Chloroacetate esterase reaction combined with immunohistochemical characterization of the specific tissue microenvironment of mast cells.

This study provides a combined histochemical method for detecting enzyme activity of chloroacetate esterase simultaneously with immunolabeling of the components of a specific tissue microenvironment on formalin-fixed, paraffin-embedded specimens. Chromogenic detection of the molecular targets within and outside the mast cells provides novel options in determining the histoarchitectonics of organ-specific mast cell populations, studying the functional significance of chloroacetate esterase and specifying the immune landscape of the tissue microenvironment.

Glucose and leukocyte esterase levels are possible biomarkers for bacterial septic arthritis.

OBJECTIVE: The objective of the study was to determine the added value of synovial fluid (SF) glucose levels and other biochemical parameters as possible biomarkers of bacterial septic arthritis (SA). MATERIALS AND METHODS: We prospectively examined adult patients with SA. As a control group, adults with uninfected joints were enrolled. SF samples were obtained, and microbiological analyses were made. SF glucose levels, pH, and leukocyte esterase were measured using a glucometer and colorimetric test strips. Blood samples were collected from both groups to determine glucose levels. RESULTS: We included eight subjects with knee ligaments lesions, six with meniscus lesions, and five with osteoarthritis as the control group, as well as 20 patients with SA. SF culture was positive in 60%. SF glucose levels from patients were lower than the controls (p = 0.0018) with the lowest concentration in patients with a positive culture (p = 0.0004). Blood and SF glucose concentration from the positive culture patients were compared (p < 0.0001). Leukocyte esterase presented the highest values in patients with a positive culture (p < 0.0001) and a more acidic pH was found compared to the control group (p < 0.0001). CONCLUSION: These biochemical parameters might be a quick and inexpensive added value for distinguishing between infective and non-infective joint disease.

OBJETIVO: Evaluar el valor añadido de los niveles de glucosa en el líquido sinovial (LS) y otros parámetros bioquímicos en el diagnostico de artritis séptica (AS). MATERIAL Y MÉTODOS: Análisis prospectivo de pacientes adultos con AS. Pacientes con articulaciones no infectadas fueron incluidos como grupo control. Se tomaron muestras de LS y sangre para la realización de análisis microbiológicos y bioquímicos en los pacientes y controles. RESULTADOS: Incluimos 8 sujetos con lesión ligamentosa de rodilla, 6 con lesiones meniscales y 5 con osteoartritis como grupo control, así como 20 pacientes con AS. El cultivo de LS fue positivo en 60%. Los niveles de glucosa en LS de pacientes con AS fueron más bajos que los controles (P = 0.0018) con la concentración más baja en pacientes con cultivo positivo (p = 0.0004). La relación de glucosa en sangre y LS de pacientes con cultivo positivo se vio afectada (p < 0.0001). La esterasa leucocitaria presentó valores más altos en pacientes con cultivo positivo (p < 0.0001); se encontró un pH más ácido en comparación con el grupo control (p < 0.0001). CONCLUSIÓN: Estos parámetros bioquímicos podrían ser un valor agregado útil, rápido y económico para distinguir entre enfermedad articular infecciosa y no infecciosa.

Synovial Biomarkers to Detect Chronic Periprosthetic Joint Infection: A Pilot Study to Compare Calprotectin Rapid Test, Calprotectin ELISA Immunoassay and Leukocyte Esterase Test.

BACKGROUND: Periprosthetic joint infection (PJI) is a devastating complication after joint replacement surgery, and making diagnosis is often far from obvious. Calprotectin was recently proposed as a promising synovial biomarker to detect PJI. To our knowledge, no comparative study exists between enzyme-linked immunosorbent assay (ELISA) and rapid calprotectin test (CalFAST). Our purpose was to compare these methods with leukocyte esterase (LE) test from synovial fluid of painful knee arthroplasty subjected to infectious workup. METHODS: Ninety-three patients were included in this prospective observational study. They underwent synovial fluid aspiration that was analyzed for cell count, microbiological culture, LE test, calprotectin rapid test, and calprotectin immunoassay dosage. The 2018 Consensus Statements criteria for PJI were used to diagnose PJI. Sensitivity, specificity, positive and negative likelihood ratio, and receiver operating characteristic were calculated for detection methods and compared. RESULTS: We categorized 39 patients as infected and 50 patients as not infected. The sensitivity comparing the ELISA test and CalFAST test was similar, 92.3% and 97.4%, respectively. LE rapid test showed 46% of sensitivity and 94% of specificity. The highest specificity was found with ELISA test (100%). Comparing the receiver operating characteristic curves by z-test, there were statistically significant differences between LE strip test and the other two methods. Otherwise, no statistically significant differences were present between ELISA and CalFAST test. CONCLUSION: Synovial calprotectin detection has high accuracy in knee PJI diagnosis, both ELISA and rapid test. LE strip test remains a good test to confirm the diagnosis of PJI in case of positivity. In clinical practice, the calprotectin rapid test can be considered an excellent point-of-care test.

