Clinical Swab Research Articles

Deciphering Bordetella pertussis epidemiology through culture-independent multiplex amplicon and metagenomic sequencing.

In this paper, we evaluated the use of targeted multiplex PCR (mPCR) amplicon sequencing and shotgun metagenomic sequencing for culture-independent typing of Bordetella pertussis directly in respiratory swabs. We first developed a novel targeted mPCR amplicon sequencing assay that can type major circulating lineages and validated its accuracy and sensitivity on 178 DNA extracts from clinical swabs. We also demonstrate the feasibility of using deep metagenomic sequencing for determining strain lineage and markers of virulence, vaccine adaptation, macrolide resistance, and co-infections. Our culture-independent typing methods applied to clinical specimens revealed the expansion of a major global epidemic lineage in Australia (termed EL4) just prior to the COVID-19 pandemic. It also detected cases of previously hidden co-infections from another Bordetella species called Bordetella holmesii. These findings offer valuable insight into the circulating pertussis lineages in Australia prior to the COVID-19 pandemic during which border closure and other interventions reduced pertussis cases to an all-time low. It also provides comparative data for future surveillance as pertussis resurgence after the COVID-19 pandemic has been reported this year in Australia and many other countries. Overall, our paper demonstrates the utility, sensitivity, and specificity of mPCR amplicon and metagenomic sequencing-based culture-independent typing of B. pertussis, which not only paves the way for culture-independent genomic surveillance of B. pertussis but also for other pathogens in the era of PCR-based diagnosis.

A Distinct Genotype, D/Ep6, Detected in Korean Female Patients: Chlamydia trachomatis Characterization from 2017 - 2018.

The objective of this study lies in identifying the dominant genotype of C. trachomatis isolated in Republic of Korea (ROK) between 2017 and 2018. A total of 504 clinical cervicovaginal swabs from patients were collected and inoculated on McCoy cell monolayers to isolate Chlamydial agents. C. trachomatis isolates were analyzed by sequencing of its ompA gene. A total of 54 C. trachomatis isolates were obtained. The constructed phylogenetic tree revealed the genotypes of isolates are D/Ep6 (48, 88.89%), D/Ep6-like (5, 9.26%), and Ja (1, 1.85%). The C. trachomatis D/Ep6-like have one single nucleotide polymorphism (SNP) compared to C. trachomatis D/Ep6, leading to E870K amino acid change. Phylogenetic analysis demonstrated globally rare C. trachomatis D/Ep6 was dominant in the ROK from 2017 to 2018. Findings in this study act as a keystone, bridging past and present molecular epidemiology of C. trachomatis in context of ROK.

Highly Sensitive and Specific Diagnosis of Enterovirus A71 by Reverse Transcription Multiple Cross‐Displacement Amplification‐Labeled Nanoparticles Biosensor

ABSTRACTEnterovirus A71 (EVA71) is a leading causative agent of hand, foot, and mouth disease, posing a significant threat to the health of young children, particularly in the Asia‐Pacific region. Currently, there is no specific antiviral drug for EVA71 infection; therefore, early and rapid diagnosis is critical for disease prevention and control. Here, we report the development of a simple, rapid, and sensitive detection method for EVA71 infection using reverse transcription‐multiple cross displacement amplification (RT‐MCDA) combined with nanoparticle‐based lateral flow biosensors (LFB). In the RT‐MCDA system, a set of 10 primers was designed to target the highly conserved region of the VP1 gene of EVA71 and amplify the genes in an isothermal amplification device. The RT‐MCDA amplification reaction products could then be identified by visual detection reagent (VDR) and LFB without the need for specialized equipment. The results demonstrated that the optimal reaction condition for the EVA71‐RT‐MCDA assay was 65℃ for 40 min. The EVA71‐RT‐MCDA assay could detect as low as 40 copies of plasmid and 50 copies of pseudotyped virus in a reaction. No cross‐reaction was found between EVA71 strains and non‐EVA71 strains. For 125 clinical anal swab samples, with EVA71‐RT‐MCDA assay, 30 samples were positive, which was in consistent with the the conventional real‐time quantitative reverse transcription polymerase chain reaction assays. The entire procedure, including a 15‐min specimen processing step, a 40‐min MCDA reaction, and result reporting within 2 min, was completed in less than 60 min. In conclusion, the EVA71‐RT‐MCDA‐LFB assay targeting the VP1 gene is a rapid, highly sensitive, simple, and specific test that could be widely applied in point‐of‐care settings and basic medical facilities in rural areas.

