confirmation of Sheep pox in 22 out of 28 suspected farms, where limited numbers of sheep with mainly erythema and papules were found, indicative of early detection. Nevertheless, to improve active surveillance and stop the outbreak, clinical inspection was reinforced by laboratory analysis in all inspected farms, even when no clinically diseased sheep were detected. Although more than 35,000 oral swabs from 335 farms were analysed by real-time PCR in pools of five, only two out of six reported outbreaks in this period were detected by laboratory analysis before clinical signs were observed. Furthermore, additional insights were gained from the extensive laboratory surveillance performed on samples collected under field conditions. No evidence of Sheep pox virus infection was found in goats. Oral swabs proved to be the sample of choice for early detection in the absence of scabs and could be tested in pools of five without extensive loss in sensitivity; serology by ELISA was not useful in outbreak detection. Finally, a non-infectious genome of the virus could be detected months after cleaning and disinfection; thus, real-time PCR results should be interpreted with caution in sentinel animals during repopulation. In conclusion, the outbreak of Sheep pox virus in Spain showed that active clinical inspection with laboratory confirmation of clinically diseased sheep via oral swab testing proved a sensitive method for detection of infected farms, providing insights in laboratory surveillance that will be helpful for other countries confronted with Sheep pox outbreaks.

Feasibility, performances and predictive value of congenital CMV neonatal screening on saliva swabs.

BACKGROUND: Early diagnosis of congenital CMV infection (cCMVI) allows for early intervention and follow-up to detect delayed hearing loss. While CMV PCR in urine is the gold standard for cCMVI diagnosis, saliva testing is often performed. OBJECTIVES: Our aim was to determine (i) if swab saliva sampling needed standardization, (ii) if a threshold value in "virus copies per million cells (Mc)" in saliva samples could improve clinical specificity, and (iii) to establish a correlation between viral load in saliva and symptomatology/outcome of cCMVI. MATERIALS AND METHODS: In our institution, universal newborn screening is performed on saliva swabs at delivery or until day 3 of life. If positive, CMV PCR in urine is done within 2 weeks to confirm or exclude cCMVI. RESULTS: Cell quantification showed that saliva swab sampling was well performed as 95.4 % samples had more than 100 cells/10 µL. There was a good correlation between saliva viral load in copies/mL and in copies/Mc (Pearson's r = 0.96, p < 0.0001). However, threshold values, established to determine a viral load level at which we could confidently identify infected newborns, did not improve positive predictive value (21.8 % for copies/mL and 21 % for copies/Mc vs 25.4 % without threshold) but instead reduced sensitivity (88 % and 85% vs 100 % without threshold). Samples collected on day 2 or 3 had better positive predictive value (38.7 %) compared to those collected on day 1 (23.8 %). Symptomatology at birth was not significantly associated with viral load in saliva at diagnosis. However, sequelae occurrence was associated with viral load in saliva (copies/Mc). DISCUSSION: Our results confirm that saliva swab is a suitable sample for universal neonatal screening. However, identifying newborns that will develop sequelae remains an issue in the management of cCMVI.

Improved passive sampling methods for wastewater to enable more sensitive detection of SARS-CoV-2 and its variants.

