The predictive value and reliability of ultrasound-guided synovial aspiration and biopsies for diagnosing periprosthetic shoulder infections.

INTRODUCTION: Preoperative diagnosis of periprosthetic shoulder infections (PSI) is difficult. Infections are mostly caused by low virulence bacteria and patients do not show typical signs of infection. The aim of this study was to determine the diagnostic value and reliability of ultrasound-guided biopsies for cultures alone and in combination with multiplex polymerase chain reaction (mPCR), serum markers, and/or synovial markers for the preoperative diagnosis of PSI in patients undergoing revision shoulder surgery. MATERIALS AND METHODS: A prospective explorative diagnostic cohort study was performed including 55 patients undergoing revision shoulder replacement surgery. A shoulder puncture was performed preoperatively before incision to collect synovial fluid for mPCR analysis and for measurement of interleukin-6, calprotectin, white blood cell count (WBC), and polymorphonuclear cells. Also prior to revision surgery, six ultrasound-guided synovial tissue biopsies were collected for culture and two for mPCR analysis. A blood sample was obtained to determine serum C-reactive protein, WBC, and erythrocyte sedimentation rate. Six routine care tissue biopsies were taken during revision surgery and served as reference standard. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV; the primary outcome measure), and accuracy were calculated for ultrasound-guided biopsies, blood and synovial markers, mPCR, and combinations thereof. RESULTS: Routine tissue cultures were positive for infection in 24 patients. Cultures from ultrasound-guided biopsies diagnosed infection in 7 of these patients, yielding a sensitivity, specificity, PPV, NPV, and accuracy of 29.2%, 93.5%, 77.8%, 63.0%, and 65.6%, respectively. The best diagnostic value was found for the combination of ultrasound-guided biopsies for culture, synovial WBC, and calprotectin with a sensitivity of 69.2%, specificity of 80.0%, PPV of 69.2%, and NPV of 80.0%. CONCLUSION: Ultrasound-guided biopsies for cultures alone and in combination with mPCR, and/or blood and/or synovial markers are not reliable enough to use in clinical practice for the preoperative diagnosis of PSI. LEVEL OF EVIDENCE: Diagnostic study level II.

Development of two multiplex PCR assays for rapid detection of eleven Gram-negative bacteria in children with septicemia.

AIM: This study aimed to develop a multiplex PCR assay for simultaneous detection of major Gram-negative etiologies of septicemia and evaluate its performance. METHODS: Multiplex PCR (mPCR) assays were developed targeting 11 bacterial strains. Species-specific primers were confirmed using known clinical isolates and standard strains. Gradient PCR was performed on each primer against its target bacterial gene to determine its optimal amplification condition. The minimum detectable DNA concentration of the two assays was evaluated by adjusting bacterial DNA concentration to 100 ng/µL and, tenfold serially diluting it up to 10 pg/µL with DNAse-free water. The diagnostic accuracy of mPCR assays was established by subjecting the assays to 60 clinical blood samples. RESULTS: Two mPCR assays were developed. Optimal primer annealing temperature of 55 °C was established and utilized in the final amplification conditions. The assays detected all targeted bacteria, with a 100 pg minimum detectable DNA concentration. Pathogens were not detected directly from whole blood, but after 4 h and 8 h of incubation, 41% (5/12) and 100% (12/12) of the bacteria were detected in culture fluids, respectively. The assays also identified Salmonella spp. and Klebsiella pneumoniae co-infections and extra pathogens (1 E. coli and 2 K. pneumoniae) compared with culture. The sensitivity and specificity of the mPCR were 100.0% (71.7-100.0) and 98.0% (90.7-99.0), respectively. The area under the ROC curve was 1.00 (1.00-1.00). CONCLUSIONS: The mPCR assays demonstrated substantial potential as a rapid tool for septicemia diagnosis alongside the traditional blood culture method. Notably, it was able to identify additional isolates, detect co-infections, and efficiently detect low bacterial DNA loads with high sensitivity, implying its value in enhancing efficiency of diagnosis of septicemia.

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.

Characterization of Tenacibaculum maritimum isolated from diseased salmonids farmed in Chile reveals high serological and genetic heterogeneity.