Multigeographic clinical assessment of a molecular diagnostic assay for detection of key codons to predict decreased susceptibility or resistance to cephalosporins in Neisseria gonorrhoeae.

Cephalosporin resistance in Neisseria gonorrhoeae has severely compromised the efficacy of World Health Organization (WHO)-recommended therapies. This study aimed to methodologically evaluate the optimized Six-CodonPlus assay, and additionally conducted a multicenter evaluation to assess its clinical application, especially for predicting antimicrobial resistance (AMR). For methodological evaluation, 397 sequence-known N. gonorrhoeae isolates were evaluated for specificity, 17 nongonococcal isolates were assessed for cross-reactivity, 159 uncultured urogenital swabs and urine samples were evaluated for sensitivity at the clinical level. For multicenter evaluation, 773 isolates with confirmed phenotypic data and 718 clinical urogenital swabs collected from four geographical cities were, respectively, utilized for the evaluation of AMR-prediction strategies and the clinical application of the assay. The assay accurately identified specific single-nucleotide polymorphisms in resistance-associated genes, the detection limits dropped to 10 copies/reaction for individual targets. The specificity reached 100% and no cross-reactivity occurred with double-target confirmation. The assay could be directly applied to clinical samples containing over 20 copies/reaction. Multicenter evaluation formulated two optimal strategies for decreased susceptibility prediction in specific scenarios, and one tactic for prediction of resistance and identification of FC428-like strains. High sensitivity of 86.84% (95% CI, 71.11-95.05) and specificity of 99.59% (95% CI, 98.71-99.89) for resistance prediction were demonstrated for ceftriaxone (CRO). Regarding N. gonorrhoeae identification among multicenter swabs, specificity reached 97.53% (95% CI, 95.49-98.69), and sensitivity reached 93.77% (95% CI, 90.04-96.22). The Six-CodonPlus assay exhibited excellent detection performance and formulated optimal AMR-related prediction strategy with regional adaptability, providing critical information for population screening and clinical treatment.

Quantification of influenza virus mini viral RNAs using Cas13.

Influenza A virus (IAV) RNA synthesis produces full-length and deletion-containing RNA molecules, which include defective viral genomes (DVG) and mini viral RNAs (mvRNA). Sequencing approaches have shown that DVG and mvRNA species may be present during infection, and that they can vary in size, segment origin, and sequence. Moreover, a subset of aberrant RNA molecules can bind and activate host pathogen receptor retinoic acid-inducible gene I (RIG-I), leading to innate immune signaling and the expression of type I and III interferons. Measuring the kinetics and distribution of these immunostimulatory aberrant RNA sequences is important for understanding their function in IAV infection. Here, we explored if IAV mvRNA molecules can be detected and quantified using amplification-free, CRISPR-LbuCas13a-based detection. We show that CRISPR-LbuCas13a can be used to measure the copy numbers of specific mvRNAs in samples from infected tissue culture cells. However, to efficiently detect mvRNAs in other samples, promiscuous CRISPR guide RNAs are required that activate LbuCas13a in the presence of multiple mvRNA sequences. One crRNA was able to detect full-length IAV segment 5 without amplification, allowing it to be used for general IAV infection detection nasopharyngeal swabs. Using CRISPR-LbuCas13a, we confirm that mvRNAs are present in ferret upper and lower respiratory tract tissue, as well as clinical nasopharyngeal swab extracts of hospitalized patients. Overall, CRISPR-LbuCas13a-based RNA detection is a useful tool for studying deletion-containing viral RNAs and it complements existing amplification-based approaches.