Wastewater-based epidemiology (WBE) can be used as a part of a long-term strategy for detecting and responding rapidly to new outbreaks of infectious disease in the community. However, wastewater collected by grab samples may miss marker presence, and composite auto-sampling throughout a day is technically challenging and costly. Tampon swabs can be used as passive collectors of wastewater markers over hours, but recovery of the captured markers is a challenge. Our goal was to improve tampon elution methods for virus detection and variant analysis to increase the likelihood of detection near the Limit of Detection (LOD) and to potentially detect new or rare variants in a new outbreak. Counts of SARS-CoV-2 N1 and N2 markers in grab samples were compared to markers eluted from tampons that had been immersed in 3 sewersheds for 4-6 h during June to December 2023. We compared tampon elution methods that used different elution volumes, pressure, and amounts of Tween 20, evaluated after automated magnetic bead purification and RT-ddPCR of SARS-CoV-2 markers. Overall, method "SwabM2" in which tampons were eluted by high pressure squeeze in a 50 mL syringe after adding 2 mL of 0.5 X TE + 0.075 % Tween-20 yielded a median four-fold higher concentration of final purified SARS-CoV-2 markers than paired grab samples and significantly more than other tested tampon elution methods (p < 0.0001). Method SwabM2 was more likely to yield enough extracted nucleic acids for sequencing and also gave higher quality variant sequences than two other tampon elution methods. Variant analysis captured the Fall 2023 transition of variants from XBB to JN and "H" lineages. In summary, we demonstrated a tampon-based wastewater collecting and elution method that yielded higher counts, more detections near the LOD, and higher quality variant sequences compared to both grab samples and other tampon-based passive-collecting wastewater methods.

Genomic Insights of a Methicillin-Resistant Biofilm-Producing Staphylococcus aureus Strain Isolated From Food Handlers.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important zoonotic pathogen associated with a wide range of infections in humans and animals. Thus, the emergence of MRSA clones poses an important threat to human and animal health. This study is aimed at elucidating the genomics insights of a strong biofilm-producing and multidrug-resistant (MDR) S. aureus MTR_BAU_H1 strain through whole-genome sequencing (WGS). The S. aureus MTR_BAU_H1 strain was isolated from food handlers' hand swabs in Bangladesh and phenotypically assessed for antimicrobial susceptibility and biofilm production assays. The isolate was further undergone to high throughput WGS and analysed using different bioinformatics tools to elucidate the genetic diversity, molecular epidemiology, sequence type (ST), antimicrobial resistance, and virulence gene distribution. Phenotypic analyses revealed that the S. aureus MTR_BAU_H1 strain is a strong biofilm-former and carries both antimicrobial resistance (e.g., methicillin resistance; mecA, beta-lactam resistance; blaZ and tetracycline resistance; tetC) and virulence (e.g., sea, tsst, and PVL) genes. The genome of the S. aureus MTR_BAU_H1 belonged to ST1930 that possessed three plasmid replicons (e.g., rep16, rep7c, and rep19), seven prophages, and two clustered regularly interspaced short palindromic repeat (CRISPR) arrays of varying sizes. Phylogenetic analysis showed a close evolutionary relationship between the MTR_BAU_H1 genome and other MRSA clones of diverse hosts and demographics. The MTR_BAU_H1 genome harbours 42 antimicrobial resistance genes (ARGs), 128 virulence genes, and 273 SEED subsystems coding for the metabolism of amino acids, carbohydrates, proteins, cofactors, vitamins, minerals, and lipids. This is the first-ever WGS-based study of a strong biofilm-producing and MDR S. aureus strain isolated from human hand swabs in Bangladesh that unveils new information on the resistomes (ARGs and correlated mechanisms) and virulence potentials that might be linked to staphylococcal pathogenesis in both humans and animals.

Sporothrix brasiliensis-specific polymerase chain reaction for the diagnosis of cat and human sporotrichosis through non-invasive samples.

Zoonotic sporotrichosis caused by Sporothrix brasiliensis is an emerging mycosis in Latin America. One of the problems to quickly treat infected animals and break the transmission chain is associated with the time-consuming gold-standard diagnosis method (culture). We aimed to evaluate a species-specific polymerase chain reaction (PCR) for the diagnosis of sporotrichosis caused by S. brasiliensis using non-invasive samples. We performed a retrospective cross-sectional study using samples collected with swabs from humans and cats with clinical suspicion of sporotrichosis. Deoxyribonucleic acid (DNA) was extracted using a commercial kit, and a species-specific PCR for S. brasiliensis detection was performed. One hundred ten samples were included. PCR showed a good concordance with culture (86% of agreement) for human and cat samples (Kappa coefficient = 0.722, and 0.727, respectively). In conclusion, our data shows that this adapted PCR using non-invasive samples can be applied to sporotrichosis diagnosis, being a good alternative mainly in regions with a lack of mycologists to identify the fungus in culture, contributing to the control of this emergent zoonosis.