The diversity of Tenacibaculum maritimum in Chile remains poorly understood, particularly in terms of antigenic and genetic diversity. This information is crucial for the future development of a vaccine against tenacibaculosis and would increase understanding of this important fish pathogen. With this aim, the biochemical, antigenic, and genetic characteristics were analysed for 14 T. maritimum isolates, recovered from diseased Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) farmed in Chile between 1998 and 2022. Biochemical analysis showed a homogeneity among all the Chilean T. maritimum isolates and all four other strains included for comparison purposes. Serological characterization using dot-blot assaying revealed antigenic heterogeneity with the use of unabsorbed antisera. The majority of isolates showed cross-reactions, identifying three main serological patterns. When the PCR-based serotyping scheme was performed, the existence of antigenic heterogeneity was confirmed. Four Atlantic salmon isolates were 4-0; and most isolates, including the rainbow trout isolate, were 3-1 (n = 9). A turbot (Scophthalmus maximus) isolate was 1-0. Using an existing Multilocus Sequence Typing system, two newly identified sequence types (ST193 and ST198) in the database were detected. ST193 encompassed nine isolates obtained from Atlantic salmon and rainbow trout, while ST198 regrouped four isolates, all retrieved from diseased Atlantic salmon in 2022. These findings highlight significant antigenic and genetic diversity among the Chilean isolates. This information is useful for epizootiology and the selection of suitable candidate strain(s) for vaccine development against tenacibaculosis caused by T. maritimum in Chilean salmon farming.

Individual genomic loci, transcript level, and biochemical profile of immune and antioxidant markers associated with genetically identified bacterial mastitis in Shami goats in Egypt.

Background: Mastitis in goats is unquestionably a grave concern, with far-reaching implications for both animal well-being and productivity, while also presenting a potential threat to public health. Aim: The study aimed to compare culture methods and multiplex PCR (m-PCR) in the detection of the most three common mastitis-causing pathogens (Staphylococcus aureus, Escherichia coli, and Streptococcus spp.) and investigate the gene expression, single nucleotide polymorphisms (SNPs), serum concentrations of immunological and antioxidant indicators linked to mastitis in Shami goats. Methods: One hundred Shami do (50 Shami goats with clinical mastitis and 50 normal goats taken as control group). The culture methods and m-PCR analysis were used to find the bacteria in the milk samples. Blood samples were obtained to assess some hemato-biochemical parameters, detect SNPs, and determine the expression of certain immunological and antioxidant indicators in the genes. Results: The culture method detected the pathogens causing mastitis in 90% of the milk samples, but m-PCR detected them in 100% of the milk samples. SNPs linked to mastitis resistance/susceptibility in examined does were detected through DNA sequencing of immunological and antioxidant indicators. The magnitude of gene expression varied significantly between the resistant and mastitis-affected groups. Significant (P ˂ 0.05) elevations were noticed in WBCs count, mainly neutsrophils count, serum levels of BHB, NEFA, triglycerides, LDL-C, AST, ALT, ALP, creatinine, total protein, globulin, Ca, K, GPx, MDA, acute phase proteins, and cytokines in mastitis affected does as compared to control. While RBCs count, PCV%, lymphocytes count, serum concentration of glucose, cholesterol, HDL-C, albumin, Na, Cl, P, GSH, SOD, and catalase significantly (P ˂ 0.05) diminished in mastitis affected does compared to healthy ones. APPs and pro-inflammatory cytokines scored high sensitivities and NPVs but TNF-α and serum amyloid A (SAA) had the highest percentages of increase. Conclusion: The study confirmed that m-PCR is the most sensitive method for bacteria identification (S. aureus, E. coli, and Strept. spp.) while SNPs in antioxidant and immunological genes may be important genetic indicators for mastitis risk or resistance in Shami does. To establish an effective management plan and forecast the most sensitive risk time for illness onset, gene expression profiles of the tested genes may also be employed as proxy biomarkers. TNF-α and SAA may be precious indicators for the detection of caprine mastitis.

