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Prolonged positive polymerase chain reaction (PCR) results, irrespective of the transmission risk, can lead to prolonged restrictions on daily activities and infection precaution interventions. Studies evaluating the duration of PCR positivity for multiple pathogens in a single patient cohort are scarce. This study aimed to evaluate and compare the durations of PCR positivity for multiple respiratory viruses among children and adolescents. This retrospective study was conducted between April 2018 and March 2024 using a multiplex PCR respiratory panel for symptomatic children and adolescents who had at least two tests within 90 days of study period, with the first PCR test positive. The rate and likelihood of persistent PCR positivity were evaluated for multiple respiratory viruses. For 1325 positive results, repeat tests were conducted within 90 days. The persistent PCR positivity rate at repeat testing decreased over time (60.6%, Days 1–15 and 21.7%, Days 76–90, after the first test). In multivariate logistic regression analysis, an increased likelihood of persistent PCR positivity was observed for rhinovirus/enterovirus and adenovirus, whereas decreased likelihood of persistent positivity was seen in influenza and seasonal coronaviruses, compared with parainfluenza viruses. Persistent PCR positivity is common for multiple respiratory viruses in symptomatic children. [ABSTRACT FROM AUTHOR]

Background: The burden and characteristics of respiratory viral infections in children hospitalized for acute respiratory tract infections (ARTIs) during the post-COVID-19 pandemic era are unclear. We analyzed the epidemiological and clinical characteristics of pediatric patients hospitalized with common respiratory virus infections before and after relaxation of non-pharmaceutical interventions in Hangzhou, China and evaluated the diagnostic value of the six-panel respiratory pathogen detection system. Methods: Six types of respiratory viruses were detected in respiratory samples from children with suspected ARTIs by multiplex real-time quantitative polymerase chain reaction (RT-qPCR). Changes in virus detection rates and epidemiological and clinical characteristics, obtained from electronic health records, were analyzed. Binary logistic regression was used to identify respiratory tract infections risk factors. Multiplex RT-qPCR and targeted next-generation sequencing results were compared in random samples. Results: Among the 11,056 pediatric samples, 3228 tested positive for one or more of six common respiratory pathogens. RSV and PIV-3 detection rates differed significantly across age groups (both P < 0.001), and were more common in younger children. PIV-1 was more common in infants, toddlers, and preschoolers than in school-age children (P < 0.001). FluB was predominantly detected in school-age children (P < 0.001). RSV-, ADV-, and PIV-1-positivity rates were higher in 2022 than in 2023. Seasonal viral patterns differed across years. RSV (OR 9.156. 95% CI 5.905–14.195) and PIV-3 (OR 1.683, 95% CI 1.133–2.501) were risk factors for lower respiratory tract infections. RSV-positivity was associated with severe pneumonia (P = 0.044). PIV-3 (OR 0.391, 95% CI 0.170–0.899), summer season (OR 1.982, 95% CI 1.117–3.519), and younger age (OR 0.938, 95% CI 0.893–0.986) influenced pneumonia severity. Multiplex RT-qPCR showed good diagnostic performance. Conclusion: After changes in COVID-19 prevention and control strategies, six common respiratory viruses in children were prevalent in 2022–2023, with different seasonal epidemic characteristics and age proclivities. RSV and PIV-3 cause lower, and FluA, FluB, and ADV more typically cause upper respiratory tract infections. Infancy and summer season influence severe pneumonia risk. Multiplex RT-qPCR is valuable for accurate and timely detection of respiratory viruses in children, which facilitates management, treatment, and prevention of ARTIs. [ABSTRACT FROM AUTHOR]

BACKGROUND: Multiplex PCR amplifies numerous targets in a single tube reaction and is essential in molecular biology and clinical diagnostics. One of its most important applications is in the targeted sequencing of pathogens. Despite this importance, few tools are available for designing multiplex primers. RESULTS: We developed primerJinn, a tool that designs a set of multiplex primers and allows for the in silico PCR evaluation of primer sets against numerous input genomes. We used primerJinn to create a multiplex PCR for the sequencing of drug resistance-conferring gene regions from Mycobacterium tuberculosis, which were then successfully sequenced. CONCLUSIONS: primerJinn provides a user-friendly, efficient, and accurate method for designing multiplex PCR primers for targeted sequencing and performing in silico PCR. It can be used for various applications in molecular biology and bioinformatics research, including the design of assays for amplifying and sequencing drug-resistance-conferring regions in important pathogens.

Abstract Background The burden and characteristics of respiratory viral infections in children hospitalized for acute respiratory tract infections (ARTIs) during the post-COVID-19 pandemic era are unclear. We analyzed the epidemiological and clinical characteristics of pediatric patients hospitalized with common respiratory virus infections before and after relaxation of non-pharmaceutical interventions in Hangzhou, China and evaluated the diagnostic value of the six-panel respiratory pathogen detection system. Methods Six types of respiratory viruses were detected in respiratory samples from children with suspected ARTIs by multiplex real-time quantitative polymerase chain reaction (RT-qPCR). Changes in virus detection rates and epidemiological and clinical characteristics, obtained from electronic health records, were analyzed. Binary logistic regression was used to identify respiratory tract infections risk factors. Multiplex RT-qPCR and targeted next-generation sequencing results were compared in random samples. Results Among the 11,056 pediatric samples, 3228 tested positive for one or more of six common respiratory pathogens. RSV and PIV-3 detection rates differed significantly across age groups (both P

The aim of this review is to provide practical guidance for the molecular diagnosis of parasitic diseases in Korea in 2024. Specifically, the prevalence of parasitic diseases, commercially available molecular diagnostic kits, and reference laboratories for molecular diagnosis are presented. It is based on the literature and the medical diagnosis device database of the Korea Disease Control and Prevention Agency. In Korea, molecular diagnostic kits are available for intestinal protozoa (Giardia lamblia, Entamoeba histolytica, Cryptosporidium hominis, and Cryptosporidium parvum), Trichomonas vaginalis, and malarial parasites. Molecular diagnosis of other parasites is also possible; however, there is no commercially available kit. Therefore, parasite samples or derivatives for molecular diagnosis should be sent to specific laboratories, the parasitology departments of medical schools, or the Division of Vectors and Parasitic Diseases, Bureau of Infectious Disease Diagnosis Control at the Korea Disease Control and Prevention Agency. In commercial diagnostic kits, multiplex real-time polymerase chain reaction (PCR) is used to rapidly and easily detect the amplified parasitic DNA. The loop-mediated isothermal amplification (LAMP) was developed to diagnose T. vaginalis and Acanthamoeba infections. Its merits are that it does not require a PCR machine and has a short test time of approximately 60 min. Although LAMP is not commercially available, it may be widely used to screen for parasitic diseases. Commercial molecular diagnostic kits for parasitic diseases are limited to the clinical setting in Korea. Available kits are used to diagnose certain intestinal protozoa, T. vaginalis, and to differentiate Plasmodium species using multiplex PCR.

Background: The optimal timing for applying the BioFire FilmArray Pneumonia Panel (FAPP) in intensive care unit (ICU) patients with hospital-acquired pneumonia (HAP) or ventilator-associated pneumonia (VAP) remains undefined, and there are limited data on its impact on antimicrobial stewardship. Methods: This retrospective study was conducted at a referral hospital in Taiwan from November 2019 to October 2022. Adult ICU patients with HAP/VAP who underwent FAPP testing were enrolled. Patient data, FAPP results, conventional microbiological testing results, and the real-world impact of FAPP results on antimicrobial therapy adjustments were assessed. Logistic regression was used to determine the predictive factors for bacterial detection by FAPP. Results: Among 592 respiratory specimens, including 564 (95.3%) endotracheal aspirate specimens, 19 (3.2%) expectorated sputum specimens and 9 (1.5%) bronchoalveolar lavage specimens, from 467 patients with HAP/VAP, FAPP testing yielded 368 (62.2%) positive results. Independent predictors for positive bacterial detection by FAPP included prolonged hospital stay (odds ratio [OR], 3.14), recent admissions (OR, 1.59), elevated C-reactive protein levels (OR, 1.85), Acute Physiology and Chronic Health Evaluation II scores (OR, 1.58), and septic shock (OR, 1.79). Approximately 50% of antimicrobial therapy for infections caused by Gram-negative bacteria and 58.4% for Gram-positive bacteria were adjusted or confirmed after obtaining FAPP results. Conclusions: This study identified several factors predicting bacterial detection by FAPP in critically ill patients with HAP/VAP. More than 50% real-world clinical practices were adjusted or confirmed based on the FAPP results. Clinical algorithms for the use of FAPP and antimicrobial stewardship guidelines may further enhance its benefits.