We aimed to evaluate a molecular method for diagnosing sporotrichosis caused by Sporothrix brasiliensis in humans and cats. We observed that the technique is in good agreement with the classic method and is a good alternative for assisting in the diagnosis and consequent control of this zoonosis.

Diagnosis of mucormycosis from nasal swabs using commercial PCR platforms; a feasible alternative?

Rhino-orbital-cerebral mucormycosis (ROCM) is linked to uncontrolled diabetes, diabetic ketoacidosis, iron overload, corticosteroid therapy, and neutropenia. This study evaluated a commercial real-time PCR system's effectiveness in detecting Mucorales from nasal swabs in 50 high-risk patients. Nasal swab PCR showed 30% positivity, compared to 8% with KOH microscopy. Despite its improved sensitivity, nasal swab PCR has limitations, highlighting the importance of established sampling methods in mucormycosis diagnosis. Participants were predominantly male (64%), with diabetes (78%) and amphotericin B use (96%). Prior COVID-19 was 42%, with 30% positive for Mucorales by PCR, compared to 8% with KOH microscopy.

Metagenomic profiling of nasopharyngeal samples from adults with acute respiratory infection.

Diagnosis of acute respiratory infections (ARIs) is challenging due to the broad diversity of potential microbial causes. We used metagenomic next-generation sequencing (mNGS) to analyze the nasopharyngeal virome of ARI patients, who had undergone testing with a clinical multiplex PCR panel (Amplisens ARVI-screen-FRT). We collected nasopharyngeal swabs from 49 outpatient adults, 32 of whom had ARI symptoms and were PCR-positive, and 4 asymptomatic controls in Kazakhstan during Spring 2021. We assessed the biodiversity of the mNGS-derived virome and concordance with PCR results. PCR identified common ARI viruses in 65% of the symptomatic cases. mNGS revealed viral taxa consisting of human, non-human eukaryotic and bacteriophage groups, comprising 15, 11 and 28 genera, respectively. Notable ARI-associated human viruses included rhinovirus (16.3%), betaherpesvirus 7 (14.3%) and Epstein-Barr virus (8.16%). The primary phage hosts were Streptococcus spp. (32.7%), Pseudomonas aeruginosa (24.5%) and Burkholderia spp. (20.4%). In total, 47% of ARIs were linked solely to bacterial pathogens, a third to viral-bacterial co-infections, and less than 10% to only viral infections by mNGS. PCR showed low concordance with mNGS, except for rhinovirus. These results underscore the importance of broad diagnostic methods and question the effectiveness of commonly used PCR panels in ARI diagnosis.

Effects of serial swabs on the nasopharyngeal mucosa: our experience in SARS-CoV2 screening.

INTRODUCTION: The purpose of the study was to analyze the effect of swabs on nasal mucosa. METHODOLOGY: Since May 2020, our department was responsible for screening coronavirus disease 2019 (COVID-19) among the employees of a company that continued its activity during the pandemic. The screening protocol consisted of two swabs per week. The samples were analyzed through objective endoscopic and subjective clinical evaluations with sino-nasal outcome test (SNOT Test) at three time points (T0, T1 - three months, T2 - six months). RESULTS: 23.76% of patients showed an increase in the SNOT score at T1, and the score decreased at T2. This could be due to the phenomenon of "adaptation" of the nasal mucosa. Endoscopic control showed that at T1, secretion, hyperemia, and edema are the most common signs. At T2, however, the crusts accounted for 52.94% of all damage. It is evident that at T1 the endoscopically detected signs of "acute" damage were more represented than at T2, while the signs of "chronic" damage increased as the number of swabs increased. CONCLUSIONS: We demonstrated that mucosal damage and perceived symptoms were absolutely acceptable compared to the diagnostic advantage obtained with serial screening.

Shotgun metagenomic sequencing of swab samples from Japanese university campuses.

