Optimization of Multiplex-PCR Technique To Determine Azf Deletions in infertility Male Patients.

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.

Development of a multiplex polymerase chain reaction assay for detection of hepatitis C virus, hepatitis B virus, and human immunodeficiency virus 1.

BACKGROUND: Hepatitis C virus (HCV), hepatitis B virus (HBV), and human immunodeficiency virus 1 (HIV-1) are the most epidemic blood-borne viruses, posing threats to human health and causing economic losses to nations for combating the infection transmission. The diagnostic methodologies that depend on the detection of viral nucleic acids are much more expensive, but they are more accurate than serological testing. AIM: To develop a rapid, cost-effective, and accurate diagnostic multiplex polymerase chain reaction (PCR) assay for simultaneous detection of HCV, HBV, and HIV-1. METHODS: The design of the proposed PCR assay targets the amplification of a short conserved region featured with a distinguishable melting profile and electrophoretic molecular weight inside each viral genome. Therefore, this diagnostic method will be appropriate for application in both conventional (combined with electrophoresis) and real-time PCR facilities. Confirmatory in silico investigations were conducted to prove the capability of the approached PCR assay to detect variants of each virus. Then, Egyptian isolates of each virus were subjected to the wet lab examination using the given diagnostic assay. RESULTS: The in silico investigations confirmed that the PCR primers can match many viral variants in a multiplex PCR assay. The wet lab experiment proved the efficiency of the assay in distinguishing each viral type through high-resolution melting analysis. Compared to related published assays, the proposed assay in the current study is more sensitive and competitive with many expensive PCR assays. CONCLUSION: This study provides a simple, cost-effective, and sensitive diagnostic PCR assay facilitating the detection of the most epidemic blood-borne viruses; this makes the proposed assay promising to be substitutive for the mistakable and cheap serological-based assays.

Epidemiological characteristics and outcome of viral respiratory tract infections in the first year after allogeneic hematopoietic cell transplantation.

Adverse outcomes of viral respiratory tract infections (RTI) have been reported in recipients of allogeneic hematopoietic cell transplantation. Using a laboratory-developed multiparameter PCR in a consecutive series of 242 patients, we found the highest incidence of viral RTI in the pre-engraftment phase. The occurrence of multiple episodes of viral RTI or viral pneumonia was significantly associated with a higher hazard of non-relapse mortality in the first year after transplantation. We observed a 90-day mortality of 19.7% after viral RTI, which was significantly different between patient groups stratified according to the ISI score.

Cytogenetics investigation in 151 Brazilian infertile male patients and genomic analysis in selected cases: experience of 14 years in a public genetic service.

OBJECTIVES: Male infertility accounts for approximately 30% of cases of reproductive failure. The characterization of genetic variants using cytogenomic techniques is essential for the adequate clinical management of these patients. We aimed to conduct a cytogenetic investigation of numerical and structural rearrangements and a genomic study of Y chromosome microdeletions/microduplications in infertile men derived from a single centre with over 14 years of experience. RESULTS: We evaluated 151 infertile men in a transversal study using peripheral blood karyotypes and 15 patients with normal karyotypes through genomic investigation by multiplex ligation-dependent probe amplification (MLPA) or polymerase chain reaction of sequence-tagged sites (PCR-STS) techniques. Out of the 151 patients evaluated by karyotype, 13 presented chromosomal abnormalities: two had numerical alterations, and 11 had structural chromosomal rearrangements. PCR-STS detected a BPY2 gene region and RBMY2DP pseudogene region microdeletion in one patient. MLPA analysis allowed the identification of one patient with CDY2B_1 and CDY2B_2 probe duplications (CDY2B and NLGN4Y genes) and one patient with BPY2_1, BPY2_2, and BPY2_4 probe duplications (PRY and RBMY1J genes).

Detection of Enteropathogens in Human Immunodeficiency Virus and Non-Human Immunodeficiency Virus-Infected Children with Acute Diarrhea in an Indonesian Tertiary Hospital Using Multiplex Real-Time Polymerase Chain Reaction.

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.

The Epidemiology of Circulating Respiratory Pathogens during the COVID-19 Pandemic.

Objective To survey the epidemiology of respiratory pathogens during the coronavirus disease 2019 (COVID-19) pandemic using multiplex polymerase chain reaction (PCR). Methods Specimens were assayed using multiplex nested PCR. Materials Specimens were obtained from outpatients who presented with symptoms of upper respiratory tract infection and asymptomatic outpatients who had contact with patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection at Tohoku Medical and Pharmaceutical University Hospital in Sendai, Japan, from November 1, 2020, to May 31, 2023. The analysis included multiple specimens collected from the same patients at different time-points. Data were collected from the electronic records after testing. Results This study included 8,335 patients (4,311 men) with a median age of 59 years old, and 11,741 total specimens were collected. At least 1 positive SARS-CoV-2 result was obtained for 1,710 (14.6%) specimens. Furthermore, 15 pathogens were identified in the positive specimens, and rhinovirus/enterovirus was detected more frequently than other viruses. We identified a larger number of SARS-CoV-2-positive specimens in patients ≥10 years old. In contrast, in patients 0-9 years old, we identified a larger number of specimens positive for rhinovirus/enterovirus than for other viruses. Conclusion In this study, we examined the epidemiology of circulating respiratory pathogens during the COVID-19 pandemic era.