Tung Nguyen Thanh,1 Sang Trieu Tien,2 Phong Nguyen Van,2 Son Dang Thai,3 Thuc Luong Cong,4 Tuan Dinh Le,5 Son Tien Nguyen,5 Tuan Tran Van,1 Hoang Huy Duong,6 Tien Minh Bui,7 Kien Trung Nguyen7 1Military Institute of Clinical Embryology and Histology, Vietnam Military Medical University, Hanoi, 100000, Vietnam; 2Department of Biology and Medical Genetics, Vietnam Military Medical University, Hanoi, 100000, Vietnam; 3Institute of Biological and Food Technology, Hanoi Open University, Hanoi, 100000, Vietnam; 4Cardiovascular Center, Military Hospital 103, Vietnam Military Medical University, Hanoi, 100000, Vietnam; 5Department of Rheumatology and Endocrinology, Military Hospital 103, Vietnam Military Medical University, Hanoi, 100000, Vietnam; 6Department of Neurology, Thai Binh University of Medicine and Pharmacy, Thai Binh, 410000, Vietnam; 7Department of Obstetrics and Gynecology, Thai Binh University of Medicine and Pharmacy, Thai Binh, 410000, VietnamCorrespondence: Sang Trieu Tien, Department of Biology and Medical Genetics, Vietnam Military Medical University, Hanoi, 100000, Vietnam, Email trieusangk83@yahoo.com.vnBackground: To optimize the multiplex polymerase chain reaction (M-PCR) technique to diagnose microdeletions of azoospermia factors (AZF) on the Y chromosome and initially apply the technique to diagnose male patients with sperm density less than 5× 106 million sperm/mL was assigned to do a test to check for AZF microdeletions on the Y chromosome.Methods: Based on the positive control samples which belong to male subjects who have had 2 healthy children without any assisted reproductive technologies, the M-PCR method was developed to detect simultaneously and accurately AZF microdeletions on 32 male patients with sperm densities below 5× 106 million sperm/mL of semen at the Department of Biology and Medical Genetics – Vietnam Military Medical University.Results: Successful optimization of the M-PCR technique including 7 reactions arranged according to each AZFabc region using 24 STS/gene on the Y chromosome. Initial application to diagnose AZF deletion on 32 azoospermic and oligospermic men reveals that AZFa deletion accounts for 6.25% (2/32); deletion of all 3 regions AZFa,b,c with 18.75% (6/32 cases); The combined deletion rate of AZFb,c is highest, accounting for 56.24% (18/32 patients).Conclusion: Successfully optimized the M-PCR technique in identifying AZF microdeletions using 24 sequence tagged sites (STS)/gene for azoospermic and oligozoospermic men. The M-PCR technique has great potential in the application of AZF deletion diagnosis.Keywords: male infertility, azoospermia factors, AZF, multiplex polymerase chain reaction, M-PCR, sequence tagged sites, STS

Background and Objectives: During the coronavirus pandemic, the overuse of antibiotics to reduce coinfections and mortality may be contributing to the rise of antimicrobial resistance. In this study, we aim to investigate the antibiotic resistance changes of Acinetobacter baumannii post-COVID-19 pandemic in Northern Iran. Materials and Methods: The current study is a cross-sectional study. Between 2022 and 2023, 2190 clinical samples were collected from patients with healthcare-associated infections (HAIs) at four hospitals in Sari, which served as corona centers after the COVID-19 pandemic. Antimicrobial sensitivity was determined using standard broth macro-dilution, and resistance genes were detected using multiplex PCR. Results: Based on the results co-amoxiclav had a resistance rate of 100%, while piperacillin/tazobactam showed the least resistance rate of 29.82%. In terms of GM MIC values, colistin was the most potent against multi-drug resistant isolates. The frequency of bla OXA-51, ampC, aphA6, and bla NDM genes were 100%, 99.12%, 90.35%, and 69.30% respectively. Conclusion: Our study revealed high multi-drug resistance rates. Piperacillin/tazobactam recommended for treating multidrug resistant Acinetobacter baumannii infections in Northern Iran. [ABSTRACT FROM AUTHOR]

Copyright of Biomédica: Revista del Instituto Nacional de Salud is the property of Instituto Nacional de Salud of Colombia and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background: To optimize the multiplex polymerase chain reaction (M-PCR) technique to diagnose microdeletions of azoospermia factors (AZF) on the Y chromosome and initially apply the technique to diagnose male patients with sperm density less than 5× 106 million sperm/mL was assigned to do a test to check for AZF microdeletions on the Y chromosome. Methods: Based on the positive control samples which belong to male subjects who have had 2 healthy children without any assisted reproductive technologies, the M-PCR method was developed to detect simultaneously and accurately AZF microdeletions on 32 male patients with sperm densities below 5× 106 million sperm/mL of semen at the Department of Biology and Medical Genetics – Vietnam Military Medical University. Results: Successful optimization of the M-PCR technique including 7 reactions arranged according to each AZFabc region using 24 STS/gene on the Y chromosome. Initial application to diagnose AZF deletion on 32 azoospermic and oligospermic men reveals that AZFa deletion accounts for 6.25% (2/32); deletion of all 3 regions AZFa,b,c with 18.75% (6/32 cases); The combined deletion rate of AZFb,c is highest, accounting for 56.24% (18/32 patients). Conclusion: Successfully optimized the M-PCR technique in identifying AZF microdeletions using 24 sequence tagged sites (STS)/gene for azoospermic and oligozoospermic men. The M-PCR technique has great potential in the application of AZF deletion diagnosis. [ABSTRACT FROM AUTHOR]

ABSTRACT Seasonal increase of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus A/B (Flu A/B), and respiratory syncytial virus (RSV) require rapid diagnostic test methods for the management of respiratory tract infections. In this study, we compared the diagnostic accuracy of Savanna RVP4 (RVP4, QuidelOrtho) with Xpert Xpress Plus SARS-CoV-2/Flu/RSV (Xpert, Cepheid). Nasopharyngeal swabs from patients treated at a tertiary care hospital (Germany) were tested for SARS-CoV-2, Flu A/B, and RSV by RVP4 to assess diagnostic accuracy (reference standard: Xpert). The intra and inter assay precision of Ct-values was assessed by repeated test in triplicates (on day 1) and duplicates (days 2–3). All patients with a physician’s order for a multiplex test for SARS-CoV-2, Flu, and RSV test were included. Duplicate swabs from the same patient, samples with a total volume ≤1 mL, or inappropriate shipment/storage were excluded. In total, 229 swabs were included between September 2023 and February 2024. The concordance between both tests was 96.5% (SARS-CoV-2), 98.7% (Flu A), and 99.6% (RSV). Flu B was not detected by both tests. The RVP4 test had a sensitivity of 85%–95% and a specificity of 100% for the detection of SARS-CoV-2, Flu A, and RSV. The intra and inter assay precision of Ct-values from RVP4 was 3% and 2% (SARS-CoV-2), 5% and 4% (Flu A), and 0% and 3% (RSV), respectively. The Savanna RVP4 has a favorable diagnostic accuracy for the detection of SARS-CoV-2, Flu A, and RSV.IMPORTANCEWe assessed the diagnostic accuracy of a new point-of-care test for the rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus A/B (Flu A/B), and respiratory syncytial virus (RSV). The new test has a concordance with the reference standard of 96.5% (SARS-CoV-2), 98.7% (Flu A), and 99.1% (RSV). The sensitivity of 85%–95% and specificity of 100% for the detection of SARS-CoV-2, Flu A, and RSV is comparable with similar nucleic acid amplification-based point of care tests but at lower costs.

Background Multiplex molecular diagnostic panels have greatly enhanced detection of gastrointestinal pathogens. However, data on the impact of these tests on clinical and patient-centered outcomes are limited. Methods We conducted a prospective, multicenter, stepped-wedge trial to determine the impact of multiplex molecular testing at 5 academic children's hospitals on children presenting to the emergency department with acute gastroenteritis. Caregivers were interviewed on enrollment and 7–10 days after enrollment to determine symptoms, risk factors, subsequent medical visits, and impact on family members. During the pre-intervention period, diagnostic testing was performed at the clinician's discretion. During the intervention period, multiplex molecular testing was performed on all children, with results available to clinicians. The primary outcome was return visits to a healthcare provider within 10 days of enrollment. Results Potential pathogens were identified by clinician-ordered tests in 19 of 571 (3.3%) in the pre-intervention period compared with 434 of 586 (74%) in the intervention period; clinically relevant pathogens were detected in 2.1% and 15%, respectively. In the multivariate model, the intervention was associated with a 21% reduction in the odds of any return visit (odds ratio, 0.79; 95% confidence interval,.70–.90) after adjusting for potential confounders. Appropriate treatment was prescribed in 11.3% compared with 19.6% during the intervention period (P =.22). Conclusions Routine molecular multiplex testing for all children who presented to the ED with acute gastroenteritis detected more clinically relevant pathogens and led to a 21% decrease in return visits. Additional research is needed to define patients most likely to benefit from testing. Clinical Trials Registration. NCT02248285. [ABSTRACT FROM AUTHOR]

