Multiplex PCR Test Research Articles

Rapid detection of SARS-CoV-2 variants by molecular-clamping technology-based RT-qPCR.

We have developed a multiplex reverse-transcription quantitative real-time PCR (RT-qPCR) testing platform for the rapid detection of SARS-CoV-2 variants using the xenonucleic acid (XNA)-based molecular-clamping technology. The XNA-based RT-qPCR assay can achieve high sensitivity with a limit of detection of about 100 copies/mL for variant detection which is much better than the next-generation sequencing (NGS) assay. Its turnaround time is about 4 hours with lower cost and a lot of Clinical Laboratory Improvement Amendments (CLIA) labs own the instrument and meet skillset requirements. This assay provides a rapid, reliable, and cost-effective testing platform for rapid detection and monitoring of known and emerging SARS-CoV-2 variants. This testing platform can be adopted by laboratories that perform routine SARS-CoV-2 PCR testing, providing a rapid and cost-effective method in lieu of NGS-based assays, for detecting, differentiating, and monitoring SARS-CoV-2 variants. This assay is easily scalable to any new variant(s) should it emerge.

Noninvasive, microbiome-based diagnosis of inflammatory bowel disease.

Despite recent progress in our understanding of the association between the gut microbiome and inflammatory bowel disease (IBD), the role of microbiome biomarkers in IBD diagnosis remains underexplored. Here we developed a microbiome-based diagnostic test for IBD. By utilization of metagenomic data from 5,979 fecal samples with and without IBD from different geographies and ethnicities, we identified microbiota alterations in IBD and selected ten and nine bacterial species for construction of diagnostic models for ulcerative colitis and Crohn's disease, respectively. These diagnostic models achieved areas under the curve >0.90 for distinguishing IBD from controls in the discovery cohort, and maintained satisfactory performance in transethnic validation cohorts from eight populations. We further developed a multiplex droplet digital polymerase chain reaction test targeting selected IBD-associated bacterial species, and models based on this test showed numerically higher performance than fecal calprotectin in discriminating ulcerative colitis and Crohn's disease from controls. Here we discovered universal IBD-associated bacteria and show the potential applicability of a multibacteria biomarker panel as a noninvasive tool for IBD diagnosis.

A-236 Rapid multiplexed PCR (polymerase chain reaction) tests for respiratory disease improve process and time to diagnosis versus laboratory-based molecular testing

Abstract Background The Infectious Disease Society of America recommends molecular testing for symptomatic patients suspected of having COVID-19. Molecular tests can be performed at the point of care or in a centralized laboratory, using different methodologies, and can be singleplex or multiplex. Limited data are available examining the use of these tests in clinical practice and impact of different test types on patient outcomes; however, common antiviral treatments for both COVID-19 and influenza are more effective when taken earlier and are not indicated for use more than 5 and 2 days after symptom onset, respectively. The aim of this study was to describe the diagnostic journey for non-inpatients who received molecular testing for respiratory pathogens in the laboratory versus those who received testing using a rapid, multiplexed real-time RT-PCR test that can be performed at the point of care. Methods De-identified administrative claims were analyzed from July 1, 2020 through September 30, 2022 for commercially insured and Medicare Advantage enrollees in the Optum Labs Data Warehouse. Patients with ≥1 medical claim for a non-inpatient visit with a procedure code for testing for SARS-CoV-2 were identified; patients who received antigen testing at index were excluded. Patients were followed for up to 90 days. Two patient groups were selected for the final study sample: 1) Patients with ≥1 claim with CPT code 0240U or 0241U with a point-of-care or laboratory place of service on the index date (proprietary laboratory analyses codes for Xpert® Xpress CoV-2/Flu/RSV, or “Xpert Xpress”); and 2) Patients with other laboratory-based molecular testing on the index date. Standardized difference of >0.10 or <-0.10 was considered a significant imbalance in proportions or means. Results In total, 51,602 patients received testing with Xpert Xpress while 317,192 received laboratory testing. Patients who were tested with Xpert Xpress were significantly older (mean 49.82 vs 42.19, SD 0.3), had a higher Charlson Comorbidity Index (mean 0.7 vs 0.35, SD 0.29), and were more likely to have a respiratory risk factor (6.09% vs 2.38%, SD 0.19). On average, patients who were tested with Xpert Xpress had significantly fewer tests for SARS-CoV-2 on the index date (mean 1.2 vs 1.58, SD -0.55), and fewer tests for SARS-CoV-2, influenza, or RSV on the index date and through the end of the 90-day fixed follow-up period (mean 1.64 vs 2.57, SD -0.45). They were also significantly more likely to be diagnosed with COVID-19 on the index date (23.31% vs 16.53%, SD 0.17). Among patients with a diagnosis for COVID-19, mean time to diagnosis was significantly shorter among patients tested with Xpert Xpress (4.85 vs 8.86 days, SD -0.27; median 0 vs 4 days). This finding was also consistent for influenza (4.05 vs 20.28 days, SD -0.74; median 0 vs 5 days). Conclusions Testing with Xpert Xpress at index resulted in fewer total tests and faster diagnosis compared to testing in the laboratory. Rapid, multiplex PCR testing provides a faster time to diagnosis and may support more appropriate and effective treatment.