Signaling Molecules in Pseudomonas aeruginosa Response to Antibiotics at Sub-Inhibitory Concentrations

Signaling Molecules are N-acylhomoserine lactones (AHLs) that form the Pseudomonas aeruginosa cell information system which determines gene expressions in a population dependent manner called quorum sensing (QS). Signal molecules, which are chemically varied, control genes expressions to antibiotics resistance, pathogenicity, motility, biofilm development, bioluminescence, secondary metabolite production, and plasmid transfer. This research was aimed to identify the signaling molecules in Pseudomonas aeruginosa response to antibiotics at sub-inhibitory concentrations. One hundred and fifty (150) clinical swab specimens were collected from urinary catheter wound and ear infection of patients and the swabs inoculated using standard microbiology method. The isolates were characterized based on the bacteriological methods such as morphology and biochemical tests. The isolates were further confirmed by species specific PCR by amplification of 16S rRNA and the amplicons were analyzed by gel electrophoresis; and further genomic sequencing was done and blast with NCBI database mining. Disc diffusion method was applied for the antibiotics susceptibility pattern. The isolates cell suspensions were analyzed by Gas Chromatography-Mass Spectrometry (GC-MS). The result of the isolation showed 22(59.46%) from wound infections, 12(32.43%) from ear infections and 3(8.11%) from urinary catheter. The isolates were Gram negative, produced β-hemolysis on blood agar and the morphology is small pigmented circular in form. The isolates showed positive result to catalase, oxidase, citrate, nitrate and indole tests. The amplification of 16S rRNA gene region resulted in the band size of 1500bp PCR product and the BLAST analysis gave 99% similarity. The resistant antibiotics susceptibility showed that Pseudomonas aeruginosa is multidrug resistant bacteria. The GC-MS results obtained from urinary catheter isolates showed four (4) signaling molecules such as N-Octanoyl-L-homoserine Lactone, N-Decanoyl-DL-Homoserine Lactone, N-Dodecanoyl-DL-Homoserine Lactone and N-Tetradecanoyl-L-Homoserine Lactone. Three (3) signaling molecules such as N-Octanoyl-L-homoserine Lactone, N-Decanoyl-DL-Homoserine Lactone and N-Tetradecanoyl-L-Homoserine Lactone were obtained from wound isolates. N-Tetradecanoyl-L-Homoserine Lactone was the only signaling molecule obtained from ear isolates. Further research should be done to develop rapid tests that could be possible to detect acyl homoserine specific to Pseudomonsa aeruginosa present to human samples and deliver results in real time. Signaling molecules inhibitors (SMI) are possible potential therapeutics that could produce the subsequent class of antibacterial drugs. The fundamental role of each signal molecule in the production of biofilms and virulent factors should also be investigated, as well as whether Pseudomonas aeruginosa needs one or more signaling molecules to form quorum sensing. Identification of the signals molecules (AHLs) of P. aeruginosa and developing signaling molecules inhibitors (SMI) can have an important prognostic, diagnostic and therapeutic value in human medicine for the treatment of P. aeruginosa infections.

Detection of OXA-23, OXA-48, OmpA and bla-TEM genes in carbapenem-resistance Acinetobacter baumannii and biofilm formation in burn patients at Al-Anbar Government

Background. Antibiotic-resistant bacteria represent a highly global health issue by causing high rates of morbidity and mortality in burn units, whereas biofilms significantly contribute to complications in burn patients. Objectives. The study investigates the prevalence of (OXA-23, OXA-48, OmpA and bla-TEM) and biofilm formation of Acinetobacter baumannii in the Burn Unit of the Al-Anbar government. Methods. A total of 120 clinical swab samples from burn patient were collected from December 2023 to April 2024 from the burn units of Ramadi Teaching Hospital and Falluja Teaching Hospital for the study. The colonies were identified using conventional and Vitek methods. Colorimetric technique (MTP) and multiplex PCR were used to detect biofilms and resistance genes. Results. Among 120 samples, 40 (33.3%) were Acinetobacter baumannii, 34 (80%) were MDR- resistant, and 6 (15%) were XDR. 90% of the isolates were resistant to Cefotaxime, and Colistin was more sensitive in all forty isolates. All 40 isolates formed significant biofilms. The percentage of positive results for OXA-23, OmpA and bla-TEM genes were 80%, 65%, and 55% of respectively. Carbapenem testing is essential to identify carbapenem-producing strains from non-producing strains due to the high prevalence of multidrug-resistant A. baumannii with the OXA-23 and OmpA genes. Conclusion. These findings highlight the urgent need for effective infection control measures and the development of new therapeutic strategies to manage these infections in burn patients.