Identification and validation of PGLS as a metabolic target for early screening and prognostic monitoring of gastric cancer.

BACKGROUND: Gastric cancer is the third leading cause of cancer-related death in the world. The purpose of the present study is to investigate the expression and prognostic significance of 6-phosphogluconolactonase (PGLS) in gastric cancer. METHODS: The protein extracted from a panel of four pairs of gastric cancer tissues and adjacent tissues, labeled with iTRAQ (8-plex) reagents, and followed by LC-ESI-MS/MS. The expressions of proteins were further validated by immunohistochemistry analysis. The expression levels of mRNA were analyzed and validated in the Oncomine database. The correlations of PGLS with prognostic outcomes were evaluated with Kaplan-Meier plotter database. RESULTS: The present study found that PGLS was significantly up-regulated in gastric cancer by using iTRAQ-based proteomics and immunohistochemistry analysis. The sensitivity of PGLS in gastric cancer was 72.9%. The high expression of PGLS was significantly correlated with TNM staging in gastric cancer (p = 0.02). The overexpression of PGLS predicts worse overall survival (OS) and post-progression survival (PPS) for gastric cancer (OS, HR = 1.48, p = 2.1e-05; PPS, HR = 1.35, p = 0.015). Specifically, the high PGLS expression predicts poor OS, PPS in male gastric cancer patients, in patients with lymph node metastasis and in patients with Her-2 (-). CONCLUSIONS: These findings suggested that PGLS was aberrantly expressed in gastric cancer and predicts poor overall survival, post-progression survival for gastric cancer patients. The present study collectively supported that PGLS is an important target for early determining and follow-up monitoring for gastric cancer.

Diagnosis of knee prosthetic joint infection; aspiration and biopsy.

BACKGROUND: Prosthetic joint infection (PJI) is a significant cause of morbidity and mortality following knee replacement surgery. The diagnosis can be challenging and is based on a combination of clinical suspicion, radiographic findings and also biochemical/ microbiological investigations. Our Aim was to review the role of aspiration and biopsy in the diagnosis of PJI in Total Knee Arthroplasty (TKA). METHOD/RESULTS: Aspirated synovial fluid should be analysed by direct culture, via blood culture bottles, EDTA bottles for cell count and 'point of care' testing such as leucocyte esterase or alpha defensin. Synovial WCC and PMN cell percentage are important steps in diagnosis of both acute and chronic PJI. A minimum of 5 deep samples using a 5 clean instrument technique should be obtained and sent for tissue culture done either blind or arthroscopic. Formal fluoroscopic guided interface biopsy has also been described with excellent results. In a recent series of 86 TKRs preoperative arthroscopic biopsy group had a sensitivity of 100%, specificity of 94.7%, positive predictive value of 87.4% and a negative predictive value of 100%. CONCLUSION: In the presence of clinical suspicion with raised biomarkers, it is recommended that aspiration +/- biopsy with synovial fluid testing is performed. Direct culture and cell count are recommended. 'Point of care tests' such as Leucocyte Esterase testing should be considered. Duration of culture, including pathogen and host factors, should be discussed with a local microbiology/ID department in the context of a formal multi-disciplinary team.

Carboxylesterases and arylacetamide deacetylase comparison in human A549, H460, and H727 pulmonary cells.

AIMS: Human carboxylesterases (CESs) and arylacetamide deacetylase (AADAC) are serine-esterase enzymes catalyzing the hydrolysis of many compounds containing esters, amides, thioesters, or acetyl groups. This study aimed to investigate the presence, kinetic parameters, and inhibition of CES1, CES2, and AADAC in A549, H460, and H727 pulmonary cells in both living cells and S9 fractions. MATERIALS AND METHODS: The p-nitrophenyl acetate (pNPA) and 4-methylumbelliferyl acetate (4-MUA) were used as non-selective esterase substrates, whereas phenacetin as selective AADAC substrate. CESs activities were also investigated in living cells by cellular bioimaging using selective fluorescent probes. KEY FINDINGS: AADAC gene was detected in A549 and H460 cells; nevertheless, arylesterase activity was not found in relative S9 fractions. Besides, CES1 and CES2 were expressed to a different extent by all lung cells, and enzymatic activities were quite overlapping each other. All enzymes exhibited a typical Michaelis-Menten saturation curve and, regarding 4-MUA, similar Km values were found in both living cells and S9 fractions. Conversely, kinetic parameters relative to the pNPA hydrolysis by S9 fractions were significantly lower than those detected in living cells. Inhibition studies revealed that 4-MUA hydrolysis was inhibited by bis-p-nitrophenyl phosphate and phenylmethanesulfonyl fluoride more than loperamide; on the contrary, pNPA hydrolysis inhibition was limited with similar inhibition profiles being obtained in both living cells and S9 fractions. The presence of carboxylesterases was definitely confirmed by cellular bioimaging. SIGNIFICANCE: These findings add information to esterase knowledge in pulmonary cells that could be used as in vitro models for toxicological and pharmacological studies.