Modelling multiplex testing for outbreak control

During the SARS-CoV-2 pandemic, polymerase chain reaction (PCR) and lateral flow device (LFD) tests were frequently deployed to detect the presence of SARS-CoV-2. Many of these tests were singleplex, and only tested for the presence of a single pathogen. Multiplex tests can test for the presence of several pathogens using only a single swab, which can allow for: surveillance of more pathogens, targeting of antiviral interventions, a reduced burden of testing, and lower costs. Test sensitivity however, particularly in LFD tests, is highly conditional on the viral concentration dynamics of individuals. To inform the use of multiplex testing in outbreak detection it is therefore necessary to investigate the interactions between outbreak detection strategies and the differing viral concentration trajectories of key pathogens. Viral concentration trajectories are estimated for SARS-CoV-2, and Influenza A/B. Testing strategies for the first five symptomatic cases in an outbreak are then simulated and used to evaluate key performance indicators. Strategies that use a combination of multiplex LFD and PCR tests achieve; high levels of detection, detect outbreaks rapidly, and have the lowest burden of testing across multiple pathogens. Influenza B was estimated to have lower rates of detection due to its modelled viral concentration dynamics. Data availability statementThe SARS-CoV-2 viral load trajectory data may be accessed by contacting Killingley et. al. The influenza A/B viral load trajectory may be accessed at https://github.com/bcowling/pediatric-vaccine-trial/tree/master/data.

A multicenter randomized control trial: Point-of-care syndromic assessment versus standard testing in urgent care center patients with acute respiratory illness.

Syndromic assessment with multiplex polymerase chain reaction (mPCR) testing in patients with acute respiratory illness (ARI) allows for simultaneous identification of multiple possible infectious etiologies. Point-of-care (POC) syndromic assessment can be conducted in a clinical setting, such as an urgent care center (UCC), without requiring certified laboratories. The primary objective of this study was to determine whether POC syndromic assessment improved patient satisfaction for patients seen at an UCC with ARI; secondary objectives included whether syndromic assessment reduced self-isolation time, increased diagnostic confidence, and reduced overall antibiotic utilization. We conducted an unblinded multicenter randomized controlled trial on UCC patients with an ARI. Patients were randomized to either SC (defined as standard UCC testing for ARI) or syndromic assessment with POC mPCR. Patients were surveyed for patient satisfaction, self-isolation plans, diagnostic confidence, and overall antibiotic utilization. Among the 360 patients enrolled, those in the syndromic assessment group were more satisfied with the time required to communicate the results (98.4%vs. 42.4%, p<0.001) on day of treatment, more likely to resume normal activities sooner (83.3%vs. 69.4%, p=0.039), and more confident in their illness cause (60.7%vs. 29.6%, p<0.001); however, the rate of antibiotic utilization did not differ (33.5%vs. 26%, p=1.0). In conclusion, our study provides evidence supporting the use of syndromic assessment in UCCs for ARI diagnosis, including patient-centered outcomes such as greater confidence in diagnosis and more efficient isolation strategies. This study did not show a difference in more clinically oriented outcomes, such as a change in antibiotic utilization. Future studies should identify clinical care pathways to improve antibiotic stewardship for likely viral syndromes and whether the increased initial cost of syndromic assessment is offset by the clinical benefits and subsequent cost savings.