Effect of X-ray radiation on Multi-drug Resistant (MDR) Enterobacteriaceae & P.aeruginosa isolated from burn unit patient. The phenotypic and molecular study, Biofilm formation

Background: Multi-drug-resistant (MDR) bacteria are a significant global health concern due to their elevated rates of morbidity and mortality in burn patients. In This study investigated the presence of multidrug-resistant pathogenic bacteria in individuals with burn injuries, the formation of biofilms, and the impact of X-ray radiation. Method: For this study Clinical swabs about120 burn patients were obtained between December 2023 and June 2024 from burn units at Ramadi and Falluja Teaching Hospitals. Both conventional and Vitek2(bioMérieux) methods were used to identify the bacterial isolates. The isolates were then divided into three multi-drug resistance groups. The Colorimetric method (MTP) detected biofilms. To detect blaOXA-23,blaOXA-48, Omp, and blaTEM resistance genes, multiplex PCR was used. This research used two lumbosacral spine X-ray levels. Two levels of radiation were used 40 (80KV) mAs and 51.2 (85KV). Result: Results of burn isolation revealed 30(27%) P.aeruginosa, 18(16%) k.pneumoniae, 7(6%) E.coli, 7(6%) P.mirabilis. Of the all isolates, 100% produced biofilm. The Multiplex-PCR analysis revealed that the blaTEM genes were the predominant ones, constituting 30% of the samples. Subsequently, blaOXA-23 constituted 24.5% of the cases, whereas Omp accounted for 22.7% and blaOXA-48 for 13.6%. The radiation impact of X-rays produced by two dosages, 40(80KV)mAs for 125msec and 51,2(85KV) mAs for 160msec, demonstrates that there is no effect or alteration in the resistance capacity of all the bacterial isolates tested. Conclusion: This study showed that burn unit isolates produce ESBLs, which is a major concern in the intensive care unit due to its high morbidity and potential mortality. These data match antibiotic phenotypic test results showing substantial cefepime resistance. All isolates forming biofilms, this may association with high pathogenicity. This demands quick intervention to manage nosocomial outbreaks. No X-ray effect was seen on susceptibility a burn unit bacterial isolate.

Enhanced Two-Step LAMP-CRISPR Assay with an Engineered Zst Polymerase for Contamination-Free and Ultrasensitive DNA Detection.

Current loop-mediated isothermal amplification (LAMP)-coupled clustered regularly interspaced short palindromic repeats (LAMP-CRISPR) biosensing in two-step or one-step formats has been applied to next-generation accurate molecular diagnosis. However, two-step LAMP-CRISPR assays intrinsically confront aerosol contamination, while one-step assays possess a compromised detection performance. To this end, we propose an enhanced two-step LAMP-CRISPR assay (ETL-CRISPR) with an engineered Zst polymerase to mediate ultrasensitive DNA detection and thoroughly eliminate aerosol contamination. Instead of supplementing any dTTP, the newly engineered Zst polymerase can efficiently polymerize four oligonucleotides (dATP, dCTP, dGTP, and dUTP), thereby enabling contamination-free and ultrasensitive ETL-CRISPR assay. By targeting the L1 gene of human papillomaviruses (HPV) 16 and the E7 gene of HPV18, our ETL-CRISPR assay achieves high specificity and single-copy level sensitivity within 1 h. Furthermore, we validated the assay by using 85 HPV clinical swab samples with an accuracy of 98.8%, which is comparable to the real-time quantitative polymerase chain reaction. Therefore, ETL-CRISPR provides a straightforward strategy for the contamination-free and ultrasensitive point-of-care diagnosis of clinical pathogens.

CHAMP: A Centrifugal Microfluidics-Based CRISPR/Cas12b-Combined Real-Time LAMP One-Pot Method for Mycoplasma pneumoniae Infection Diagnosis.