Purpose: Diarrhea is one of the leading causes of mortality in children living in developing countries. The etiology of acute diarrhea in each healthcare center varies depending on place, time, and population. This study aimed to identify pathogen patterns in human immunodeficiency virus (HIV)-infected and non-HIV children suffering from acute diarrhea, using multiplex real time reverse transcriptase polymerase chain reaction (RT-PCR), in an Indonesian tertiary hospital. Methods: This cross-sectional study was conducted at Dr. Cipto Mangunkusumo National Hospital from March 2019 to April 2020. Results: The study showed that multiplex RT-PCR results were positive in 58.9% of the specimens, with more positive results in HIV-infected children than in non-HIV-infected children (70% vs. 54.7%). Altogether 72 enteropathogens were detected from all specimens. Enteropathogens in non-HIV children with acute diarrhea consisted of bacteria (70.6%) and viruses (29.4%) with a predominance of enteroaggregative Escherichia coli (25.4%), followed by Campylobacter spp. (11.8%), enteropathogenic E. coli (9.8%), Norovirus GII (7.8%), and Clostridium difficile (7.8%). Enteropathogens in HIV-infected children consisted of viruses (57.1%), bacteria (28.6%), and parasites (14.3%) comprising Norovirus GII (24%), Cryptosporidium spp. (14.3%), Campylobacter spp. (14.3%), Norovirus GI (14.3%), and Astrovirus (14.3%). Cryptosporidium spp. was the only parasite found in this study and was found only in HIV-infected children. In non-HIV children with acute diarrhea, most pathogens were invasive bacteria, while in HIV-infected children, more viral and parasite infections occurred, primarily caused by opportunistic pathogens. Conclusion: The pattern of enteropathogens can help clinicians determine further examinations and appropriate empirical antimicrobial therapy for the patient. [ABSTRACT FROM AUTHOR]

Various harmful microalgae coexist in the marine environment, seriously affecting the survival of aquatic animals, the sustainable development of fisheries, and pose a great threat to the safety of aquatic food. Consequently, monitoring these harmful microalgae in the marine aquaculture environment is an urgent issue that needs to be addressed. This paper focused on developing a multiplex polymerase chain reaction (MPCR) assay for the simultaneous detection of representative harmful microalgae commonly distributed in the aquaculture environment along the Chinese coast, including Karenia mikimotoi, Alexandrium tamarense, Chattonella marina, Prorocentrum minimum and Heterosigma akashiwo. First, the internal transcribed spacer and large subunit rDNA of target algal species were selected as target for the design of MPCR primers, the specificity of which was subsequently verified. Next, the MPCR system was established and the reaction conditions were optimized, including primer concentration, dNTP concentration, Mg2+ concentration, the concentration of Taq DNA polymerase, and annealing temperature. Specificity test showed that the established MPCR did not cross-react with all of the control algal species and thus can be used for specific detection of target algal species. The sensitivity test showed that the established MPCR could still detect all of the target algae when the concentration of genomic DNA was as low as 1 ng µL–1. Moreover, the MPCR could still detect K. mikimotoi when the concentration of genomic DNA was as low as 1 pg µL–1. The stability test indicated that the performance of the developed MPCR was not affected by the interfering algal species. In addition, the practicality of the established MPCR assay was evaluated using spiked water samples. The tests with the spiked samples indicated that the detection limit of MPCR for the all of the target algal species was approximately 4 cells mL−1, which can meet the requirement for the warning of HABs caused by them. In conclusion, the MPCR assay established here is characterized with strong specificity and good stability and is expected to be promising for daily monitoring of harmful microalgae in the aquaculture environment as an important alternative to traditional morphology-based methods. [ABSTRACT FROM AUTHOR]

Antibiotics are frequently prescribed to children with pneumonia, although viruses are responsible for most cases. We aimed to evaluate the impact of multiplex polymerase chain reaction (mPCR) on antibiotic use. We conducted a prospective study of children under 14 years of age admitted for suspected viral pneumonia, from October 2019 to June 2022 (except March–November 2020). A mPCR respiratory panel (FilmArray® 2plus, bioMérieux, Marcy-l'Étoile, France) was performed within 72 h of admission. Patients with positive reverse transcription PCR for respiratory syncytial virus, influenza, or SARS-CoV-2 were excluded. We compared the patients with historical controls (2017–2018) who had suspected viral pneumonia but did not undergo an aetiological study. We included 64 patients and 50 controls, with a median age of 26 months. The respiratory panel detected viral pathogens in 55 patients (88%), including 17 (31%) with co-infections. Rhinovirus/enterovirus (n = 26) and human metapneumovirus (n = 22) were the most common pathogens, followed by adenovirus and parainfluenza (n = 10). There were no statistically significant differences in the total antibiotic consumption (83% of cases and 86% of controls) or antibiotics given for ≥72 h (58% vs. 66%). Antibiotics were prescribed in 41% of the cases and 72% of the controls at discharge (p = 0.001). Ampicillin was the most commonly prescribed antibiotic among the patients (44% vs. 18% for controls, p = 0.004), while azithromycin was the most commonly prescribed among the controls (19% vs. 48% for patients and controls, respectively; p = 0.001). Our findings underscore the need for additional interventions alongside molecular diagnosis to reduce antibiotic usage in paediatric community-acquired pneumonia. [ABSTRACT FROM AUTHOR]

Background and Objectives: During the coronavirus pandemic, the overuse of antibiotics to reduce coinfections and mortality may be contributing to the rise of antimicrobial resistance. In this study, we aim to investigate the antibiotic resistance changes of Acinetobacter baumannii post-COVID-19 pandemic in Northern Iran. Materials and Methods: The current study is a cross-sectional study. Between 2022 and 2023, 2190 clinical samples were collected from patients with healthcare-associated infections (HAIs) at four hospitals in Sari, which served as corona centers after the COVID-19 pandemic. Antimicrobial sensitivity was determined using standard broth macro-dilution, and resistance genes were detected using multiplex PCR. Results: Based on the results co-amoxiclav had a resistance rate of 100%, while piperacillin/tazobactam showed the least resistance rate of 29.82%. In terms of GM MIC values, colistin was the most potent against multi-drug resistant isolates. The frequency of blaOXA-51, ampC, aphA6, and blaNDM genes were 100%, 99.12%, 90.35%, and 69.30% respectively. Conclusion: Our study revealed high multi-drug resistance rates. Piperacillin/tazobactam recommended for treating multidrug resistant Acinetobacter baumannii infections in Northern Iran.

Dakun Wang,1 Emery Haley,2 Natalie Luke,2 Mohit Mathur,3 Richard A Festa,3 Xinhua Zhao,4 Lori A Anderson,5 Jennifer L Allison,6 Kristen L Stebbins,6 Manuel Jose Diaz,6 David Baunoch7 1Department of Writing, Stat4Ward, Pittsburgh, PA, USA; 2Department of Clinical Research, Pathnostics, Irvine, CA, USA; 3Department of Medical Affairs, Pathnostics, Irvine, CA, USA; 4Department of Statistical Analysis, Stat4Ward, Pittsburgh, PA, USA; 5L. Anderson Diagnostic Market Access Consulting, San Diego, CA, USA; 6DispatchHealth, Denver, CO, USA; 7Pathnostics, Irvine, CA, 92618, USACorrespondence: David Baunoch, Pathnostics, 15545 Sand Canyon Suite 100, Irvine, CA, 92618, USA, Tel +1 714-966-1221, Fax +1 714-966-1231, Email dbaunoch@pathnostics.comIntroduction: This study compared microbial compositions of midstream and catheter urine specimens from patients with suspected complicated urinary tract infections to determine if emerging and fastidious uropathogens are infecting the bladder or are contaminants.Methods: Urine was collected by in-and-out catheter (n = 1000) or midstream voiding (n = 1000) from 2000 adult patients (≥ 60 years of age) at 17 DispatchHealth sites across 11 states. The two groups were matched by age (mean 81 years), sex (62.1% female, 37.9% male), and ICD-10-CM codes. Microbial detection was performed with multiplex polymerase chain reaction (M-PCR) with a threshold for “positive detection” ≥ 10,000 cells/mL for bacteria or any detection for yeast. Results were divided by sex.Results: In females, 28 of 30 microorganisms/groups were found by both collection methods, while in males 26 of 30 were found by both. There were significant overlaps in the detection and densities of classical uropathogens including Escherichia coli, Enterococcus faecalis, and Klebsiella pneumoniae, as well as emerging uropathogens including Actinotignum schaalii and Aerococcus urinae. In females, detection rates were slightly higher in midstream voided compared to catheter-collected (p = 0.0005) urine samples, while males showed the opposite trend (p < 0.0001). More polymicrobial infections were detected in midstream voided compared to catheter-collected samples (64.4% vs 45.7%, p < 0.0001) in females but the opposite in males (35.6% vs 47.0%, p = 0.002).Discussion: In-and-out catheter-collected and midstream voided urine specimens shared significant similarities in microbial detections by M-PCR, with some differences found for a small subset of organisms and between sexes.Conclusion: Non-invasive midstream voided collection of urine specimens for microbial detection and identification in cases of presumed UTI does not result in significantly more contamination compared to in-and-out catheter-collected specimens. Additionally, organisms long regarded as contaminants should be reconsidered as potential uropathogens.Keywords: urinary tract infection, standard urine culture, diagnostic testing, multiplex polymerase chain reaction, catheter, midstream voided