Detection of gastrointestinal pathogens in stool samples using a rapid multiplex PCR test at a large tertiary pediatric hospital

Acute gastrointestinal infections (AGIs) and diarrheal disease disproportionately impact children. The purpose of this study was to characterize the clinical and epidemiological characteristics of pediatric patients who had stool samples tested using the BIOFIRE® FILMARRAY® Gastrointestinal Panel at Nationwide Children's Hospital in Columbus, Ohio from January 2016 to December 2020. Detection rates were estimated by pathogen. Poisson and logistic regression were used to assess trends and identify risk factors for detection. A total of 12,783 patients had 17,611 stool samples tested during the study period. Nearly half of stool samples (47.3 %) tested positive for at least one pathogen. Enteropathogenic Escherichia coli, Clostridioides difficile, and norovirus were the most commonly detected. Overall annual detection rates for most pathogens increased from 2016 to 2019 and declined in 2020. This paper presents an analysis of five years of data that characterizes detection, hospitalizations, and deaths of children tested for AGI pathogens by sociodemographics.

Risk factors and clinical outcomes associated with multiple as opposed to single pathogens detected on the gastrointestinal disease polymerase chain reaction assay

BackgroundThe use of gastrointestinal disease multiplex polymerase chain reaction (GI PCR) testing has become common for suspected gastrointestinal infection. Patients often test positive for multiple pathogens simultaneously through GI PCR, although the clinical significance of this is uncertain.MethodsThis retrospective cohort study investigated risk factors and clinical outcomes associated with detection of multiple (as opposed to single) pathogens on GI PCR. We included adult patients who underwent GI PCR testing from 2020 to 2023 and had one or more pathogens detected. We compared patients with multiple versus those with single pathogens and hypothesized that immunosuppression would be a risk factor for detection of multiple pathogens. We further hypothesized that, during the 90 days after GI PCR testing, patients with multiple pathogens would have worse clinical outcomes such as increased rates of emergency department (ED) visits, death, hospitalization, or ambulatory care visits.ResultsGI PCR was positive in 1341 (29%) of tested patients; 356 patients had multiple pathogens and 985 had one pathogen. The most common pathogens included Enteropathogenic Escherichia coli (EPEC, 27%), norovirus (17%), and Enteroaggregative E. coli (EAEC, 14%) in both multi- and singly positive patients. Immunosuppression was not associated with multiple pathogens (adjusted odds ratio [aOR] 1.35, 95% CI 0.96, 1.86). The factors most associated with multiple pathogens were Hispanic ethnicity (OR 1.86, 95% CI 1.42, 2.45) and chronic kidney disease (OR 1.69, 95% CI 1.13, 2.49). Patients with multiple pathogens were more likely to have ED visits during the 90 days after GI PCR testing (40% vs. 32%, p < 0.01), but they were not more likely to die, be hospitalized, or to have ambulatory medical visits.ConclusionsImmunosuppression was not associated with detection of multiple as opposed to single pathogens on GI PCR testing. There were worse clinical outcomes associated with detection of multiple pathogens, although these effects were modest.

Multiplex real-time PCR in non-invasive respiratory samples to reduce antibiotic use in community-acquired pneumonia: a randomised trial

We assessed whether multiplex real-time PCR plus conventional microbiological testing is safe and more effective than conventional microbiological testing alone for reducing antibiotic use in community-acquired pneumonia (CAP). In this randomised trial, we recruited adults hospitalised with CAP at four Spanish hospitals. Patients were randomly assigned (1:1) to undergo either multiplex real-time PCR in non-invasive respiratory samples plus conventional microbiological testing or conventional microbiological testing alone. The primary endpoint was antibiotic use measured by days of antibiotic therapy (DOT). Between February 20, 2020, and April 24, 2023, 242 patients were enrolled; 119 were randomly assigned to multiplex real-time PCR plus conventional microbiological testing and 123 to conventional microbiological testing alone. All but one of the patients allocated to multiplex real-time PCR plus conventional microbiological testing underwent PCR, which was performed in sputum samples in 77 patients (65.2%) and in nasopharyngeal swabs in 41 (34.7%). The median DOT was 10.04 (IQR 7.98, 12.94) in the multiplex PCR plus conventional microbiological testing group and 11.33 (IQR 8.15, 16.16) in the conventional microbiological testing alone group (difference −1.04; 95% CI, −2.42 to 0.17; p = 0.093). No differences were observed in adverse events and 30-day mortality. Our findings do not support the routine implementation of multiplex real-time PCR in the initial microbiological testing in hospitalised patients with CAP. Clinicaltrials.gov registration: NCT04158492.