The Mycoplasma pneumoniae outbreak poses health risks to community residents. However, it still has limitations for current clinical diagnostic methods (qPCR nucleic acid assay or IgM immunoassay), including specialized handling, expensive equipment, prolonged turnaround time, and false positives and negatives, highlighting the need to improve clinical diagnostic methods. Herein, we present a novel centrifugal microfluidics-based method for rapidly diagnosing M. pneumoniae infections (CHAMP system). This user-friendly method combines CRISPR/Cas12b and real-time loop-mediated isothermal amplification (LAMP) in a one-pot reaction, offering high sensitivity, specificity, and simplicity for methodology. By adding fully automated nucleic acid magnetic bead-extracted samples to a prepackaged centrifugal microfluidics chip, 48 samples can be automated tested simultaneously within 15 to 60 min at 60 °C. 427 clinical nasopharyngeal swab specimens were used for validation, demonstrating good positive and negative predictive values and good diagnostic sensitivity, specificity, and significant time savings. This method is particularly suitable for detecting low nucleic acid copies of M. pneumoniae samples.

Development of a triplex RT-RAA-LFA assay for the rapid differential diagnosis of porcine epidemic diarrhea virus, porcine deltacoronavirus and transmissible gastroenteritis virus

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and transmissible gastroenteritis virus (TGEV) are three clinically common coronaviruses causing diarrhea in pigs, with indistinguishable clinical signs and pathological changes. Rapid, portable and reliable differential diagnosis of these three pathogens is crucial for the prompt implementation of appropriate control measures. In this study, we developed a triplex nucleic acid assay that combines reverse transcription recombinase-aided amplification (RT-RAA) with lateral flow assay (LFA) by targeting the most conserved genomic region in the ORF1b genes of PEDV, PDCoV and TGEV. The entire detection process of the triplex RT-RAA-LFA assay included 10-min nucleic acid amplification at 42 °C and 5-min visual LFA readout at room temperature. The assay could specifically differentiate PEDV, PDCoV and TGEV without cross-reaction with any other major swine pathogens. Sensitivity analysis showed that the triplex RT-RAA-LFA assay was able to detect the viral RNA extracted from the spiked fecal samples with the minimum of 1 × 100 TCID50 PEDV, 1 × 104 TCID50 PDCoV, and 1 × 102 TCID50 TGEV per reaction, respectively. Further analysis showed that the 95 % detection limit (LOD) of triplex RT-RAA-LFA for PEDV, PDCoV, and TGEV were 22, 478, and 205 copies of recombinant plasmids per reaction, respectively. The diagnostic performance of triplex RT-RAA-LFA was compared with that of PEDV, PDCoV and TGEV respective commercial real-time RT-PCR kits by testing 114 clinical rectal swab samples in parallel. The total diagnostic coincidence rates of triplex RT-RAA-LFA with real-time RT-PCR kits of PEDV, PDCoV and TGEV were 100 %, 99.1 % and 99.1 %, respectively, and their Kappa values were 1.00, 0.958 and 0.936, respectively. Collectively, the RT-RAA-LFA assay is a powerful tool for the rapid, portable, visual, and synchronous differential diagnosis of PEDV, PDCoV, and TGEV.

From Lab to Home: Ultrasensitive Rapid Detection of SARS-CoV-2 with a Cascade CRISPR/Cas13a-Cas12a System Based Lateral Flow Assay.

Currently, CRISPR/Cas-based molecular diagnostic techniques usually rely on the introduction of nucleic acid amplification to improve their sensitivity, which is usually more time-consuming, susceptible to aerosol contamination, and therefore not suitable for at-home molecular testing. In this research, we developed an advanced CRISPR/Cas13a-Cas12a-based lateral flow assay that facilitated the ultrasensitive and rapid detection of SARS-CoV-2 RNA directly from samples, without the need for nucleic acid amplification. This method was called CRISPR LFA enabling at-home RNA testing (CLEAR). CLEAR used a novel cascade mechanism with specially designed probes that fold into hairpin structures, enabling visual detection of SARS-CoV-2 sequences down to 1 aM sensitivity levels. More importantly, CLEAR had a positive coincidence rate of 100% and a negative coincidence rate of 100% for clinical nasopharyngeal swabs from 16 patients. CLEAR was particularly suitable for at-home molecular testing, providing a low-cost, user-friendly solution that can efficiently distinguish between different SARS-CoV-2 variants. CLEAR overcame the common limitations of high sensitivity and potential contamination associated with traditional PCR-based systems, making it a promising tool for widespread public health application, especially in environments with limited access to laboratory resources.