Background: Multiplex polymerase chain reaction (M-PCR) has increased sensitivity for microbial detection compared with standard urine culture (SUC) in cases diagnosed as urinary tract infections (UTIs), leading to questions whether detected microbes are likely causative of UTIs or are incidental findings. Objective: To compare infection-associated biomarker levels against M-PCR and SUC results in symptomatic cases with a presumptive diagnosis of a UTI by a urologist. Design, setting, and participants: Participants were ≥60 yr old and presented to urology clinics between January and April 2023 with symptoms of UTIs (n = 583). Urine microbial detection was by M-PCR and SUC. Three infection-associated biomarkers (neutrophil gelatinase-associated lipocalin, interleukin-8, and interleukin-1β) were measured by enzyme-linked immunosorbent assay. Symptomatic cases with elevated biomarkers, detection of uropathogens, and a specialist clinical diagnosis of a UTI were considered definitive UTI cases. Outcome measurements and statistical analysis: Distributions were compared using two-sample Wilcoxon rank sum test, with two-tailed p values of

We evaluated the impact of the FilmArray blood culture identification (BCID) panel on the time taken to administer effective antibiotics and the clinical outcomes of bloodstream infections. We retrospectively screened patients with bloodstream infections who underwent BCID testing and compared them to a historical control group that received conventional culture testing. A total of 144 and 214 patients who underwent BCID and conventional cultures, respectively, were compared. The 30-day mortality (BCID: 9.7% vs. conventional method: 10.7%, p = 0.755), time to effective antibiotic administration (3 h for both BCID and conventional method, p = 0.789), and time to appropriate antibiotic administration did not differ significantly between the groups. BCID was not significantly associated with 30-day mortality after adjusting for the Pitt bacteremia score and the Charlson comorbidity index (adjusted OR = 0.833, CI; 0.398–1.743). Compared with conventional methods, BCID reduced the time to administration of effective antibiotics in cases of carbapenem-resistant Enterobacterales (CRE) (39 h vs. 93 h, p = 0.012) and vancomycin-resistant enterococci (VRE) (50 h vs. 92 h, p < 0.001) bacteremia. BCID did not affect the clinical outcomes of overall bloodstream infections; however, it contributed to the early administration of effective antibiotics in cases of CRE and VRE bacteremia. [ABSTRACT FROM AUTHOR]

Background: Acute respiratory infections (ARIs) are an important cause of pediatric mortality–morbidity worldwide, the most common etiology being viral. This study aims to identify causative organisms for ARIs admitted in pediatric intensive care unit (PICU), when multiplex polymerase chain reaction (PCR) testing of respiratory secretions was sent; any seasonal trends detect microbiological correlation when co-infections. Subjects and Methods: This was a retrospective observational study, from July 2021 to December 2022, of children aged 1 month–18 years, whose multiplex PCR tests (nasopharyngeal, endotracheal [ET] secretion or bronchoscopic alveolar lavage [BAL]) were sent when admitted for ARI to tertiary care PICU. Results: In the study period, 372 of 1492 medical PICU admissions were ARI. Multiplex PCR of 81 respiratory secretions was sent, of which 69 (85%) were positive. Multiplex pcr sample positivity : 83% for nasopharyngeal aspirate, 78% for ET secretions, 100% for BAL samples. Forty-one percent of samples detected >1 organism. Respiratory syncytial virus (RSV)-A was the most common virus (18); other organisms included adenovirus (n = 5), influenza (n = 9), parainfluenza (n = 5), rhinovirus: 13, Pneumocystis Jerovecci (PCP): 4, Streptococcus pneumoniae: 17, pertussis: 1, and Haemophilus influenzae B: 9. ARIs were seen throughout the year with peaks in monsoon season and a peak in cases of ARI due to RSV from July to October. Of co-infections with bacteria in ET secretions and BAL samples via multiplex PCR, bacterial culture reports were sterile. Conclusions: Multiplex PCR detected organisms in 85% of ARI patients tested. Most of the ARIs getting admitted to PICU were viral in origin. RSV was the most common virus isolated showing peak from July to October, local monsoon season. With extended viral and bacterial PCR being available, mixed infections/colonization with uncertain significance are being detected. [ABSTRACT FROM AUTHOR]

Background and Objectives: Acinetobacter baumannii, an opportunistic pathogen, is related to hospital-acquired infections and increased mortality. This study aimed to develop the loop-mediated isothermal amplification (LAMP) test for the fast-detecting of A. baumannii isolates as well as determining genetic relatedness for these isolates via the REP-PCR technique. Materials and Methods: LAMP primers and multiplex PCR primers were designed for recognizing A. baumannii isolates harboring the blaSHV-1, blaPER-1, blaTEM-1, AMPC, qnr, and aac (6)-1 genes, were collected (October 2020 to February 2021) from Shahid Motahari Hospital, Tehran, Iran. Combination disc test (CDT) results were used to assess the phenotypic identification of isolates from ESBL producers. The sensitivity of the LAMP method was evaluated using a range of serial dilutions of genomic DNA. Results were compared between the LAMP technique, and multiplex PCR. The genetic diversity of clinical isolates was determined by REP-PCR. Results: Among one hundred A. baumannii samples and based on the combined disc test, 56% of isolates were ESBL producers. The sensitivity of the LAMP technique for the identification of A. baumannii was 4.06 ng/μl whilst the multiplex PCR was (16.2 ng/μl). Regarding multiplex PCR, (68%) of the isolates were blaSHV-1 positive, (40%) blaPER-1, (85%) aac (6)-1, AMPC (67%), blaTEM-1 (63%), and (15%) qnr respectively. While in LAMP, (69%) of isolates were bla positive, (86%) aac (6')-1, and (20%) qnr. The results of AMPC, blaTEM-1, and blaPER-1 genes showed 100% compatibility between multiplex PCR and LAMP assays. The results of REP-PCR indicated there were 17 clones, clone A at 14% was the most prevalent of the isolates. Conclusion: Wherever equipment and financial constraints are crucial, the LAMP test offers a better and more potent detection rate for the identification of A. baumannii isolates than multiplex PCR. Furthermore, the genetic diversity of A. baumannii in these clinical isolates showed frequent commonality of genotypes. [ABSTRACT FROM AUTHOR]

In this cohort study of 800 children attending a pediatric emergency department at Oulu University Hospital, Finland with fever or respiratory symptoms, the cycle threshold values of point-of-care multiplex polymerase chain reaction testing for respiratory viruses were not associated with hospitalization, respiratory support, or need for intensive care. [ABSTRACT FROM AUTHOR]

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Background and Aim: Bovine coccidiosis, caused by the protozoa Eimeria, is an important parasitic cattle disease that affects animal health and has economic impact worldwide. This study was conducted to report the first molecular prevalence and genetic diversity of Eimeria spp. in dairy cattle in Khon Kaen province, Thailand, and to identify the risk factors associated with Eimeria spp. infection. Materials and Methods: From July 2020 to October 2021, 296 fecal samples were collected from dairy cattle divided into three age groups, including 1-year-old cattle. Eimeria spp. were identified by multiplex polymerase chain reaction (PCR) amplifying 18S RNA gene and confirmed by DNA sequencing. Information regarding all associated risk factors was collected using questionnaires and analyzed using logistic regression tests in the Statistical Package for the Social Sciences program. Results: Polymerase chain reaction results showed that 104 (35.13%) of 296 samples were positive for Eimeria spp. The

Abstract Background Escherichia coli infections are common in Xinjiang, a major region of cattle and sheep breeding in China. Therefore, strategies are required to control E. coli. The aim of this study was to investigate the phylogenetic groups, virulence genes, and antibiotic resistance characteristics of E. coli isolates. Methods In this study, 116 tissue samples were collected from the organs of cattle and sheep that were suspected of having E. coli infections between 2015 and 2019. Bacteria in the samples were identified using a biochemical identification system and amplification of 16S rRNA, and the phylogenetic groupings of E. coli isolates were determined by multiplex polymerase chain reactions. In addition, PCR detection and analysis of virulence factors, antibiotic resistance genes, and drug‐resistant phenotypes of E. coli isolates were performed. Results A total of 116 pathogenic E. coli strains belonging to seven phylogenetic groups were isolated, with the majority of isolates in groups A and B1. Among the virulence genes, curli‐encoding crl had the highest detection rate of 97.4%, followed by hemolysin‐encoding hlyE with the detection rate of 94.82%. Antimicrobial susceptibility test results indicated that the isolates had the highest rates of resistance against streptomycin (81.9%). Conclusion These characteristics complicate the prevention and treatment of E. coli‐related diseases in Xinjiang.

Listeria monocytogenes is a rare cause of bacterial meningitis, particularly beyond the neonatal period. This article describes a 16-month-old girl who presented with a new-onset febrile status epilepticus, and subsequently developed altered mentality and lethargy. L. monocytogenes was detected on a point-of-care, multiplex polymerase chain reaction using the cerebrospinal fluid. On day 3, she developed a rapidly progressive hydrocephalus. Her consciousness improved after placement of an external ventricular drain. After 3-week antibiotic therapy, she was successfully discharged without residual complications.