Human Herpesvirus 6-A Rare Aetiologic Agent for CNS Infections in Immunocompetent Individuals or an Underestimation?

Background: Human herpesvirus 6 (HHV-6) is considered a ubiquitous virus, with many countries reporting a seroprevalence of more than 80-90% among the general population. However, this virus is unique among herpesviruses in its ability to integrate into the genetic material of the host's cells. Thus, there are three ways by which HHV-6 can cause an active infection-primary infection, reactivation of a latent acquired infection, or activation of iciHHV-6 (inherited chromosomally integrated HHV-6). Whole blood quantitative polymerase chain reaction (qPCR) is very useful in distinguishing between iciHHV-6 and primary infection/reactivation. Our aim is to assess the role of HHV-6 in the aetiology of central nervous system (CNS) infections in adults and children, to describe all HHV-6-positive cases in an attempt to determine the susceptible population and to identify potential risk factors that can be linked to HHV-6 meningoencephalitis. Methods: We performed a retrospective study involving patients that were admitted to Prof. Dr. Matei Bals National Institute of Infectious Diseases, Bucharest, Romania, with a diagnosis of meningitis or encephalitis. We only selected the clinical records of patients that had a multiplex PCR Biofire® FilmArray® meningitis/encephalitis panel. Results: We report a 5% HHV-6 positivity in the cerebrospinal fluid (CSF) of patients with CNS infections tested with a commercial multiplex PCR M/E (meningitis/encephalitis) panel. Additionally, 2% to 4% of the total study population (n = 100) had active HHV-6 infections, which denotes 40 to 80% of the HHV-6-positive samples. We did not observe any statistically significant correlation between HHV-6 positivity in the CSF and variables such as age, sex, or comorbidities, including obesity, diabetes, hypertension, immunosuppression, or oncologic disease. Therefore, no risk factors could be linked with HHV-6 positivity in the CSF. Conclusions: although multiplex qualitative PCR is highly useful for providing rapid results and identifying nearly every pathogen that can cause meningitis/encephalitis, we have to be aware of this type of test's limitations. All patients with HHV-6 detectable in their CSF via a multiplex PCR test should also undergo qPCR testing from both CSF and blood to prevent over-diagnosing HHV-6 CNS infections, to avoid unnecessary antiviral treatments, and ensure the accurate identification of the true diagnosis.

SARS-CoV-2 co-detection with other respiratory pathogens-descriptive epidemiological study

BackgroundCo-detection of respiratory pathogens with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is poorly understood. This descriptive epidemiological study aimed to determine the effect of the interaction of different respiratory pathogens on clinical variables. MethodsWe retrospectively reviewed the results of comprehensive multiplex polymerase chain reaction (PCR) testing from November 2020 to March 2023 to estimate respiratory pathogen co-detection rates in Shinjuku, Tokyo. We evaluated the interactions of respiratory pathogens, particularly SARS-CoV-2, between observed and expected co-detection. We estimated the trend of co-detection with SARS-CoV-2 in terms of age and sex and applied a multiple logistic regression model adjusted for age, testing period, and sex to identify influencing factors between co-detection and single detection for each pathogen. ResultsAmong 57,746 patients who underwent multiplex PCR testing, 10,516 (18.2%) had positive for at least one of the 22 pathogens. Additionally, 881 (1.5%) patients were confirmed to have a co-detection. SARS-CoV-2 exhibited negative interactions with adenovirus, coronavirus, human metapneumovirus, parainfluenza virus, respiratory syncytial virus, and rhino/enterovirus. SARS-CoV-2 co-detection with other pathogens occurred most frequently in patients of the youngest age group (0–4 years). A multiple logistic regression model indicated that younger age was the most influential factor for SARS-CoV-2 co-detection with other respiratory pathogens. ConclusionThe study highlights the prevalence of SARS-CoV-2 co-detection with other respiratory pathogens in younger age groups, necessitating further exploration of the clinical implications and severity of SARS-CoV-2 co-detection.

Viral Etiology Associated With Acute Respiratory Tract Infection Patients in Bangkok, Thailand.