Application of a multiplex molecular pneumonia panel and real-world impact on antimicrobial stewardship among patients with hospital-acquired and ventilator-associated pneumonia in intensive care units.

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.

Multiplex PCR and Antibiotic Use in Children with Community-Acquired Pneumonia.

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.

Evaluation of chronic diarrhea in patients newly diagnosed with HIV infection through the FilmArray® gastrointestinal panel.

INTRODUCTION: The treatment and diagnosis of chronic diarrhea in the immunocompromised patient depends on the ability to rapidly detect the etiologic agents. AIMS: Our aim was to evaluate the results of the FilmArray® gastrointestinal panel in patients newly diagnosed with HIV infection that presented with chronic diarrhea. MATERIAL AND METHODS: Utilizing nonprobability consecutive convenience sampling, 24 patients were included that underwent molecular testing for the simultaneous detection of 22 pathogens. RESULTS: In 24 HIV-infected patients with chronic diarrhea, enteropathogen bacteria were detected in 69% of the cases, parasites in 18%, and viruses in 13%. Enteropathogenic Escherichia coli and enteroaggregative Escherichia coli were the main bacteria identified, Giardia lamblia was found in 25%, and norovirus was the most frequent viral agent. The median number of infectious agents per patient was three (range of 0 to 7). The biologic agents not identified through the FilmArray® method were tuberculosis and fungi. CONCLUSIONS: Several infectious agents were simultaneously detected through the FilmArray® gastrointestinal panel in patients with HIV infection and chronic diarrhea.

Clinical Impact of Multiplex Molecular Diagnostic Testing in Children With Acute Gastroenteritis Presenting to an Emergency Department: A Multicenter Prospective Study.

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.

The Impact of the Rapid Blood Culture Identification Panel on Antibiotic Treatment and Clinical Outcomes in Bloodstream Infections, Particularly Those Associated with Multidrug-Resistant Micro-Organisms.

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.

A retrospective study on the status of sexually transmitted co-infections in university hospitals in Korea from 2017 to 2021.

Background: The prevalence of sexually transmitted infections (STIs) remains high worldwide. Despite the worldwide increase in the incidence of STIs every year, there are few reports on the frequency of STIs with different pathogens according to age and gender. Accordingly, a study was conducted to determine trends in co-infection with STIs by age and gender in Cheonan, South Korea from 2017 to 2021. Objectives: To identify trends by age or sex in co-infection of STIs in this region. Design: A retrospective study was conducted on clinical samples examined at Dankook University Hospital from January 2017 to November 2021. A total of 3297 specimens were collected from patients visiting Dankook University Hospital (Cheonan, Korea), and statistical analysis was performed on patients ranging in age from 1 day to 93 years. Methods: Multiplex polymerase chain reaction, the most efficient method to diagnose a bacterial infection, was performed using an MJ Research PTC-200 Thermal Cycler (Marshall Scientific, Richmond, VA, USA) and a Seeplex STD Detection Kit (Seegene, Seoul, Republic of Korea). The co-infection rate with STI pathogens was analyzed according to age and sex. Results: Of the 3297 clinical samples, 1017 (30.9%) tested positive for sexually transmitted pathogens, ranging from one to six co-infections. Analysis of the co-infection rate by age revealed that the average age gradually decreased as the total number of co-infection pathogens increased. The co-infection percentage and age distribution of STIs differed according to sex. Co-infection was more prevalent in female patients. Furthermore, co-infection in male patients occurred frequently in the 30-39-year-old group, while those in female patients occurred in the 20-29- and 30-39-year-old groups. Conclusion: Our statistical analysis showed that STI co-infections were more common among younger than older people. Therefore, it helps in recognizing STIs at a young age and provides possible indicator data to prevent STIs at a young age. In addition, further research is needed on co-infection in other regions.

Emerging and Fastidious Uropathogens Were Detected by M-PCR with Similar Prevalence and Cell Density in Catheter and Midstream Voided Urine Indicating the Importance of These Microbes in Causing UTIs.

Introduction: 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.

Comparison Shows that Multiplex Polymerase Chain Reaction Identifies Infection-associated Urinary Biomarker-positive Urinary Tract Infections That Are Missed by Standard Urine Culture.