The potential of residual clinical Group B Streptococcus swabs for assessing the vaginorectal microbiome in late pregnancy

The maternal pregnancy microbiome (including genitourinary and gut) has been linked to important pregnancy/birth and later childhood health outcomes. However, such sampling as part of large population cohort studies is logistically and financially challenging. Many countries routinely collect vaginal or vaginal-rectal swabs in late pregnancy for Group B Streptococcus (GBS) screening, but their utility for population-based research is still unclear. As part of planning for the Generation Victoria population-based cohort study beginning in pregnancy, we assessed the utility and reliability of residual clinical GBS vaginal/vaginal-rectal swabs for generating late pregnancy microbiome data. We carried out a two-phased pilot study. Phase one assessed the level of microbial diversity apparent in ‘residual’ clinical vaginal/vaginal-rectal swabs post clinical testing and storage for 7–10 days at 4 °C (routine clinical practice). Phase two directly assessed the impact of storage time and temperature on the microbial composition of vaginal/vaginal-rectal swabs collected specifically for research purposes. The microbiota composition in the ‘residual’ clinical swabs aligned with published studies. The ‘research’ swabs, stored at 4 °C for up to ten days, showed minimal changes in microbiota profile, compared to swabs examined on the day of collection. In contrast, significant variation in diversity was seen in swabs stored at room temperature for up to 48 h. Residual clinical material from swabs collected primarily for GBS screening in late pregnancy represent a reliable and abundant source of material for assessing the late pregnancy maternal microbiome for research purposes. This represents a low-burden opportunity for population-representative pregnancy studies to assess the potential of late pregnancy microbiome for prediction and understanding maternal and child health outcomes.

Sequence-specific nanoparticle barcode strategy for multiplex human enterovirus typing

Human enteroviruses (HEV) can cause a range of diseases from mild to potentially life-threatening. Identification and genotyping of HEV are crucial for disease management. Existing typing methods, however, have inherent limitations. Developing alternative methods to detect HEV with more virus types, high accuracy, and sensitivity in an accessible manner presents a technological and analytical challenge. Here, a sequence-specific nanoparticle barcode (SSNB) method is presented for simultaneous detection of 10 HEV types. This method significantly increases sensitivity, enhancing detection by 10-106 times over the traditional multiplex hybrid genotyping (MHG) method, by resolving cross-interference between the multiple primer sets. Furthermore, the SSNB method demonstrates a 100% specificity in accurately distinguishing between 10 different HEV types and other prevalent clinical viruses. In an analysis of 70 clinical throat swab samples, the SSNB method shows slightly higher detection rate for positive samples (50%) compared to the RT-PCR method (48.6%). Additionally, further assessment of the typing accuracy for samples identified as positive by SSNB using sequencing method reveals a concordance rate of 100%. The combined high sensitivity and specificity level of the methodology, together with the capability for multiple type analysis and compatibility with clinical workflow, make this approach a promising tool for clinical settings.

Ratiometric Readout of Bacterial Infections via a Lyophilized CRISPR-Cas12a Sensor with Color-Changeable Bioluminescence.

The healthcare burden imposed by bacterial infections demands robust and accessible diagnostic methods that can be performed outside hospitals and centralized laboratories. Here, we report Pathogen Assay with Ratiometric Luminescence (PEARL), a sensitive and easy-to-operate platform for detecting pathogenic bacteria. The PEARL leveraged a color-changeable CRISPR-Cas12a sensor and recombinase polymerase amplification to elicit ratiometric bioluminescence responses to target inputs. This platform enabled robust and visualized identification of attomolar bacteria genome deoxyribonucleic acid according to the color changes of the reactions. In addition, the components of the color-changeable Cas12a sensor could be lyophilized for 3 month storage at ambient temperature and then be fully activated with the amplicons derived from crude bacterial lysates, reducing the requirements for cold-chain storage and tedious handling steps. We demonstrated that the PEARL assay is applicable for identifying the infections caused by Pseudomonas aeruginosa in different clinical specimens, including sputa, urines, and swabs derived from wounds. These results revealed the potential of PEARL to be used by untrained personnel, which will facilitate decentralized pathogen diagnosis in community- and resource-limited regions.