Background Acute respiratory infections (ARIs) are a significant public health concern globally. After the relaxation of COVID-19 containment measures, there has been an increase in respiratory tract infections. However, the epidemiological data on circulating respiratory pathogens after the COVID-19 pandemic in Bangkok, Thailand are interesting. We conducted a study on the respiratory pathogens detected in patients with ARIs in 2023. Methodology This retrospective study utilizes archived nasopharyngeal swab samples from patients with ARIs of all ages at Bangpakok 9 Hospital between January and December 2023. These samples were collected when physicians ordered multiplex polymerase chain reaction testing as part of the diagnostic investigation. All samples were tested for 23 types of respiratory viruses and bacteria using multiplex reverse transcription-polymerase chain reaction (QIAstat-Dx, Qiagen) testing. Results Of 321 patients, viral pathogens were found in 78.5% of cases, with 21.5% remaining unidentified. Most patients (47%) were aged between two months to five years.The most common pathogen identified was rhino/enterovirus (28.0%), followed by human parainfluenza virus (15.1%), influenza virus (12.0%), respiratory syncytial virus (9.9%), human metapneumovirus (9.5%), adenovirus (9.2%), bocavirus (8.0%), and coronavirus (5.5%). Interestingly, the prevalence of SARS-CoV-2 infection was relatively low at 2.8%. Moreover, viral co-infection was observed in 25% of cases. Monthly distribution revealed the fluctuating prevalence of detected respiratory pathogens co-circulation throughout the year. In addition, the proportion of identified pathogens varied among patients across all age groups. Conclusions Our study reported the high prevalence of respiratory pathogens in ARI patients of all ages after the COVID-19 pandemic. The most prevalent respiratory pathogens among ARI cases were viruses, particularly rhino/enterovirus. The data hold significance for physician awareness regarding diagnosis, treatment, and the implementation of infection control strategies in cases of ARIs.

Unveiling the dynamics of respiratory infections revealed by multiplex PCR testing during the COVID-19 pandemic in Taiwan, 2020–2023

BackgroundThe emergence of SARS-CoV-2 in late 2019 sparked the global COVID-19 pandemic, leading to varied vaccine policies worldwide. The evolving patterns of respiratory pathogens, aside from SARS-CoV-2, during the pandemic have had a significant impact on the development of vaccine strategies. MethodsThis study explores the landscape of respiratory pathogens, encompassing SARS-CoV-2, respiratory syncytial virus (RSV), and influenza viruses, through a retrospective analysis of data obtained from the BioFire Respiratory Panel 2.1 (RP 2.1) at China Medical University Hospital (Taichung, Taiwan) spanning from January 2020 to November 2023. ResultsAmong the 7950 respiratory samples studied, pediatric cases exhibited higher positivity (64.9%, 2488/3835) and mixed detection rates (43.8%, 1090/2488) than adults. Annual mixed detection rates increased (27.9–48%). Prevalence analysis revealed diverse patterns across age groups, with higher rates in pediatrics. Notably, human rhinovirus/enterovirus predominated (48.1%). Mixed detection illustrated viral co-detections, notably with parainfluenza viruses and adenovirus. Government policies and pandemic dynamics influenced infection patterns, with RSV resurgence after May 2022. Age-specific RSV detection demonstrated a shift, influencing vaccine considerations. Amid global vaccine initiatives, RSV's increasing trend in adults warrants attention. ConclusionsThis comprehensive analysis emphasizes the importance of multiplex PCR testing in shaping targeted vaccination strategies during evolving respiratory pathogen landscapes.

Should multiplex PCR testing be integrated into antimicrobial stewardship programs for paediatric community-acquired pneumonia in the emergency department?

Should multiplex PCR testing be integrated into antimicrobial stewardship programs for paediatric community-acquired pneumonia in the emergency department?

Impact of respiratory pathogens detection by a rapid multiplex polymerase chain reaction assay on the management of community-acquired pneumonia for children at the paediatric emergency department. A randomized controlled trial, the Optimization of Pneumonia Acute Care (OPTIPAC) study