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-1ß) 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 <0.05 considered statistically significant. Results and limitations: In cases with M-PCR-positive/SUC-negative results (n = 80), all median biomarker levels were significantly higher (p < 0.0001) than in cases with M-PCR-negative/SUC-negative results (n = 107). Two or more biomarkers were positive in 76% of M-PCR-positive/SUC-negative specimens. Limitation was an inability to examine associations between each individual organism and inflammation. Conclusions: A significant number of M-PCR-positive/SUC-negative cases had elevated levels of infection-related urinary biomarkers, especially when infection was caused by organisms other than Escherichia coli. This is a strong indication that microbes detected by M-PCR, which would be missed by SUC, are associated with UTIs. Patient summary: We compared infection-associated biomarkers in patients diagnosed with urinary tract infections (UTIs) against the detection of microorganisms by standard urine culture (SUC) and multiplex polymerase chain reaction (M-PCR). We found that most patients with microorganisms detected by M-PCR, which were missed by SUC, had elevated markers of inflammation, indicating that these organisms were likely causative of UTIs.

Investigation of etiology of community-acquired pneumonia in hospitalized patients in a tertiary hospital of São Paulo City, Brazil.

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.

Comparison of loop-mediated isothermal amplification, multiplex PCR, and REP- PCR techniques for identification of carbapenem-resistant Acinetobacter baumannii clinical isolates.

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 bla SHV-1 , bla PER-1 , bla TEM-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 bla SHV-1 positive, (40%) bla PER-1, (85%) aac (6')-1, AMPC (67%), bla TEM-1 (63%), and (15%) qnr respectively. While in LAMP, (69%) of isolates were bla SHV-1 positive, (86%) aac (6')-1, and (20%) qnr. The results of AMPC, bla TEM-1 , and bla PER-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.

Improving Patient Outcomes While Reducing Empirical Treatment with Multiplex-Polymerase-Chain-Reaction/Pooled-Antibiotic-Susceptibility-Testing Assay for Complicated and Recurrent Urinary Tract Infections.

This study compared rates of empirical-therapy use and negative patient outcomes between complicated and recurrent urinary tract infection (r/cUTI) cases diagnosed with a multiplex polymerase chain reaction or pooled antibiotic susceptibility testing (M-PCR/P-AST) vs. standard urine culture (SUC). Subjects were 577 symptomatic adults (n = 207 males and n = 370 females) presenting to urology/urogynecology clinics between 03/30/2022 and 05/24/2023. Treatment and outcomes were recorded by the clinician and patient surveys. The M-PCR/P-AST (n = 252) and SUC (n = 146) arms were compared after patient matching for confounding factors. The chi-square and Fisher's exact tests were used to analyze demographics and clinical outcomes between study arms. Reduced empirical-treatment use (28.7% vs. 66.7%), lower composite negative events (34.5% vs. 46.6%, p = 0.018), and fewer individual negative outcomes of UTI-related medical provider visits and UTI-related visits for hospitalization/an urgent care center/an emergency room (p < 0.05) were observed in the M-PCR/P-AST arm compared with the SUC arm. A reduction in UTI symptom recurrence in patients ≥ 60 years old was observed in the M-PCR/P-AST arm (p < 0.05). Study results indicate that use of the M-PCR/P-AST test reduces empirical antibiotic treatment and negative patient outcomes in r/cUTI cases.

Clinical Outcomes of Acutely Ill Children According to Cycle Threshold Values of Respiratory Viruses Detected by Multiplex PCR Testing.

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.

A Study on Glycopeptide-Resistant Genotypes among Clinical Isolates of Enterococcus with Van B Phenotype.

Background: Enterococci, although remarked as harmless commensals of the intestinal tract of humans and animals, have become a significant source of infection in hospitalized patients worldwide.The present study aimed to isolate and identify enterococci from clinical samples and to determine the genotypic characteristics of vancomycin-resistant enterococcus isolates. Materials and Methods: A total of 774 isolates of enterococci from clinical samples were identified to the species level, and their anti-microbial susceptibility pattern was determined by Kirby Bauer Disk Diffusion method and Vitek 2 automated system. Screening for vancomycin resistance was performed by using brain heart infusion agar containing 6 micrograms/ml of vancomycin. The minimum inhibitory concentration (MIC) of vancomycin was determined by the agar dilution method and Epsilometer test (E test). Genotyping was carried out for resistant isolates by multiplex polymerase chain reaction. Results: Thirty (3.88%) isolates were resistant to vancomycin by agar screen method. Five isolates showed intermediate resistance with anMIC of 8-16 micrograms/ml for vancomycin by agar dilution. By the E test, two isolates displayed an MIC of ≥32 micrograms/ml for vancomycin and one isolate showed an MICof ≥32 micrograms/ml for teicoplanin.Van A was the common genotype isolated. The present study reports isolates of Enterococcus faecalis with a susceptible MIC for glycopeptide and the presence of the Van A gene. Conclusion: Heterogeneous resistance among clinical isolates of Enterococcus faecalis was observed in our study. The predominant phenotype and genotype detected among clinical isolates were Van A.

First report on the molecular prevalence and associated risk factors of Eimeria spp. in dairy cattle in Khon Kaen, Thailand.

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.