Successful Mycobacterium tuberculosis culture isolation from tongue swabs: Results from both experimentally infected and clinical swabs from pulmonary tuberculosis patients

This study explored Mycobacterium tuberculosis (MTB) growth from tongue swabs, both experimentally infected after sampling from healthy controls, or sampled from patients with smear-microscopy confirmed pulmonary tuberculosis (PTB). For both, we evaluated the performance of NALC-NaOH/MGIT960 (MGIT), Kudoh-Ogawa (KO), and cetylpyridinium chloride-Löwenstein-Jensen (CPC/LJ) culture processing methods. Experimentally spiked swabs from 20 participants exhibited 94.4% MTB growth when inoculated within 7 days of CPC exposure, declining significantly after 14-21 days (p<0.00001). KO-processed specimens showed 100% MTB growth, with a non-significant reduction after storage (94.1%; p=0.21), and all spiked swabs yielded growth in MGIT. In the field evaluation on 99 PTB patients, MGIT isolated MTB from 89% of tongue swabs, with an 8% contamination rate, compared to 99% MGIT positivity from sputum. Solid media had lower positivity, 62% for KO and 49% for CPC/LJ, suggesting MGIT as optimal for growing MTB from tongue swabs. Further testing of presumptive PTB patients is recommended.

Isolation and identification of Pseudomonas aeruginosa obtained from dogs and cats in Great Cairo regarding status of phenotypic antimicrobial resistance pattern

Background Companion animals; dog and cat are closely associated with the daily life of humans and may be implemented in the transmission of many microorganisms to their adopters. Pseudomonas aeruginosa (P. aeruginosa) is a suited opportunistic and harsh to treat pathogen due to its rife environmental distribution, unique intrinsic and acquired resistance to numerous antimicrobials’ categories. Objective The current study targeted to survey the existence of P. aeruginosa in laboratory samples obtained from diseased dogs and cats. The study also investigated the susceptibility and resistance of recovered isolates against antimicrobials. Materials and methods A total of 315 samples gathered from veterinary laboratories in Great Cairo governorates; fecal, ear, eye, respiratory, wounds and urine samples were previously collected from diseased dogs and cats. The samples were examined bacteriologically and biochemically to isolate P. aeruginosa. The isolates were assayed for their sensitivity and resistance versus 25 antimicrobials belonging to various categories. Results and conclusion Fifty- eight P. aeruginosa isolates (18.41%) were obtained from 315 dogs (44/233, 18.88%) and cats’ (14/82, 17.07%) clinical swabs. The isolates were confirmed biochemically and via VITEK 2 compact system. All isolates showed alpha-type of hemolysis and pigment production. The obtained P. aeruginosa isolates revealed a multidrug resistance pattern by 70.45% in dog isolates while cat isolates demonstrated a higher ratio 78.57%. P. aeruginosa isolates were highly resistant to cephalosporins, trimethoprim/sulfamethoxazole, and intermediate resistant to erythromycin fosfomycin. On the other hand imipenem, amikacin, azithromycin then gentamycin and ciprofloxacin were the most efficient on P. aeruginosa isolates. The study included that P. aeruginosa isolates obtained from canine and feline clinical samples collected from Great Cairo laboratories were characterized by high and intermediate levels of antimicrobial resistance. However, this pattern was directed to some classes of antibiotics, which are not authorized for veterinary use, which could expose an early warning mark and give the need for ongoing monitoring.

P29 Long-read sequencing provides unprecedented insight into the skin and wound microbial landscape