ObjectivesThe pathogen of community-acquired pneumonia (CAP) in children is typically uncertain during initial treatment, leading to systematic empiric antibiotic use. This study investigates if having rapid multiplex PCR results in the emergency department (ED) improves empiric treatment. MethodsOPTIPAC, a French multicentre study (2016–2018), enrolled patients consulting for CAP at the paediatric ED in 11 centres. Patients were randomized to either receive a multiplex PCR test plus usual care or usual care alone and followed for 15 days. The primary outcome was the appropriateness of initial antimicrobial management, determined by a blinded committee. ResultsOf the 499 randomized patients, 248 were tested with the multiplex PCR. Appropriateness of the antibiotic treatment was higher in the PCR group (168/245, 68.6% vs. 120/249, 48.2%; Relative risk 1.42 [1.22–1.66]; p < 0.0001), chiefly by reducing unnecessary antibiotics in viral pneumonia (RR 3.29 [2.20–4.90]). No adverse events were identified. DiscussionThe multiplex PCR assay result at the ED improves paediatric CAP's antimicrobial stewardship, by both reducing antibiotic prescriptions and enhancing treatment appropriateness.

Evaluation of the Bio-Speedy Meningitis/Encephalitis Panel in the Diagnosis of Central Nervous System Infections

Infections of the central nervous system (CNS) can lead to severe outcomes if not accurately diagnosed and treated. The broad spectrum of pathogens involved in CNS infections can make diagnosis challenging. Polymerase chain reaction (PCR) -based multiplex molecular diagnostic panels can rapidly and simultaneously detect multiple neuropathogens in cerebrospinal fluid (CSF). This study was aimed to assess the Bio-Speedy Meningitis/Encephalitis RT-PCR MX-17 panel (Bioeksen, İstanbul, Türkiye), a novel multiplex PCR test, in diagnosing CNS infections. The panel can detect a range of pathogens, including Escherichia coli K1, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus agalactiae, enterovirus (EV), herpes simplex virus (HSV) 1 and 2, HHV-6, HHV-7, HHV-8, human parechovirus (HPeV), varicella zoster virus (VZV), cytomegalovirus (CMV) and Cryptococcus gatti/neoformans in CSF samples. This retrospective study included 128 CSF samples from 128 patients sent to Bursa Uludağ University Health Application and Research Center Microbiology Laboratory between June 2022 and July 2023 to search for CNS infectious agents. Patient clinical, radiological and laboratory data were collected from the Hospital Information Record System (HIRS). Bacterial pathogens were identified through culture, while viral pathogens were detected in CSF samples using the Fast Track Diagnostics (FTD) multiplex RT-PCR panel (Fast Track Diagnostics Ltd., Luxembourg) for HSV-1, HSV-2, VZV, EV, mumps virus and HPeV. The stored CSF samples were then tested using the BioSpeedy panel and the results were compared with those of the culture and the FTD panel. Pathogens that were detected were considered positive if they were consistent with the patient's symptoms and CSF characteristics according to infectious disease and pediatric infectious disease specialists. Pathogens detected but not supported by the patient's symptoms and CSF characteristics were classified as uncertain clinical relevance (UCR). Out of the 128 patients tested for CNS infectious agents, 44 (34.4%) were diagnosed with a CNS infection. The overall pathogen detection rate with all methods was 43.2% (19/44). The Bio-Speedy panel identified pathogens in 29.5% (13/44) of the patients, followed by the FTD panel (20.5%, 9/44) and culture (9.1%, 4/44). Four bacteria were identified with culture, three of which were also detected by the Bio-Speedy panel. Additionally, six bacteria were identified with Bio-Speedy panel, that were not identified by culture. The FTD panel identified nine viruses, four of which were also identified by Bio-Speedy. In total, the Bio-Speedy panel detected 13 of the 19 positive pathogens (nine bacteria and four viruses: [S.pneumoniae (n= 3), VZV (n= 3), N.meningitidis (n= 2), H.influenzae (n= 2), L.monocytogenes (n= 1), E.coli (n= 1) ve EV (n= 1)]. However, the Bio-Speedy panel identified 15 pathogens [S.pneumoniae (n= 1), E.coli (n= 1), C.gatti/neoformans (n= 1), CMV (n= 8), HHV-6 (n= 3) ve HHV-7 (n= 1)] considered as UCR. The Bio-Speedy identified the causative pathogens in the highest percentage (29.5%) of patients with confirmed CNS infections. Nevertheless, test results should be interpreted based on patient characteristics to ensure appropriate patient management. Using multiple methods and multiplex tests may improve diagnostic accuracy for CNS infections.

Time-Dependent Shifts in Intestinal Bacteriome, Klebsiella Colonization and Incidence of Antibiotic-Resistance Genes after Allogeneic Hematopoietic Stem Cell Transplantation.