We obtained shotgun metagenome sequences from swab samples obtained through 3-minute swabbing of different surfaces and the air within buildings at three university campuses in part of the Greater Tokyo Area in Japan. These data aid in understanding built environment microbial communities and elucidate various microbial profiles across different locations.

SARS-CoV-2 Positivity in Foreign-Born Adults: A Retrospective Study in Verona, Northeast Italy.

We compared SARS-CoV-2 positivity between the foreign-born adult working population and Italians living in the Verona area to investigate whether being a foreign-born adult could confer an increased risk of infection or lead to a diagnostic delay. The present study included 105,774 subjects, aged 18-65 years, tested for SARS-CoV-2 by nasopharyngeal swabs and analyzed at the University Hospital of Verona between January 2020 and September 2022. A logistic regression model was used, controlling for gender, age, time of sampling, and source of referral. A higher proportion of SARS-CoV-2 positivity in Italian (30.09%) than in foreign-born (25.61%) adults was reported, with a higher proportion of SARS-CoV-2 positivity in men than women in both cohorts analyzed. The difference in swab positivity among Italian and foreign-born adults was the highest in people aged 18-29 years (31.5% vs. 23.3%) and tended to disappear thereafter. Swab positivity became comparable between Italian and foreign-born adults during the vaccination campaign. Multivariable analysis confirmed the lower risk of swab positivity among foreign-born adults (OR = 0.85, 95% CI 0.82-0.89). In the Verona area, foreign-born adults showed a lower rate of SARS-CoV-2 positivity than the native population, likely because of underdiagnosis. Hence, public health should increase attention toward these particularly vulnerable populations.

Comparing Occurrence of Bovine Respiratory Pathogens Detected by High-Throughput Real-Time PCR in Nasal Swabs and Non-Endoscopic Bronchoalveolar Lavage Samples from Dairy and Veal Calves.

This study aimed to enhance our understanding of the agreement between two sampling methods for the detection of bovine respiratory disease (BRD) pathogens in calves using high-throughput real-time qPCR (ht-RT-qPCR). In total, 233 paired nasal swab (NS) and non-endoscopic bronchoalveolar lavage (nBAL) samples were collected from 152 calves from 12 Danish cattle herds. In 202 of the observations, the calves were examined using a standardized clinical protocol. Samples were tested for three viruses (bovine respiratory syncytial virus, bovine corona virus, and influenza D virus) and six bacteria (Histophilus somni, Mannheimia haemolytica, Mycoplasma bovis, Mycoplasma species, Pasteurella multocida, and Truepurella pyogenes). The results showed age-related differences in disease and pathogen occurrence, with the highest detection rates in calves aged 35 days or older. Poor to moderate agreement was found between the NS and nBAL results. The presence of Mannheimia haemolytica in both NS and nBAL in younger calves and in nBAL in older calves was associated with clinical BRD. There was a potential link between BRD and influenza D virus in older calves, although it was only found in one herd in a small sample size. Overall, NS was a relatively poor predictor of pathogens in the lower respiratory tract. The present study confirms the complexity of pathogen detection in BRD, with marked influences of age and the sampling method on pathogen detection and disease associations.

A Four-Gene Panel in Rectal Swab Samples as a Biomarker for Colorectal Cancer Screening.