Abstract Introduction and aims It is becoming increasingly evident that the microbial inhabitants of our skin play a key role in cutaneous health and disease. However, our understanding of the contribution of the complex skin microbiome to wound healing outcome remains incomplete. This is, in part, due to the historic challenges around traditional sampling and profiling Methods, which often fail to capture the abundance and diversity of the skin microbiome. Recent advances in microbiome sequencing technologies now enable more comprehensive characterization of skin bacterial composition and host-microbe interactions. Thus, the aim of this study was to utilize a novel long-read sequencing platform to provide species-level microbiome characterization and deliver important insight into the drivers of pathological skin repair. Methods Microbial DNA samples were collected from the skin and wounds of healthy volunteers and patients with chronic wounds (n = 40 per group). Long-read nanopore sequencing was used to profile microbiome samples, while RNA-Seq enabled generation of donor transcriptomic profiles. A multifaceted approach was used to integrate these datasets to investigate the complex interplay between host skin pathology and the microbiome. Results We first identified perturbations in the skin microbiome of patients with chronic wounds, which was significantly less diverse than healthy skin and colonized with key bacteria associated with pathogenicity. These alterations in the skin microbiome also correlated with transcriptomic changes, such as upregulation of genes associated with inflammation, stress and poor healing. Interestingly, long-read sequencing data displayed poor correlation with routine clinical microbiology swabs, while specific clinical characteristics (e.g. glycaemic control and wound duration) were strongly linked to wound microbiome composition. Conclusions Collectively, these data provide crucial new insight into the skin and wound microbial landscape, demonstrating a potential role for specific wound bacteria in modulating pathological skin repair.

Dual structure-switching aptamer-mediated signal amplification cascade for SARS-CoV-2 detection

Since the outbreak of the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) at the end of 2019, the spread of the virus has posed a significant threat to public health and the global economy. This work proposed a one-step, dual-structure-switching aptamer-mediated signal amplification cascade for rapid and sensitive detection of the SARS-CoV-2 nucleocapsid protein. This system consisted of two DNA aptamers with structure-switching functionality and fuel DNA, where a cascade of strand hybridization and displacement triggered fluorescence generation and signal amplification. This aptamer-based amplification cascade required neither an amplification stage using enzymes nor pre-processing steps such as washing, viral isolation, and gene extraction. The assay could distinguish SARS-CoV-2 from other respiratory viruses and detect up to 1.0 PFU/assay of SARS-CoV-2 within 30 min at room temperature. In 35 nasopharyngeal clinical samples, the assay accurately assessed 25 positive and 10 negative clinical swab samples, which were confirmed using quantitative polymerase chain reaction. The strategy reported herein can help detect newly emerging pathogens and biomarkers of various diseases in liquid samples. In addition, the developed detection system consisting of only DNA and fluorophores can be widely integrated into liquid biopsy platforms for disease diagnosis.

Establishment of a real-time fluorescent quantitative PCR detection method and phylogenetic analysis of BoAHV-1

BackgroundInfectious bovine rhinotracheitis (IBR), caused by Bovine alphaherpesvirus-1 (BoAHV-1), is an acute, highly contagious disease primarily characterized by respiratory tract lesions in infected cattle. Due to its severe pathological damage and extensive transmission, it results in significant economic losses in the cattle industry. Accurate detection of BoAHV-1 is of paramount importance. In this study, we developed a real-time fluorescent quantitative PCR detection method for detecting BoAHV-1 infections. Utilizing this method, we tested clinical samples and successfully identified and isolated a strain of BoAHV-1.1 from positive samples. Subsequently, we conducted a genetic evolution analysis on the isolate strain’s gC, TK, gG, gD, and gE genes.ResultsThe study developed a real-time quantitative PCR detection method using SYBR Green II, achieving a detection limit of 7.8 × 101 DNA copies/μL. Specificity and repeatability analyses demonstrated no cross-reactivity with other related pathogens, highlighting excellent repeatability. Using this method, 15 out of 86 clinical nasal swab samples from cattle were found to be positive (17.44%), which was higher than the results obtained from conventional PCR detection (13.95%, 12/86). The homology analysis and phylogenetic tree analysis of the gC, TK, gG, gD, and gE genes of the isolated strain indicate that the JL5 strain shares high homology with the BoAHV-1.1 reference strains. Amino acid sequence analysis revealed that gC, gE, and gG each had two amino acid mutations, while the TK gene had one synonymous mutation and one H to Y mutation, with no amino acid mutations observed in the gD gene. Phylogenetic tree analysis indicated that the JL5 strain belongs to the BoAHV-1.1 genotype and is closely related to American strains such as C33, C14, and C28.ConclusionsThe established real-time fluorescent quantitative PCR detection method exhibits good repeatability, specificity, and sensitivity. Furthermore, genetic evolution analysis of the isolated BoAHV-1 JL-5 strain indicates that it belongs to the BoAHV-1.1 subtype. These findings provide a foundation and data for the detection, prevention, and control Infectious Bovine Rhinotracheitis.