Dose-intensive cytostatic therapy and antibiotic treatment in allogeneic hematopoietic stem cell transplantation (allo-HSCT) cause severe abnormalities in a composition of gut microbiota as well as the emergence of antibiotic resistance. The data on the longitudinal recovery of major bacterial phyla and the expansion of genes associated with antibiotic resistance are limited. We collected regular stool samples during the first year after allo-HSCT from 12 adult patients with oncohematological disorders after allo-HSCT and performed 16SrRNA sequencing, multiplex PCR, conventional bacteriology and CHROMagar testing. We observed a decline in Shannon microbiota diversity index as early as day 0 of allo-HSCT (p = 0.034) before any administration of antibiotics, which persisted up to 1 year after transplantation, when the Shannon index returned to pre-transplant levels (p = 0.91). The study confirmed the previously shown decline in Bacillota (Firmicutes) genera and the expansion of E. coli/Shigella, Klebsiella and Enterococci. The recovery of Firmicutes was slower than that of other phyla and occurred only a year post-transplant. A positive correlation was observed between the expansion of E. coli/Shigella genera and blaKPC, blaCTX-M-1 and blaTEM (p < 0.001), Klebsiella spp. and blaOXA-48-like, blaNDM, blaCTX-M-1, blaTEM, and blaSHV (p < 0.001), Pseudomonas spp. and blaNDM (p = 0.002), Enterococcus spp. and blaOXA-48-like, blaNDM, blaCTX-M-1, blaSHV (p < 0.01). The correlation was observed between the expansion of Enterobacterales and and carbapenemase-positive CHROMagar samples (p < 0.001). Samples positive for carbapenem-resitant bacteria were at their maximum levels on day +30, and were gradually diminishing one year after allo-HSCT. From day +30 to +60, all isolated K. pneumoniae strains in fecal samples proved to be resistant to the main antibiotic groups (carbapenems, aminoglycosides, fluoroquinolones, third-generation cephalosporins). One year after HSCT, we documented the spontaneous decolonization of K. pneumoniae. The sensitivity of molecular biology techniques in the search for total and antibiotic-resistant Klebsiella seems to be superior to common bacteriological cultures. Future studies should be focused on searching for novel approaches to the efficient reconstitution and/or maintenance of strictly anaerobic microbiota in oncological patients.

Genetic Diversity of Ralstonia solanacearum Causing Tobacco Bacterial Wilt in Fujian Province and Identification of Biocontrol Streptomyces sp.

Tobacco bacterial wilt is a highly destructive soilborne disease caused by the Ralstonia solanacearum species complex, exhibiting a significant risk to global flue-cured tobacco cultivation and resulting in substantial economic loss. In this study, 77 isolates were collected from three prominent flue-cured tobacco cultivation areas in Fujian, China (Nanping, Sanming, and Longyan), in 2021 and 2022. The isolated strains were classified through phylotype-specific multiplex polymerase chain reaction (Pmx-PCR) and physiological tests. The analysis showed that all the strains were associated with phylotype I, race 1, and biovar III. Subsequent phylogenetic analysis using partial egl gene sequences classified the 77 isolates into 5 distinct sequevars: 13, 15, 16, 17, and 34. Notably, a remarkable predominance of sequevar 15 was observed in Fujian Province, while sequevar 16 was first reported on tobacco in China, which was identified in other plants, expanding the understanding of its host range and distribution in the country. In addition, a Streptomyces strain extracted from the rhizosphere soil of tobacco was found to inhibit the growth of multiple sequevars of tobacco R. solanacearum, indicating its broad-spectrum antagonistic properties. Furthermore, pot experiments showed that the strain St35 effectively controlled tobacco bacterial wilt. The isolate St35 was conclusively identified as Streptomyces gancidicus according to the morphological and genetic features. In summary, the present study demonstrated the genetic diversity and distribution of tobacco R. solanacearum strains in the Fujian province of China, as well as the identification of a candidate biological control agent for the management of tobacco bacterial wilt.

Prevalence and predictors of antibiotic prescription among patients hospitalized with viral lower respiratory tract infections in Southern Province, Sri Lanka.