Vaginal microbiota molecular profiling and diagnostic performance of artificial intelligence-assisted multiplex PCR testing in women with bacterial vaginosis: a single-center experience.

Background: Bacterial vaginosis (BV) is a most common microbiological syndrome. The use of molecular methods, such as multiplex real-time PCR (mPCR) and next-generation sequencing, has revolutionized our understanding of microbial communities. Here, we aimed to use a novel multiplex PCR test to evaluate the microbial composition and dominant lactobacilli in non-pregnant women with BV, and combined with machine learning algorithms to determine its diagnostic significance. Methods: Residual material of 288 samples of vaginal secretions derived from the vagina from healthy women and BV patients that were sent for routine diagnostics was collected and subjected to the mPCR test. Subsequently, Decision tree (DT), random forest (RF), and support vector machine (SVM) hybrid diagnostic models were constructed and validated in a cohort of 99 women that included 74 BV patients and 25 healthy controls, and a separate cohort of 189 women comprising 75 BV patients, 30 intermediate vaginal microbiota subjects and 84 healthy controls, respectively. Results: The rate or abundance of Lactobacillus crispatus and Lactobacillus jensenii were significantly reduced in BV-affected patients when compared with healthy women, while Lactobacillus iners, Gardnerella vaginalis, Atopobium vaginae, BVAB2, Megasphaera type 2, Prevotella bivia, and Mycoplasma hominis were significantly increased. Then the hybrid diagnostic models were constructed and validated by an independent cohort. The model constructed with support vector machine algorithm achieved excellent prediction performance (Area under curve: 0.969, sensitivity: 90.4%, specificity: 96.1%). Moreover, for subjects with a Nugent score of 4 to 6, the SVM-BV model might be more robust and sensitive than the Nugent scoring method. Conclusion: The application of this mPCR test can be effectively used in key vaginal microbiota evaluation in women with BV, intermediate vaginal microbiota, and healthy women. In addition, this test may be used as an alternative to the clinical examination and Nugent scoring method in diagnosing BV.

Multiple-Drug Resistant Shiga Toxin-Producing E. coli in Raw Milk of Dairy Bovine.

INTRODUCTION: Raw milk may contain pathogenic microorganisms harmful to humans, e.g., multidrug-resistant Escherichia coli non-O157:H7, which can cause severe colitis, hemolytic uremia, and meningitis in children. No studies are available on the prevalence of Shiga toxin-producing E. coli (STEC O157:H7) in sick or healthy dairy animals in the Khyber Pakhtunkhwa Province of Pakistan. AIM: This study aimed to isolate, characterize, and detect antibiotic resistance in STEC non-O157:H7 from unpasteurized milk of dairy bovines in this province. MATERIALS AND METHODS: We collected raw milk samples (n = 800) from dairy farms, street vendors, and milk shops from different parts of the Khyber Pakhtunkhwa Province. E. coli was isolated from these samples followed by latex agglutination tests for serotyping. The detection of STEC was conducted phenotypically and confirmed by the detection of virulence genes genotypically. An antibiogram of STEC isolates was performed against 12 antibiotics using the disc diffusion method. RESULTS: A total of 321 (40.12%) samples were found to be positive for E. coli in this study. These samples were processed for the presence of four virulence genes (Stx1, Stx2, ehxA, eae). Forty samples (5.0%) were STEC-positive. Of these, 38%, 25%, 19%, and 18% were positive for Stx1, Stx2, ehxA, and eae, respectively. Genotypically, we found that 1.37% of STEC isolates produced extended-spectrum beta-lactamase (ESBL) and contained the blaCTX M gene. Resistance to various antibiotics ranged from 18% to 77%. CONCLUSION: This study highlights the risk of virulent and multidrug-resistant STEC non-O157:H7 in raw milk and the need for proper quality surveillance and assurance plans to mitigate the potential public health threat.

Characterization of Actinobacillus pleuropneumoniae biovar 2 isolates reportedly reacted with the serovar 4 antiserum, and development of a multiplex PCR for O-antigen typing.