Background: Community-Acquired Pneumonia (CAP) is the primary cause of hospitalization in the United States and the third leading cause of death in Brazil. The gold standard for diagnosing the etiology of CAP includes blood culture, Gram-stained sputum, and sputum culture. However, these methods have low sensitivity. No studies investigating the etiology of CAP have been conducted in Brazil in the last 20-years, and the empirical choice of antimicrobials is mainly based on the IDSA guidelines. This is the first national study with this aim, and as a result, there's potential for the Brazilian consensus to be impacted and possibly modify its guidelines rather than adhering strictly to the IDSA's recommendations. Methods: The aim of this study is to identify the main microorganisms implicated in CAP by employing a multiplex Polymerase Chain Reaction (mPCR) at the foremost public hospital in Brazil. All patients who were admitted to the emergency department and diagnosed with severe CAP underwent an mPCR panel using nasopharyngeal and oropharyngeal swabs, with the aim of detecting 13 bacterial and 21 viral pathogens. Results: A total of 169 patients were enrolled in the study. The mPCR panel identified an etiological agent in 61.5% of patients, with viruses being the most common (42.01%), led by Rhinovirus, followed by Influenza and Coronavirus (non-SARS-CoV-2). Bacterial agents were identified in 34.91% of patients, with S. pneumoniae being the most common, followed by H. influenzae, M. catarrhalis, and S. aureus. Additionally, we found that the prescription for 92.3% of patients could be modified, with most changes involving de-escalation of antibiotics and antiviral therapy. Conclusion: Our study revealed different etiological causes of CAP than those suggested by the Brazilian guidelines. Using molecular diagnostic tests, we were able to optimize treatment by using fewer antibiotics.

Background and Objectives: Acinetobacter baumannii, an opportunistic pathogen, is related to hospital-acquired infections and increased mortality. This study aimed to develop the loop-mediated isothermal amplification (LAMP) test for the fast-detecting of A. baumannii isolates as well as determining genetic relatedness for these isolates via the REP-PCR technique. Materials and Methods: LAMP primers and multiplex PCR primers were designed for recognizing A. baumannii isolates harboring the blaSHV-1, blaPER-1, blaTEM-1, AMPC, qnr, and aac (6)-1 genes, were collected (October 2020 to February 2021) from Shahid Motahari Hospital, Tehran, Iran. Combination disc test (CDT) results were used to assess the phenotypic identification of isolates from ESBL producers. The sensitivity of the LAMP method was evaluated using a range of serial dilutions of genomic DNA. Results were compared between the LAMP technique, and multiplex PCR. The genetic diversity of clinical isolates was determined by REP-PCR. Results: Among one hundred A. baumannii samples and based on the combined disc test, 56% of isolates were ESBL producers. The sensitivity of the LAMP technique for the identification of A. baumannii was 4.06 ng/μl whilst the multiplex PCR was (16.2 ng/μl). Regarding multiplex PCR, (68%) of the isolates were blaSHV-1 positive, (40%) blaPER-1, (85%) aac (6́)-1, AMPC (67%), blaTEM-1 (63%), and (15%) qnr respectively. While in LAMP, (69%) of isolates were blaSHV-1 positive, (86%) aac (6')-1, and (20%) qnr. The results of AMPC, blaTEM-1, and blaPER-1 genes showed 100% compatibility between multiplex PCR and LAMP assays. The results of REP-PCR indicated there were 17 clones, clone A at 14% was the most prevalent of the isolates. Conclusion: Wherever equipment and financial constraints are crucial, the LAMP test offers a better and more potent detection rate for the identification of A. baumannii isolates than multiplex PCR. Furthermore, the genetic diversity of A. baumannii in these clinical isolates showed frequent commonality of genotypes.

Background and Aim: Bovine coccidiosis, caused by the protozoa Eimeria, is an important parasitic cattle disease that affects animal health and has economic impact worldwide. This study was conducted to report the first molecular prevalence and genetic diversity of Eimeria spp. in dairy cattle in Khon Kaen province, Thailand, and to identify the risk factors associated with Eimeria spp. infection. Materials and Methods: From July 2020 to October 2021, 296 fecal samples were collected from dairy cattle divided into three age groups, including <3-month-old calves, 3-month-old to 1-year-old calves, and >1-year-old cattle. Eimeria spp. were identified by multiplex polymerase chain reaction (PCR) amplifying 18S RNA gene and confirmed by DNA sequencing. Information regarding all associated risk factors was collected using questionnaires and analyzed using logistic regression tests in the Statistical Package for the Social Sciences program. Results: Polymerase chain reaction results showed that 104 (35.13%) of 296 samples were positive for Eimeria spp. The <3-month-old calves (46.51%) had the highest infection rate. Moreover, multiplex PCR identified five species of Eimeria, namely, Eimeria bovis (32.69%), Eimeria zuernii (18.26%), Eimeria alabamensis (5.76%), Eimeria ellipsoidalis (3.84%), and Eimeria cylindrica (2.88%). An association was observed between risk factors and Eimeria spp. incidence (p < 0.05). DNA sequencing revealed the similarity of each Eimeria spp. with 91%-100% nucleotide identity. Phylogenetic tree analysis demonstrated the close relationships of clusters of E. bovis and E. zuernii, E. ellipsoidalis, and E. cylindrica and another cluster of E. alabamensis. Conclusion: The results confirm that Eimeria spp. are commonly found in dairy cattle, especially calves. The molecular test could be powerful for species identification. This study also provides epidemiological information for developing future strategies to control bovine coccidiosis. [ABSTRACT FROM AUTHOR]

Background: When children have a preoperative fever, anesthesiologists must help determine whether to postpone or proceed with surgery, as fever may be a sign of upper respiratory tract infection (URTI). Such infections are a known risk factor for perioperative respiratory adverse events (PRAEs), which are still one of the prime causes of anesthetic mortality and morbidity in pediatric patients. Since the COVID‐19 pandemic, preoperative assessments have become drastically more complex as hospitals strive to balance practicality and safety. In our facility, if pediatric patients presented with preoperative fever, we used the FilmArray® Respiratory Panel 2.1 to determine whether to postpone or proceed with surgery. Methods: This is a single‐center retrospective observational study evaluating the efficacy of the FilmArray® Respiratory Panel 2.1 as a preoperative screening test. This study included pediatric patients scheduled for elective surgeries between March 2021 and February 2022. FilmArray was used if a patient had a preoperative fever (determined by axillary temperature, ≥38°C for <1‐year‐old, ≥37.5°C for ≥1‐year‐old) between hospital admission and before surgery. We excluded patients if they had apparent symptoms of URTI. Results: In the FilmArray positive group, 11 of 25 (44%) cases developed subsequent symptoms after surgery was canceled. No patients in the negative group developed symptoms. The proportion of the development of subsequent symptoms between the FilmArray positive and negative groups was statistically significant (p <.001, odds ratio: 29.6, 95% confidence interval: [3.80–1356.01]). Conclusions: Our retrospective observational study revealed that 44% of the FilmArray positive group subsequently developed symptoms, and no PRAEs were observed in the FilmArray negative group. We suggest that FilmArray could be useful as a screening test for pediatric patients with preoperative fever. [ABSTRACT FROM AUTHOR]

We developed and assessed the performance of a new multiplex real-time PCR assay for the detection of all Chlamydia species and simultaneous differentiation of Chlamydia psittaci and Chlamydia pneumoniae--two important human respiratory pathogens--in human clinical specimens. Next-generation sequencing was used to identify unique targets to design real-time PCR assays targeting all Chlamydia species, C. psittaci, and C. pneumoniae. To validate the assay, we used a panel of 49 culture isolates comprising seven C. psittaci genotypes, eight C. pneumoniae isolates, seven other Chlamydia species, and 22 near-neighbor bacterial and viral isolates, along with 22 specimens from external quality assessment (EQA) panels and 34 nasopharyngeal and oropharyngeal swabs and cerebrospinal fluid, stool, and sputum specimens previously identified as positive or negative for C. psittaci or C. pneumoniae. The assays were 100% specific, with limits of detection of 7.64-9.02 fg/µL. The assay results matched with historical assay results for all specimens, except for one owing to the increased sensitivity of the new C. psittaci assay; the results of the EQA specimens were 100% accurate. This assay may improve the timely and accurate clinical diagnosis of Chlamydia infections and provide a greater understanding of the burden of disease caused by these agents. [ABSTRACT FROM AUTHOR]

To accurately identify the genes and pathways involved in the initiation of the Myxococcus xanthus multicellular developmental program, we have previously reported a method of growing vegetative populations as biofilms within a controllable environment. Using a modified approach to remove up to ~90% rRNAs, we report a comprehensive transcriptional analysis of the M. xanthus developmental cycle while comparing it with the vegetative biofilms grown in rich and poor nutrients. This study identified 1522 differentially regulated genes distributed within eight clusters during development. It also provided a comprehensive overview of genes expressed during a nutrient-stress response, specific development time points, and during development initiation and regulation. We identified several differentially expressed genes involved in key central metabolic pathways suggesting their role in regulating myxobacterial development. Overall, this study will prove an important resource for myxobacterial researchers to delineate the regulatory and functional pathways responsible for development from those of the general nutrient stress response.