Antimicrobial overprescription is common for lower respiratory tract infections (LRTI), as viral and bacterial infections generally present with similar clinical features. Overprescription is associated with downstream antimicrobial resistance. This study aims to identify the prevalence and predictors of antibiotic prescription among patients hospitalized with viral LRTI. A prospective cohort study was conducted among patients aged ≥1 year hospitalized with viral LRTI in a tertiary care hospital in Southern Province, Sri Lanka from 2018-2021. Demographic, clinical, and laboratory data were recorded. Nasopharyngeal and blood samples were collected for multiplex polymerase chain reaction testing for 21 respiratory pathogens and procalcitonin (PCT) detection, respectively. Demographic and clinical features associated with antibiotic prescription were identified using Chi Square and t-tests; significant variables (p<0.05) were further included in multivariable logistic regression models. The potential impact of biomarker testing on antibiotic prescription was simulated using standard c-reactive protein (CRP) and PCT cut-offs. Of 1217 patients enrolled, 438 (36.0%) had ≥1 respiratory virus detected, with 48.4% of these patients being male and 30.8% children. Influenza A (39.3%) and human rhinovirus/ enterovirus (28.3%) were most commonly detected. A total of 114 (84.4%) children and 266 (87.8%) adults with respiratory viruses were treated with antibiotics. Among children, neutrophil percentage (median 63.6% vs 47.6%, p = 0.04) was positively associated with antibiotic prescription. Among adults, headache (60.6% vs 35.1%, p = 0.003), crepitations/crackles (55.3% vs 21.6%, p<0.001), rhonchi/wheezing (42.9% vs 18.9%, p = 0.005), and chest x-ray opacities (27.4% vs 8.1%, p = 0.01) were associated with antibiotic prescription. Access to CRP and procalcitonin test results could have potentially decreased inappropriate antibiotic prescription in this study by 89.5% and 83.3%, respectively. High proportions of viral detection and antibiotic prescription were observed among a large inpatient cohort with LRTI. Increased access to point-of-care biomarker testing may improve antimicrobial prescription.

Gastrointestinal Panel Performance for the Diagnosis of Acute Gastroenteritis in Pediatric Patients.

Various methods are used to identify the causative organisms of acute gastroenteritis (AGE) in children. The gastrointestinal (GI) panel has the potential to detect up to 22 pathogens rapidly through the multiplex real-time PCR test. We studied the impact of the GI panel on clinical management in the pediatric population. A retrospective study was conducted to collect data on GI panel results and clinical details of inpatient children presenting with AGE at King Hamad University Hospital, Kingdom of Bahrain, over the course of one year. One hundred nine samples were collected. The GI panel was positive in 96 samples (88.1%), with the majority detected in the toddler age group. Forty-one (42.7%) samples were positive for at least one organism. Salmonella was the most frequently encountered bacteria as a single isolate, 10/55 (18.2%), while enteropathogenic Escherichia coli was the most common co-infected organism, 16/41 (39%). Norovirus was the most common virus among the viruses. Bacterial detection peaked from July to October, while viral detection plateaued throughout the year. The GI panel and stool culture were positive for the same organism in 17 samples, versus one sample with a different organism. Sixty-two (56.9%) samples had a positive GI panel but negative stool cultures and stool analysis, and half of those detected viruses. The GI panel was positive in 86.2% of severely ill patients; the majority were bacteria. Bacterial detection was associated with a higher CRP compared to viruses. The GI panel is an informative tool for detecting the causative pathogen of AGE in children. However, it can detect multiple organisms, indicating a possible carrier status, which points toward future studies.

A Rapid and Inexpensive PCR Test for Mastitis Diagnosis Based on NGS Data.

Mastitis is a common mammary gland disease of dairy cattle caused by a wide range of organisms including bacteria, fungi and algae. Mastitis contributes to economic losses of dairy farms due to reduced yield and poor quality of milk. Since the correct identification of pathogens responsible for the development of mastitis is crucial to the success of treatment, it is necessary to develop a quick and accurate test to distinguish the main pathogens causing this disease. In this paper, we describe the development of a test based on the multiplex polymerase chain reaction (PCR) method allowing for the identification of Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Staphylococcus aureus. When creating our test, we relied on the results from new generation sequencing (NGS) for accurate determination of species affiliation. The multiplex PCR test was verified on 100 strains including veterinary samples, ATCC and Polish Collection of Microorganisms (PCM) reference strains. The obtained results indicate that this test is accurate and displays high specificity. It may serve as a valuable molecular tool for the detection of major mastitis pathogens.