We have analyzed the capsule (CPS) and the lipopolysaccharide O-Antigen (O-Ag) biosynthesis loci of twelve Spanish field isolates of Actinobacillus pleuropneumoniae biovar 2, eleven of them previously typed serologically as serovar 4 and one non-typable (NT) (Maldonado et al., 2009, 2011). These isolates have the common core genes of the type I CPS locus, sharing >98% identity with those of serovar 2. However, the former possesses the O-Ag locus as serovar 4, and the latter possesses the O-Ag locus as serovar 7. The main difference found between the CPS loci of the 11 isolates and that of serovar 2 reference strain S1536 are two deletions, one of an 8 bp sequence upstream of the coding sequence and one of 111 bp sequence at the 5' end of the cps2G gene. The deletion mutations mentioned lead to a defect in the production of CPS in these isolates, which contributed to their previous mis-identification. In order to complement the serotyping of A. pleuropneumoniae in diagnostics and epidemiology, we have developed a multiplex PCR for the comprehensive O-Ag typing of all A. pleuropneumoniae isolates.

Diagnostic Accuracy of Cerebrospinal Fluid Multiplex Polymerase Chain Reaction Panel Testing in Patients With Suspected Central Nervous System Infections: A Multi-Center Study in the United Arab Emirates.

Background Delays in diagnosis and treatment of central nervous system (CNS) infections can lead to significant morbidity and mortality among children and adults. Prior antibiotic treatment is a major hurdle to accurate diagnosis due to falsely negative cerebrospinal fluid (CSF) cultures in partially treated patients. Increasingly, molecular diagnostic methods using multiplex polymerase chain reaction (mPCR) testing on CSF samples are being utilized in clinical practice for timely and accurate diagnosis. However, there is no data regarding the diagnostic accuracy or clinical impact of CSF mPCR testing in the Middle East region. We sought to compare the diagnostic accuracy of an automated mPCR CSF panel with routine CSF culture, the current gold standard, in the United Arab Emirates (UAE).  Methods This single-gated, multi-center, diagnostic accuracy study included patients from birth onwards who were admitted to any of the three participating hospitals with an initial diagnosis of meningitis or encephalitis, between January 2017 and March 2021, and had CSF samples collected for mPCR and culture. Sociodemographic, clinical, and molecular data were collected for all. Results A total of 353 CSF samples were collected from patients from 0-90 years old hospitalized for suspected CNS infection. Children constituted 51% of the study population, and males were slightly over-represented (55.2%). Pathogens were detected by mPCR in 78 (22%) CSF samples, of which 19 (24%) were bacteria and 59 (76%) were viruses. No fungal pathogens were detected. Enteroviruses were the most prevalent CNS pathogen among our cohort (40%), followed by herpes simplex virus type 2 (HSV-2) (12.5%). Children constituted 69% of positive samples for enterovirus, while HSV-2 was exclusively detected among adults. Using CSF culture as the diagnostic gold standard, the mPCR panel demonstrated high specificity (100%) and sensitivity (96.3%) in diagnosing CNS infection among all age groups. mPCR testing demonstrated a high overall percentage of agreement (OPA) with CSF culture (98.9%). Patients with bacterial meningitis had a significantly longer hospitalization (p=0.004) and duration of antibiotic therapy (p=0.001) compared to those with viral meningitis. Three CSF samples were negative on mPCR testing but positive on culture. These pathogens included: methicillin-sensitive Staphylococcus aureus(MSSA), Bacillus cereus, and Mycobacterium Tuberculosis (MTB). In addition, 13 patients had negative CSF cultures but tested positive on CSF mPCR. These pathogens included Streptococcus pneumoniae (seven patients), Haemophilus influenzae (three patients), Streptococcus agalactiae (two patients), and Escherichia coli (one patient). All discordant results were confirmed by reviewing the patient's clinical presentation, CSF analysis, clinical course, and final diagnosis. Conclusion CSF mPCR panel is a highly sensitive and specific diagnostic tool for the diagnosis of CNS infections among all age groups in the UAE. Routine use of CSF mPCR panels can decrease healthcare costs by reducing the length of stay and can also aid antibiotic stewardship efforts by reducing antibiotic overuse in patients with viral CSF infections. CSF culture and mPCR complement each other by identifying CNS pathogens in patients with prior antibiotic exposure who would otherwise be missed if relying on CSF culture alone. However, concomitant CSF culture samples should be sent to avoid missing unusual CNS pathogens.