Amaç:Enfeksiyöz gastroenterit salgınlarla seyredebilen küresel bulaşıcı hastalıktır. Akut gastroenteritlerin nedenleri arasında çok çeşitli bakteri, virüs ve parazitler bulunur. Çalışmamızda bölgemizdeki gastroenterit vakalarına neden olan bakteriyel etkenlerin dağılımının yanı sıra yaş ve mevsim gibi epidemiyolojik özelliklerini belirlemek, konvansiyonel metotlarla tespit edilmesi zor olan Campylobacter türlerinin multipleks gerçek zamanlı polimeraz zincir reaksiyonu yöntemiyle gerçek sıklığına ışık tutmak amaçlandı. Gereç ve Yöntem:Ocak 2016 ve Ağustos 2019 tarihleri arasında hastanemizin çeşitli kliniklerinden bakteriyel etkenlerin saptanması amacıyla mikrobiyoloji laboratuvarına gönderilen 7659 dışkı örneğinin bakteri paneli sonuçları geriye dönük olarak incelendi. Bulgular:Değerlendirilen olguların %21,7’si pozitif, %78,3’ü negatif saptandı. Pozitif örneklerin %92’sinde dört etkenden herhangi biri pozitif iken %7,9’unda 2 etken, %0,1’inde ise 3 etken birlikte pozitif saptandı. Tüm yaşlarda en çok tekli pozitif bulunan etken toplamda Campylobacter (%35,8) idi. Diğerleri sırayla Shiga toksin (%23,6 ), Shigella/ Enteroinvaziv E. coli (%22,3) ve Salmonella (%18,3) idi. Çocuklarda en sık Campylobacter (%43,6), erişkinlerde ise Shigella (%30,6 ) pozitif idi. Pozitiflik en sık yaz aylarında (%37,6) iken en az (%17,8) kış aylarında saptandı. Yaz aylarındaki en sık pozitif olan etken Campylobacter (%38,7), kış aylarında ise Shigella/ Enteroinvaziv E. coli (%39,7) olarak bulundu. Sonuç:Konvansiyonel yöntemlerle yapılan dışkı kültürlerinde özellikle Campylobacter türlerinin tanımlanmasında zorluklar yaşanmaktadır. Çalışmamız Campylobacter türlerinin neden olduğu gastroenteritlerin gerçek sıklığını belirlemek ve diğer etkenlerin bölgemizdeki dağılımının epidemiyolojik faktörlerden nasıl etkilendiğini göstermek açısından literatüre katkı sağlayacaktır. Diğer taraftan moleküler temelli yöntemler tanı açısından her ne kadar altın standart olarak kabul edilmese de, erken tanıya katkıda bulunarak ampirik tedaviye yön verebilir.

Background: Acute respiratory infections (ARIs) are an important cause of pediatric mortality–morbidity worldwide, the most common etiology being viral. This study aims to identify causative organisms for ARIs admitted in pediatric intensive care unit (PICU), when multiplex polymerase chain reaction (PCR) testing of respiratory secretions was sent; any seasonal trends detect microbiological correlation when co-infections. Subjects and Methods: This was a retrospective observational study, from July 2021 to December 2022, of children aged 1 month–18 years, whose multiplex PCR tests (nasopharyngeal, endotracheal [ET] secretion or bronchoscopic alveolar lavage [BAL]) were sent when admitted for ARI to tertiary care PICU. Results: In the study period, 372 of 1492 medical PICU admissions were ARI. Multiplex PCR of 81 respiratory secretions was sent, of which 69 (85%) were positive. Multiplex pcr sample positivity : 83% for nasopharyngeal aspirate, 78% for ET secretions, 100% for BAL samples. Forty-one percent of samples detected >1 organism. Respiratory syncytial virus (RSV)-A was the most common virus (18); other organisms included adenovirus (n = 5), influenza (n = 9), parainfluenza (n = 5), rhinovirus: 13, Pneumocystis Jerovecci (PCP): 4, Streptococcus pneumoniae: 17, pertussis: 1, and Haemophilus influenzae B: 9. ARIs were seen throughout the year with peaks in monsoon season and a peak in cases of ARI due to RSV from July to October. Of co-infections with bacteria in ET secretions and BAL samples via multiplex PCR, bacterial culture reports were sterile. Conclusions: Multiplex PCR detected organisms in 85% of ARI patients tested. Most of the ARIs getting admitted to PICU were viral in origin. RSV was the most common virus isolated showing peak from July to October, local monsoon season. With extended viral and bacterial PCR being available, mixed infections/colonization with uncertain significance are being detected.

Background: Prevotella is a Gram-negative anaerobic bacilli. The phenotypic characteristics of the various species of Prevotella are similar, which often makes it difficult in routine differentiation and identification of all the species. Aim: The purpose of the study was to detect and compare presence of Prevotella intermedia, Prevotella nigrescens, Prevotella melaninogenica, and Prevotella loescheii in subgingival plaque samples of chronic periodontitis and healthy individuals. Materials and Methods: Two hundred and thirty-six subjects were considered consisting of chronic periodontitis (128) and healthy (108) individuals. Subgingival plaque sample was collected in reduced transport fluid and analyzed. DNA extraction and polymerase chain reaction (PCR) were performed for genus Prevotella followed by positive samples were considered for the detection of selected species through multiplex PCR using specific primers. Results: Out of 236 samples, 94.1% were positive for genus Prevotella. Out of 222 cases P. nigrescens showed the highest number of cases positive (59.5%) followed by P. melaninogenica (57.2%), P. intermedia (55.4%), and P. loescheii (40.1%). Species were analyzed individually between chronic periodontitis and healthy, P. intermedia, P. nigrescens, and P. loescheii showed greater positivity in healthy compared to chronic periodontitis. Positivity for P. melaninogenica was high in chronic periodontitis compared to healthy. Conclusion: The number of positive cases for species, when correlated with clinical parameters showed an increase in mean score for all clinical parameters assessed, suggesting the presence of variation in the prevalence of Prevotella species and geographic variation do exist in oral microflora. Findings suggest that they can be normal commensals and opportunistic.

Emulsions offer the means to miniaturize and parallelize high-throughput screening but require a robust method to localize activity-based fluorescent probes in each droplet. Multiplexing probes in droplets is impractical, though highly desirable for identifying library members that possess very specific activity. Here, we present multiplexed probe immobilization on library beads for emulsion screening. During library bead preparation, we quantitated ∼106 primers per bead by fluorescence in situ hybridization, however emulsion PCR yielded only ∼103 gene copies per bead. We leveraged the unextended bead-bound primers to hybridize complementary probe-oligonucleotide heteroconjugates to the library beads. The probe-hybridized bead libraries were then used to program emulsion in vitro transcription/translation reactions and analyzed by FACS to perform multiplexed activity-based screening of trypsin and chymotrypsin mutant libraries for novel proteolytic specificity. The approach's modularity should permit a high degree of probe multiplexing and appears extensible to other enzyme classes and library types.

Antibiotics are frequently prescribed to children with pneumonia, although viruses are responsible for most cases. We aimed to evaluate the impact of multiplex polymerase chain reaction (mPCR) on antibiotic use. We conducted a prospective study of children under 14 years of age admitted for suspected viral pneumonia, from October 2019 to June 2022 (except March–November 2020). A mPCR respiratory panel (FilmArray® 2plus, bioMérieux, Marcy-l’Étoile, France) was performed within 72 h of admission. Patients with positive reverse transcription PCR for respiratory syncytial virus, influenza, or SARS-CoV-2 were excluded. We compared the patients with historical controls (2017–2018) who had suspected viral pneumonia but did not undergo an aetiological study. We included 64 patients and 50 controls, with a median age of 26 months. The respiratory panel detected viral pathogens in 55 patients (88%), including 17 (31%) with co-infections. Rhinovirus/enterovirus (n = 26) and human metapneumovirus (n = 22) were the most common pathogens, followed by adenovirus and parainfluenza (n = 10). There were no statistically significant differences in the total antibiotic consumption (83% of cases and 86% of controls) or antibiotics given for ≥72 h (58% vs. 66%). Antibiotics were prescribed in 41% of the cases and 72% of the controls at discharge (p = 0.001). Ampicillin was the most commonly prescribed antibiotic among the patients (44% vs. 18% for controls, p = 0.004), while azithromycin was the most commonly prescribed among the controls (19% vs. 48% for patients and controls, respectively; p = 0.001). Our findings underscore the need for additional interventions alongside molecular diagnosis to reduce antibiotic usage in paediatric community-acquired pneumonia.