BACKGROUND: The dysregulation of gene expression is one of the key molecular features of colorectal cancer (CRC) development. This study aimed to investigate whether such dysregulation is reflected in rectal swab specimens of CRC patients and to evaluate its potential as a non-invasive approach for screening. METHODS: We compared the expression level of 14 CRC-associated genes in tumor and adjacent non-tumor tissue of CRC patients and examined the correlation of their levels in tissue with paired rectal swab specimens. The level of these 14 genes in rectal swab specimens was compared among patients with CRC or polyp and control subjects, and the diagnostic potential of each dysregulated gene and the gene panel were evaluated. RESULTS: The expression of CXCR2, SAA, COX1, PPARδ, PPARγ, Groγ, IL8, p21, c-myc, CD44 and CSF1 was significantly higher in CRC, and there was a significant correlation in the levels of most of them between the CRC and rectal swab specimens. In the training study, we showed that CD44, IL8, CXCR2 and c-myc levels were significantly higher in the rectal swab specimens of the CRC patients. Such result was confirmed in the validation study. A panel of these four genes was developed, and ROC analysis showed that this four-gene panel could identify CRC patients with an AUC value of 0.83 and identify overall polyp and precancerous adenoma patients with AUC values of 0.6522 and 0.7322, respectively. Finally, the predictive study showed that the four-gene panel demonstrated sensitivities of 63.6%, 76.9% and 88.9% in identifying overall polyp, precancerous adenoma and CRC patients, respectively, whereas the specificity for normal subjects was 72.2%. CONCLUSION: The expression of CRC-associated genes in rectal swab specimens reflects the dysregulation status in colorectal tissue, and the four-gene panel is a potential non-invasive biomarker for early precancerous adenoma and CRC screening.

Comparison of cervicovaginal fluid extracellular vesicles isolated from paired cervical brushes and vaginal swabs.

Endometriosis is a common gynaecological condition, with a long diagnostic delay. Surgery is required to confirm a diagnosis, highlighting the need for a non-invasive biomarker. Extracellular vesicles (EVs) may have a role in endometriosis pathogenesis, yet there is limited EV biomarker literature available. This study aimed to investigate the feasibility of isolating cervico-vaginal fluid EVs sampled using cervical brushes and vaginal swabs and to compare these methods. After providing informed consent, patients undergoing surgery for suspected endometriosis had cervical brush and vaginal swab samples collected under general anaesthetic. Isolated EVs were characterised through negative stain transmission electron microscopy (TEM), Western blotting (TSG101, CD63, Calnexin, ApoB, Albumin), tunable resistive pulse sensing (TRPS), microBCA assays and RT-qPCR of miRNAs. PCR was performed on samples prior to EV isolation to assess bacteria present in samples. Cervical brush and vaginal swab EVs were intact vesicles with limited co-isolated contaminants. Cervical brushes had higher concentrations of particles compared to match vaginal swabs, although both samples had low concentrations. Protein and miRNA yield were similar between matched samples. PCR demonstrated only a small amount DNA within samples was bacterial (>0.5%). Cervico-vaginal fluids EVs were successfully isolated from cervical brushes and vaginal swabs, demonstrating a new method of sampling reproductive EVs. EV yield from both sample types was low. Similar protein and miRNA levels suggest either sampling method may be suitable for biomarker studies.

Development of selective isolation media for detecting the genera Actinomyces and Schaalia from oral specimens containing indigenous bacteria.

To isolate specific bacteria from samples constituting the microbiota, it is essential to employ selective media that suppress the growth of resident bacteria other than specific target bacteria. Selective media for clinically important Actinomyces (including Schaalia, which was previously taxonomically classified as part of the genus Actinomyces) have been limited because they have been designed for a limited range of species within the genus and require ingredients which are difficult to prepare and handle. This study aimed to develop a selective medium [referred to as Actinomyces and Schaalia Selective Medium (ASSM)] for the isolation of a broad range of Actinomyces and Schaalia species from samples mixed with resident bacteria. The composition of ASSM includes yeast extract, agar, brain heart infusion (BHI), levofloxacin (LVFX), fosfomycin (FOM), colistin (CL) and metronidazole (MNZ). Evaluation of the medium using 24 swab samples serially collected from the roots of the teeth of a healthy individual for whom metagenome sequencing data of a saliva sample are publicly available revealed that ASSM adjusted to concentrations of LVFX 0.5 mg l-1, FOM 5 mg l-1, CL 1 mg l-1 and MNZ 2 mg l-1 and cultured anaerobically at 35 °C for 7 days enabled the isolation of Actinomyces species from 37.5 % of the samples. The inclusion of CL and MNZ in ASSM can also be useful for samples harbouring other bacterial species. The selective isolation medium is expected to contribute to studies investigating the relationship between these bacteria and their pathogenesis or disease.