The Prevalence and Association of Different Uropathogens Detected by M-PCR with Infection-Associated Urine Biomarkers in Urinary Tract Infections.

Background: Many emerging uropathogens are currently identified by multiplex polymerase chain reaction (M-PCR) in suspected UTI cases. Standard urine culture (SUC) has significantly lower detection rates, raising questions about whether these organisms are associated with UTIs and truly cause inflammation. Objective: To determine if microbes detected by M-PCR were likely causative of UTI by measuring inflammatory biomarkers in the urine of symptomatic patients. Design Setting and Participants: Midstream voided urine was collected from subjects ≥60 years presenting to urology clinics with symptoms of UTI (n = 1132) between 01/2023 and 05/2023. Microbe detection was by M-PCR and inflammation-associated biomarker (neutrophil gelatinase-associated lipocalin, interleukin 8, and interleukin 1ß) was by enzyme-linked immunosorbent assay. Biomarker positivity was measured against individual and groups of organisms, E. coli and non-E. coli cases, emerging uropathogens, monomicrobial and polymicrobial cases. Outcome Measurements and Statistical Analysis: Distributions were compared using 2-sample Wilcoxon Rank Sum test with 2-tailed p-values < 0.05 considered statistically significant. Results and Limitations: M-PCR was positive in 823 (72.7%) specimens with 28 of 30 (93%) microorganisms/groups detected. Twenty-six of twenty-eight detected microorganisms/groups (93%) had ≥2 biomarkers positive in >66% of cases. Both non-E. coli cases and E. coli cases had significant biomarker positivity (p < 0.05). Limitations were that a few organisms had low prevalence making inferences about their individual significance difficult. Conclusion: The majority of microorganisms identified by M-PCR were associated with active inflammation measured by biomarker positivity, indicating they are likely causative of UTIs in symptomatic patients. This includes emerging uropathogens frequently not detected by standard urine culture.

Prevalence, Toxin Genes, and Antibiotic Resistance Profiles of Bacillus cereus Isolates from Spices in Antalya and Isparta Provinces in Türkiye.

Bacillus cereus is a pathogenic bacterium commonly found in nature and can produce toxins that cause food poisoning. This study aimed to detect the prevalence of B. cereus group bacteria in 50 unpackaged and 20 packaged spice samples frequently used as flavoring in Turkish cuisine, as well as investigate the presence of toxin genes and antibiotic resistance in the isolates. A total of 48 B. cereus group bacteria were isolated from 27 of 70 (38.57%) spice samples. The prevalence of B. cereus group bacteria in packaged (25%, 5/20) and unpackaged (44%, 22/50) spice samples did not differ significantly (P ˃ 0.05). All B. cereus group isolates were identified as B. cereus sensu stricto (B. cereus) using molecular methods. The hemolytic activity tests revealed that the most strains (44/48, 91.67%) are ß-hemolytic. The distributions of toxin genes in isolates were investigated by PCR. It was determined that all isolates were identified to have 2-8 toxin genes, except B. cereus SBC3. The three most common toxin genes were found to be nheA (47/48, 97.92%), nheB (46/48, 95.83%), and entFM (46/48, 95.83%). All B. cereus isolates were susceptible to linezolid and vancomycin, while 35.42% (17/48) showed resistance to erythromycin. Multi-drug resistance (MDR) was detected in 8.3% (4/48) of B. cereus isolates, while 33.33% of the isolates showed multiple antibiotic resistance (MAR) index values higher than 0.2. The findings indicate that B. cereus may pose a health risk in packaged and unpackaged spices if present in high quantities. Therefore, the presence of B. cereus strains in both packaged and unpackaged spices should be monitored regarding consumer health and product safety.

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.

Seasonal Prevalence of Respiratory Pathogens Among Children in the United Arab Emirates: A Multicenter Cross-Sectional Study in the Pre-COVID-19 Era.