Background and Objectives: In clinical diagnostics, molecular methods are used to detect Mycobacterium tuberculosis bacilli (MTB) and to distinguish them from non-tuberculous mycobacteria (NTM). They are also used to make the right treatment decision for the patient as soon as possible. The aim of this study was to establish a rapid and novel multiplex PCR (mPCR) assay for the detection and differentiation of MTB and NTM in a single tube. Materials and Methods: 100 sputum samples positive for acid-fast bacilli (AFB) were included in this study. Mycobacterial culture, biochemical tests, and antibiotic susceptibility testing were performed on samples. After alkaline decontamination, total DNA was extracted from the samples. A primer pair targeting the rpoB gene, encoding the beta-subunit of RNA polymerase, was used to detect MTB and NTM, amplifying a 235-bp fragment of MTB and a 136-bp sequence of NTM. A pair of primers targeting a 190-bp fragment of the IS6110 region of MTB was also used to confirm the results. The sensitivity and specificity of the mPCR assay were evaluated using DNA extracted from standard strains. The amplified products were then analyzed by conventional agarose gel electrophoresis. Results: Of 100 AFB smear-positive sputum samples, 92 MTB DNA, 7 NTM DNA, and one mixed-infection sample were identified in a single tube using mPCR assay. There was no correlation between the AFB degree of smear positivity and PCR results. Of seven NTM isolates, 6 (86%) were resistant to rifampin, isoniazid, and ethambutol, the three first-line anti-tuberculosis drugs. Conclusion: A single-tube mPCR assay based on the rpoB gene provides a rapid and reliable means of detecting and differentiating MTB and NTM in sputum specimens. [ABSTRACT FROM AUTHOR]

Background: Escherichia coli infections are common in Xinjiang, a major region of cattle and sheep breeding in China. Therefore, strategies are required to control E. coli. The aim of this study was to investigate the phylogenetic groups, virulence genes, and antibiotic resistance characteristics of E. coli isolates. Methods: In this study, 116 tissue samples were collected from the organs of cattle and sheep that were suspected of having E. coli infections between 2015 and 2019. Bacteria in the samples were identified using a biochemical identification system and amplification of 16S rRNA, and the phylogenetic groupings of E. coli isolates were determined by multiplex polymerase chain reactions. In addition, PCR detection and analysis of virulence factors, antibiotic resistance genes, and drug‐resistant phenotypes of E. coli isolates were performed. Results: A total of 116 pathogenic E. coli strains belonging to seven phylogenetic groups were isolated, with the majority of isolates in groups A and B1. Among the virulence genes, curli‐encoding crl had the highest detection rate of 97.4%, followed by hemolysin‐encoding hlyE with the detection rate of 94.82%. Antimicrobial susceptibility test results indicated that the isolates had the highest rates of resistance against streptomycin (81.9%). Conclusion: These characteristics complicate the prevention and treatment of E. coli‐related diseases in Xinjiang. [ABSTRACT FROM AUTHOR]

Background: Prevotella is a Gram-negative anaerobic bacilli. The phenotypic characteristics of the various species of Prevotella are similar, which often makes it difficult in routine differentiation and identification of all the species. Aim: The purpose of the study was to detect and compare presence of Prevotella Intermedia, Prevotella nigrescens, Prevotella melaninogenica, and Prevotella loescheii in subgingival plaque samples of chronic periodontitis and healthy individuals. Materials and Methods: Two hundred and thirty-six subjects were considered consisting of chronic periodontitis (128) and healthy (108) individuals. Subgingival plaque sample was collected in reduced transport fluid and analyzed. DNA extraction and polymerase chain reaction (PCR) were performed for genus Prevotella followed by positive samples were considered for the detection of selected species through multiplex PCR using specific primers. Results: Out of 236 samples, 94.1% were positive for genus Prevotella. Out of 222 cases P. nigrescens showed the highest number of cases positive (59.5%) followed by P. melaninogenica (57.2%), P. intermedia (55.4%), and P. loescheii(40.1%). Species were analyzed individually between chronic periodontitis and healthy, P. intermedia, P. nigrescens, and P. loescheii showed greater positivity in healthy compared to chronic periodontitis. Positivity for P. melaninogenica was high in chronic periodontitis compared to healthy. Conclusion: The number of positive cases for species, when correlated with clinical parameters showed an increase in mean score for all clinical parameters assessed, suggesting the presence of variation in the prevalence of Prevotella species and geographic variation do exist in oral microflora. Findings suggest that they can be normal commensals and opportunistic. [ABSTRACT FROM AUTHOR]

It is urgent to develop techniques that can simultaneously detect multiple microalgae, due to the diversity of harmful algal blooms (HABs)-forming algal species. The target algae species in this study are Heterosigma akashiwo, Prorocentrum donghaiense and Karenia mikimotoi. These algae are the dominant species that cause HABs in the East China Sea, and the multiple detection technique focusing on these three algae is not common. Therefore, this study established a multiplex polymerase chain reaction(mPCR) to diagnose the three algae, which is simple and low cost. First, the corresponding specific primers were designed based on the D1-D2 region of the large subunit (LSU) ribosomal DNA sequence. Then, mPCR was established and the reaction conditions were optimized. And the specificity, sensitivity, and stability of mPCR were evaluated. The result of specificity test showed that the established mPCR had good specificity for the target microalgae and did not cross-react with eighteen non-target microalgae. The sensitivity of experiment was 3.3 × 10−1 ng μL−1, and the established mPCR was not affected by the interfering microalgae. Moreover, the practicability evaluation of mPCR by using the simulated natural water samples showed that the detection limit of target microalgae was 100 cells mL−1, which could meet the demand for early warning of HABs. In summary, the established mPCR is characterized by strong specificity, good stability, and multiple analysis to detect H. akashiwo, P. donghaiense, and K. mikimotoi. [ABSTRACT FROM AUTHOR]

In low-resource settings with high tuberculosis (TB) burdens, lack of rapid diagnostic methods for detection and differentiation of Mycobacterium tuberculosis complex (MTBC) is a major challenge affecting TB management. This study utilized comparative genomic analyses of MTBC lineages; M. tuberculosis, M. africanum Lineages 5/6 and M. bovis to identify lineage-specific genes. Primers were designed for the development of a Multiplex PCR assay which was successful in differentiating the MTBC lineages. There was no cross-reaction with other respiratory pathogens tested. Validation of the assay using clinical samples was performed with sputum DNA extracts from 341 clinically confirmed active TB patients. It was observed that 24.9% of cases were caused by M. tuberculosis, while M. africanum L5 & L6 reported 9.0% and 14.4%, respectively. M. bovis infection was the least frequently detected lineage with 1.8%. Also, 27.0% and 17.0% of the cases were PCR negative and unspeciated, respectively. However, mixed-lineage TB infections were recorded at a surprising 5.9%. This multiplex PCR assay will allow speciation of MTBC lineages in low-resource regions, providing rapid differentiation of TB infections to select appropriate medication at the earliest possible time point. It will also be useful in epidemiological surveillance studies providing reliable information on the prevalence of TB lineages as well as identifying difficult to treat cases of mixed-lineage tuberculosis infections. [ABSTRACT FROM AUTHOR]

Aim: We aimed to assess whether detection of respiratory bacteria by multiplex polymerase chain reaction (PCR) testing associates with clinical outcomes in acutely ill children. Methods: This cross‐sectional study enrolled children under the age of 18 with a suspected respiratory infection treated in a paediatric emergency department of Oulu University Hospital, Finland from January 2015 through December 2015. Nasopharyngeal samples were routinely analysed for 16 respiratory viruses and later, after storage, analysed with a multiplex PCR panel for seven respiratory bacteria. Results: At least one bacterial pathogen was detected in 600 out of the 1195 children (50%). The mean age was 3.3 (SD 3.7) years and 54% were boys. Atypical bacteria were associated with a risk of pneumonia (adjusted odds ratio [aOR] 14.1, 95% CI 3.98–50.1). Co‐detection of rhinovirus with Streptococcus pneumoniae was not associated with risk of pneumonia (aOR 2.39, 95% CI 0.78–7.30). Detection of Streptococcus pneumoniae, Haemophilus influenzae or both was not associated with the risk of hospital admission or prescription of antibiotics. Conclusion: Nasopharyngeal detection of atypical bacteria in acutely ill children was associated with a markedly increased risk of pneumonia. The clinical utility of wide testing for other respiratory bacteria needs further evaluation. [ABSTRACT FROM AUTHOR]

PCR tests for viral identification, performed on nasopharyngeal secretions, have experienced a major boom in the last few years. Their use is very frequent, but their indications are still not well defined, especially in Paediatric Intensive Care Units (PICU). These tests are used for the microbiological diagnosis of lower respiratory infections but can be used in other situations. The aim of the study was to investigate the effect of viral identification on antibiotic therapy management. We conducted a single-centre retrospective study from 1 October 2017 to 31 December 2019. This study included all consecutive FilmArray® Respiratory Panel tests performed in patients hospitalised in a PICU. Patients were identified using the microbiology laboratory prospective database and data were extracted from the medical record. 544 tests corresponding to 408 patients were included. The main reasons for testing were pneumonia (34%) and bronchiolitis (24%). In 70% of cases, at least one virus was identified, with Human Rhinovirus (56%) and Respiratory Syncytial Virus (28%) being the two predominant. Bacterial co-infection was present in 25% of cases. Viral identification was not associated with reduced antibiotic therapy. On multivariate analysis, antibiotic management was significantly associated with clinical gravity, CRP value or radiology findings regardless of virus identification. Viral identification has an epidemiological value, but antibiotic prescription relies on other factors. [ABSTRACT FROM AUTHOR]