Background Viral respiratory infections in children pose a significant burden on healthcare facilities globally. In the United Arab Emirates (UAE) these account for 15% of all healthcare encounters among children. However, the seasonal prevalence and molecular epidemiology of respiratory viral infections in the UAE remains unknown. We sought to determine trends in seasonal viral prevalence in order to monitor disease activity and optimize the timing of Respiratory Syncytial Virus (RSV) prophylaxis among high-risk infants in the UAE. Methods This cross-sectional multicenter study included children 0-18 years of age who presented to a large private healthcare group in Dubai, UAE, and had upper respiratory samples collected for multiplex polymerase chain reaction (mPCR) testing between January 1st and December 31st, 2019. Sociodemographic, clinical, and molecular data were examined for children who tested positive for any pathogen on the mPCR panel. Results A total of three thousand and ninety-eight infants and children had mPCR assays performed during the study period, of which 2427 (78.3%) were positive for any respiratory pathogen. The median age of our sample population was 39 months and 56.8% were male. Emergency room was the most common site (34.7%) of sample collection and the vast majority of children presented with fever (85.3%). Rhinovirus/enterovirus was the most prevalent viral infection (45%) throughout the year and peaked in September, followed by Influenza (20.2%), and RSV (17.1%). RSV season, defined as an infection prevalence of >10%, occurred from August to December with a peak in October. Adenovirus (15.6%) infections peaked in June and accounted for 43% of hospitalizations in our study (p<0.05). Viral co-infections with RSV and rhinovirus/enterovirus were most common and observed in 19.9 % of children. Conclusion Rhinovirus/enterovirus is the most prevalent viral pathogen throughout the calendar year among the pediatric population in the UAE. RSV season begins earlier than reported in other countries regionally, hence RSV prophylaxis should be initiated in August to optimize protection among high-risk infants.

The Development of a Multiplex PCR Assay for Fast and Cost-Effective Identification of the Five Most Significant Pathogenic Prototheca Species.

A multiplex PCR system (m-PCR) has been developed to accurately differentiate the five most important pathogenic Prototheca species, including the three species associated with infection in dairy cattle (P. ciferrii, P. blaschkeae, and P. bovis) and the two species associated with human infections (P. wickerhamii and P. cutis). The method is low-cost since it employs a simple "heat-shock" method in a TE buffer for DNA extraction. Furthermore, it requires only primers, a Taq polymerase, an agarose gel, and a molecular weight marker for identification. The method was based on published Prototheca cytochrome B sequences and was evaluated using reference strains from each of the five Prototheca species. The validity of the method was confirmed by identifying 50 strains isolated from milk samples. The specificity was tested in silico and with experimental PCR trials, showing no cross-reactions with other Prototheca species, as well as with bacteria, fungi, cows, algae, animals, or humans. The method could detect mixed infections involving two or three Prototheca species, providing a rapid test that delivers results within three hours.

Elevated UTI Biomarkers in Symptomatic Patients with Urine Microbial Densities of 10,000 CFU/mL Indicate a Lower Threshold for Diagnosing UTIs.

The literature lacks consensus on the minimum microbial density required for diagnosing urinary tract infections (UTIs). This study categorized the microbial densities of urine specimens from symptomatic UTI patients aged ≥ 60 years and correlated them with detected levels of the immune response biomarkers neutrophil gelatinase-associated lipocalin (NGAL), interleukin-8 (IL-8), and interleukin-1-beta (IL-1ß). The objective was to identify the microbial densities associated with significant elevation of these biomarkers in order to determine an optimal threshold for diagnosing symptomatic UTIs. Biobanked midstream voided urine samples were analyzed for microbial identification and quantification using standard urine culture (SUC) and multiplex-polymerase chain reaction (M-PCR) testing, while NGAL, IL-8, and IL-1ß levels were measured via enzyme-linked immunosorbent assay (ELISA). NGAL, IL-8, and IL-1ß levels were all significantly elevated at microbial densities ≥ 10,000 cells/mL when measured via M-PCR (p < 0.0069) or equivalent colony-forming units (CFUs)/mL via SUC (p < 0.0104) compared to samples with no detectable microbes. With both PCR and SUC, a consensus of two or more elevated biomarkers correlated well with microbial densities > 10,000 cells/mL or CFU/mL, respectively. The association between ≥10,000 cells and CFU per mL with elevated biomarkers in symptomatic patients suggests that this lower threshold may be more suitable than 100,000 CFU/mL for diagnosing UTIs.