Malaria antigen detection through rapid diagnostic tests (RDTs) is widely used to diagnose malaria and estimate prevalence. To support more sensitive next-generation RDT development and screen asymptomatic malaria, we developed and evaluated the Q-Plex™ Human Malaria Array (Quansys Biosciences, Logan, UT), which quantifies the antigens commonly used in RDTs-Plasmodium falciparum-specific histidine-rich protein 2 (HRP2), P. falciparum-specific lactate dehydrogenase (Pf LDH), Plasmodium vivax -specific LDH (Pv LDH), and Pan malaria lactate dehydrogenase (Pan LDH), and human C-reactive protein (CRP), a biomarker of severity in malaria. At threshold levels yielding 99.5% or more diagnostic specificity, diagnostic sensitivities against polymerase chain reaction-confirmed malaria for HRP2, Pf LDH, Pv LDH, and Pan LDH were 92.7%, 71.5%, 46.1%, and 83.8%, respectively. P. falciparum culture strains and samples from Peru indicated that HRP2 and Pf LDH combined improves detection of P. falciparum parasites with hrp2 and hrp3 deletions. This array can be used for antigen-based malaria screening and detecting hrp2/3 deletion mutants of P. falciparum.

ObjectiveA growing body of evidence suggests that testing for influenza virus alone is more appropriate than multiplex respiratory viral panel (RVP) testing for general populations of patients with respiratory tract infections. We aimed to decrease the proportion of RVPs out of total respiratory viral testing ordered during influenza season.MethodsWe implemented two consecutive interventions: reflex testing for RVPs only after a negative influenza test, and noninterruptive clinical decision support (CDS) including modifications of the computerized physician order entry search behavior and cost display. We conducted an interrupted time series of RVPs and influenza polymerase chain reaction tests pre- and postintervention, and performed a mixed-effects logistic regression analysis with a primary outcome of proportion of RVPs out of total respiratory viral tests. The primary predictor was the intervention period, and covariates included the provider, clinical setting, associated diagnoses, and influenza incidence.ResultsFrom March 2013 to April 2019, there were 24,294 RVPs and 26,012 influenza tests (n = 50,306). Odds of ordering an RVP decreased during the reflex testing period (odds ratio: 0.432, 95% confidence interval: 0.397-0.469), and decreased more dramatically during the noninterruptive CDS period (odds ratio: 0.291, 95% confidence interval: 0.259-0.327).DiscussionThe odds of ordering an RVP were 71% less with the noninterruptive CDS intervention, which projected 4,773 fewer RVPs compared with baseline. Assuming a cost equal to Medicare reimbursement rates for RVPs and influenza tests, this would generate an estimated averted cost of $1,259,474 per year.ConclusionNoninterruptive CDS interventions are effective in reducing unnecessary and expensive testing, and avoid typical pitfalls such as alert fatigue.

In light of the current biodiversity crisis, molecular barcoding has developed into an irreplaceable tool. Barcoding has been considerably simplified by developments in high throughput sequencing technology, but still can be prohibitively expensive and laborious when community samples of thousands of specimens need to be processed. Here, we outline an Illumina amplicon sequencing approach to generate multilocus data from large collections of arthropods. We reduce cost and effort up to 50-fold, by combining multiplex PCRs and DNA extractions from pools of presorted and morphotyped specimens and using two levels of sample indexing. We test our protocol by generating a comprehensive, community wide dataset of barcode sequences for several thousand Hawaiian arthropods from 14 orders, which were collected across the archipelago using various trapping methods. We explore patterns of diversity across the Archipelago and compare the utility of different arthropod trapping methods for biodiversity explorations on Hawaii, highlighting undergrowth beating as highly efficient method. Moreover, we show the effects of barcode marker, taxonomy and relative biomass of the targeted specimens and sequencing coverage on taxon recovery. Our protocol enables rapid and inexpensive explorations of diversity patterns and the generation of multilocus barcode reference libraries across whole ecosystems.

BackgroundHIV-infected cell lines are widely used to study latent HIV infection, which is considered the main barrier to HIV cure. We hypothesized that these cell lines differ from each other and from cells from HIV-infected individuals in the mechanisms underlying latency.ResultsTo quantify the degree to which HIV expression is inhibited by blocks at different stages of HIV transcription, we employed a recently-described panel of RT-ddPCR assays to measure levels of 7 HIV transcripts ("read-through," initiated, 5' elongated, mid-transcribed/unspliced [Pol], distal-transcribed [Nef], polyadenylated, and multiply-sliced [Tat-Rev]) in bulk populations of latently-infected (U1, ACH-2, J-Lat) and productively-infected (8E5, activated J-Lat) cell lines. To assess single-cell variation and investigate cellular genes associated with HIV transcriptional blocks, we developed a novel multiplex qPCR panel and quantified single cell levels of 7 HIV targets and 89 cellular transcripts in latently- and productively-infected cell lines. The bulk cell HIV transcription profile differed dramatically between cell lines and cells from ART-suppressed individuals. Compared to cells from ART-suppressed individuals, latent cell lines showed lower levels of HIV transcriptional initiation and higher levels of polyadenylation and splicing. ACH-2 and J-Lat cells showed different forms of transcriptional interference, while U1 cells showed a block to elongation. Single-cell studies revealed marked variation between/within cell lines in expression of HIV transcripts, T cell phenotypic markers, antiviral factors, and genes implicated in latency. Expression of multiply-spliced HIV Tat-Rev was associated with expression of cellular genes involved in activation, tissue retention, T cell transcription, and apoptosis/survival.ConclusionsHIV-infected cell lines differ from each other and from cells from ART-treated individuals in the mechanisms governing latent HIV infection. These differences in viral and cellular gene expression must be considered when gauging the suitability of a given cell line for future research on HIV. At the same time, some features were shared across cell lines, such as low expression of antiviral defense genes and a relationship between productive infection and genes involved in survival. These features may contribute to HIV latency or persistence in vivo, and deserve further study using novel single cell assays such as those described in this manuscript.

BACKGROUND:Most genetically modified (GM) plants contain a promoter, P35S, from the plant virus, Cauliflower mosaic virus (CaMV), and many have a terminator, TNOS, derived from the bacterium, Agrobacterium tumefaciens. Assays designed to detect GM plants often target the P35S and/or TNOS DNA sequences. However, because the P35S promoter is derived from CaMV, these detection assays can yield false-positives from non-GM plants infected by this naturally-occurring virus. RESULTS:Here we report the development of an assay designed to distinguish CaMV-infected plants from GM plants in a single multiplexed quantitative PCR (qPCR) reaction. Following initial testing and optimization via PCR and singleplex-to-multiplex qPCR on both plasmid and plant DNA, TaqMan qPCR probes with different fluorescence wavelengths were designed to target actin (a positive-control plant gene), P35S, P3 (a CaMV-specific gene), and TNOS. We tested the specificity of our quadruplex qPCR assay using different DNA extracts from organic watercress and both organic and GM canola, all with and without CaMV infection, and by using commercial and industrial samples. The limit of detection (LOD) of each target was determined to be 1% for actin, 0.001% for P35S, and 0.01% for both P3 and TNOS. CONCLUSIONS:This assay was able to distinguish CaMV-infected plants from GM plants in a single multiplexed qPCR reaction for all samples tested in this study, suggesting that this protocol is broadly applicable and readily transferrable to any interested parties with a qPCR platform.

Deletion 9p syndrome is a rare chromosomal abnormality with a wide spectrum of manifestations such as craniofacial dysmorphism, congenital anomalies, and psychomotor delay. We report a case of a seven-year-old girl with simultaneous 9p24.3 deletion and 8p23.3 duplication detected using multiplex ligation-dependent probe amplification (MLPA). Chromosomal and cytogenetic analyses using MLPA are effective in assessing genetic abnormalities in patients with developmental delay and mental retardation. We found breakpoints at 9p24.3 and duplication in the 8p23.3 region, leading to a wide variety of manifestations including speech delay, upslanting palpebral fissures, hypertelorism, epicanthal fold, high arched eyebrows, flat nasal bridge, thin upper lip, and cleft palate. Simultaneous detection of 9p24.3 deletion and 8p23.3 duplication has been rarely reported. Clinical phenotypes of our patient resembled the features of Nicolaides-Baraitser syndrome, which might have been primarily caused by the haploinsufficiency of SMARCA2 (SWI/SNF-related, matrix associated, actin-dependent regulator of chromatin, subfamily A, member 2) gene located at 9p24.3.