Development of Multiplex PCR and Melt-Curve Analysis for the Molecular Identification of Four Species of the Mullidae Family, Available in the Market.

The authentication of food products and the verification of their identity are of major importance for consumers. Food fraud through mislabeling is an illegal practice consisting of the substitution of an expensive food product by a relatively cheaper one, misleading false labelling of their origin and adulteration in processed or frozen products. This issue is particularly of high importance concerning fish and seafood, which are easily adulterated primarily due to difficult morphological identification. Fish species of the Mullidae family are considered among the most high-valued seafood products traded in Greece and Eastern Mediterranean in general, in terms of the price and demand. Specifically, the red mullet (Mullus barbatus) and the striped red mullet (Mullus surmuletus) are both indigenous in the Aegean (FAO Division 37.3.1) and the Ionian (FAO Division 37.2.2) Seas, with high levels of consumers' preferences. However, they could be easily adulterated or misidentified by the invasive Aegean Sea Lessepsian migrator goldband goatfish (Upeneus moluccensis) as well as by the imported West African goatfish (Pseudupeneus prayensis). Keeping this in mind, we designed two novel, time-saving and easy-to-apply multiplex PCR assays and one multiple Melt-Curve analysis real-time PCR for the identification of these four species. These methodologies are based on species-specific primers targeting single nucleotide polymorphisms (SNPs) detected via sequencing analysis of the mitochondrial cytochrome C oxidase subunit I (CO1) and of the cytochrome b (CYTB) genes in newly collected individuals, with additional comparison with congeneric and conspecific haplotypes obtained from the GenBank database. Both methodologies, targeting CO1 or CYTB, utilize one common and four diagnostic primers, producing amplicons of different length that are easily and reliably separated on agarose gel electrophoresis, yielding a single clear band of diagnostic size for each species or a certain Melt-Curve profile. The applicability of this cost-effective and fast methodology was tested in 328 collected specimens, including 10 cooked samples obtained from restaurants. In the vast majority (327 out of the 328) of the specimens tested, one single band was produced, in agreement with the expected products with a single exception a M. barbatus sample that was identified as M. surmuletus, the identity of which was confirmed using sequencing, indicating erroneous morphological identification. The developed methodologies are expected to contribute to the detection of commercial fraud in fish authentication.

Atypical Staphylococcal Septic Arthritis in a Native Hip: A Case Report and Review.

Septic arthritis is a synovial fluid and joint tissue infection with significant morbidity and mortality risk if not diagnosed and treated promptly. The most common pathogen to cause septic arthritis is Staphylococcus aureus, a Gram-positive bacterium. Although diagnostic criteria are in place to guide the diagnosis of staphylococcal septic arthritis, there is a lack of adequate sensitivity and specificity. Some patients present with atypical findings which make it difficult to diagnose and treat in time. In this paper, we present the case of a patient with an atypical presentation of recalcitrant staphylococcal septic arthritis in a native hip complicated by uncontrolled diabetes mellitus and tobacco usage. We review current literature on diagnosing S. aureus septic arthritis, novel diagnostic technique performance to guide future research and assist clinical suspicion, and current S. aureus vaccine development for at-risk patients.

Occurrence of diarrheagenic Escherichia coli pathotypes from raw milk and unpasteurized buttermilk by culture and multiplex polymerase chain reaction in southwest Iran.

BACKGROUND: In developing countries including Iran, there are limited data on diarrheagenic Escherichia coli (DEC) contamination in milk and unpasteurized buttermilks. This study aimed to determine the occurrence of DEC pathotypes by culture and multiplex polymerase chain reaction (M-PCR) in some dairy products from southwest Iran. METHODS AND RESULTS: In this cross-sectional study (September to October 2021), 197 samples (87 unpasteurized buttermilk and 110 raw cow milk) were collected from dairy stores of Ahvaz, southwest Iran. The presumptive E. coli isolates were primarily identified using biochemical tests and then confirmed by PCR of uidA gene. The occurrence of 5 DEC pathotypes: enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) were investigated using M-PCR. Overall, 76 (76/197, 38.6%) presumptive E. coli isolates were identified by biochemical tests. Using uidA gene, only 50 isolates (50/76, 65.8%) were confirmed as E. coli. DEC pathotypes were detected in 27 of 50 (54.0%) E. coli isolates (74.1%, 20/27 from raw cow milk and 25.9%, 7/27 from unpasteurized buttermilk). The frequency of DEC pathotypes was as follows: 1 (3.7%) EAEC, 2 (7.4%) EHEC, 4 (14.8%) EPEC, 6 (22.2%) ETEC, and 14 (51.9%) EIEC. However, 23 (46.0%) E. coli isolates had only the uidA gene and were not considered DEC pathotypes. CONCLUSION: Possible health risks for Iranian consumers can be attributed to the presence of DEC pathotypes in dairy products. Hence, serious control and prevention efforts are needed to stop the spread of these pathogens.

Changes in the Epidemiology of Respiratory Pathogens in Children during the COVID-19 Pandemic.

Since the outbreak of the COVID-19 pandemic, a significant decrease in non-COVID-19 respiratory illnesses were observed, suggesting that the implementation of measures against COVID-19 affected the transmission of other respiratory pathogens. The aim of this study was to highlight the changes in the epidemiology of respiratory pathogens in children during the COVID-19 pandemic. All children with Severe Acute respiratory illness admitted to the pediatric departments between January 2018 and December 2021 with negative COVID-19 PCR, were enrolled. The detection of respiratory pathogens was made by the Film Array Respiratory Panel. A total of 902 respiratory specimens were tested. A significantly lower positivity rate during the COVID-19 period was found (p = 0.006), especially in infants under 6 months (p = 0.008). There was a substantial absence of detection of Respiratory Syncytial Virus and Influenza A during the winter season following the outbreak of the pandemic (p < 0.05; p = 0.002 respectively). An inter-seasonal resurgence of Respiratory Syncytial Virus was noted. Human Rhinovirus was detected throughout the year, and more prevalent in winter during COVID-19 (p = 0.0002). These changes could be explained by the impact of the implementation of preventive measures related to the COVID-19 pandemic on the transmission of respiratory pathogens in children.

Genetic diversity of wild rice accessions (Oryza rufipogon Griff.) in Guangdong and Hainan Provinces, China, and construction of a wild rice core collection.

Oryza rufipogon Griff. is a valuable germplasm resource for rice genetic improvement. However, natural habitat loss has led to the erosion of the genetic diversity of wild rice populations. Genetic diversity analysis of O. rufipogon accessions and development of the core collection are crucial for conserving natural genetic diversity and providing novel traits for rice breeding. In the present study, we developed 1,592 SNPs by multiplex PCR and next-generation sequencing (NGS) technology and used them to genotype 998 O. rufipogon accessions from 14 agroclimatic zones in Guangdong and Hainan Provinces, China. These SNPs were mapped onto 12 chromosomes, and the average MAF value was 0.128 with a minimum of 0.01 and a maximum of 0.499. The O. rufipogon accessions were classified into ten groups. The mean Nei's diversity index and Shannon-Wiener index (I) were 0.187 and 0.308, respectively, in all populations, indicating that O. rufipogon accessions had rich genetic diversity. There were also differences in the genetic diversity of O. rufipogon resources in the 14 regions. Hainan populations possessed higher levels of genetic diversity, whereas the Guangzhou population had lower levels of genetic diversity than did the other populations. Phylogenetic analysis revealed that the genetic relationship among the distribution sites of O. rufipogon was closely related to geographical location. Based on genetic distance, a core collection of 299 accessions captured more than 99% of the genetic variation in the germplasm. This study provides insights into O. rufipogon conservation, and the constructed core collection provides valuable resources for future research and genomics-assisted breeding of rice.