Utility of bedside leucocyte esterase testing to rule out septic arthritis.

INTRODUCTION: Suspected septic arthritis is a common presentation to EDs. The underlying diagnosis is often non-infective pathology. Differentiating between aetiologies is difficult. A bedside test with high negative predictive value (NPV) may allow safe discharge of patients, reduce the time in the ED, hospital admission and associated costs. This study aims to evaluate the NPV of bedside leucocyte esterase (LE) in the assessment of these patients. METHODS: A prospective multicentre observational study of ED adult patients referred to orthopaedics with suspected native joint septic arthritis between October 2015 and April 2016. At three hospital sites in the Bristol region, the results of the LE test exposed to aspirated synovial fluid were recorded along with Gram stain, culture, haematinics and length of stay. A positive LE test was considered 2+ or 3+ leucocytes based on the test strip colour. Data were analysed to establish sensitivity, specificity, NPV and positive predictive value (PPV) against the gold standard 48-hour culture. We determined the potential number of inpatient bed-days that might be avoided using this bedside test. RESULTS: Eighty patients underwent joint aspiration. Five cases had positive 48-hour culture. All (5/5) infected cases showed ≥2+ LE, sensitivity of 100% (95% CI 47.8% to 100%) while the Gram stain was positive in only one case (sensitivity 20%, 95% CI 0.51% to 71.6%). Twenty-three LE were read negative or 1+, all with negative 48-hour culture results, resulting in an NPV of 100% (95% CI 82.1% to 1.00%) for a negative LE test. Specificity of a positive LE test was 30.7% (95% CI 20.5% to 42.45%) with PPV of 8.77% (95% CI 7.64% to 10.1%). It was calculated that 57 orthopaedic bed-days could have potentially been saved by immediately discharging those with a negative LE test. CONCLUSIONS: LE point-of-care testing for suspected septic arthritis of native joints has a high NPV. Implementation of LE may facilitate more rapid discharge of patients with negative results. This test has the potential to reduce diagnostic uncertainty and costs to the healthcare system.

Detection of Carboxylesterase 1 and Chlorpyrifos with ZIF-8 Metal-Organic Frameworks Using a Red Emission BODIPY-Based Probe.

In this work, a red emission fluorescent probe CBZ-BOD@zeolitic imidazolate framework-8 (ZIF-8) was fabricated based on metal-organic frameworks (MOFs) for detecting carboxylesterase 1 (CES1). The small molecule probe CBZ-BOD was first synthesized and then used to prepare the functionalized MOF material. ZIF-8 was chosen as an encapsulation shell to improve the detection properties of CBZ-BOD. Using this unique porous materials, ultrasensitive quantification of CES1 and chlorpyrifos was successfully realized. The low detection limit and high fluorescence quantum yield were calculated as 1.15 ng/mL and 0.65 for CBZ-BOD@ZIF-8, respectively. CBZ-BOD@ZIF-8 has good biocompatibility and was successfully applied to monitor the activity of CES1 in living cells. A molecular docking study was used to explore the binding of CES1 and CBZ-BOD, finding that CES1 can bind with the probe before and after hydrolysis. This type of materialized probe can inspire the development of fluorescent tools for further exploration of many pathological processes.

What Is the Optimal Timing for Reading the Leukocyte Esterase Strip for the Diagnosis of Periprosthetic Joint Infection?

BACKGROUND: The leucocyte esterase (LE) strip test often is used to diagnose periprosthetic joint infection (PJI). In accordance with the manufacturer's directions, the LE strip test result is read 3 minutes after exposing it to joint fluid, but this has not been supported by robust research. Moreover, we have noted that the results of the LE strip test might change over time, and our previous studies have found that centrifugation causes the results of the LE strip test to degrade. Still, there is no evidence-based recommendation as to when to read the LE strip test to maximize diagnostic accuracy, in general, and the best reading times for the LE strip test before and after centrifugation need to be determined separately, in particular. QUESTIONS/PURPOSES: (1) What is the optimal timing for reading LE strip test results before centrifugation to diagnose PJI? (2) What is the optimal timing for reading LE strip test results after centrifugation to diagnose PJI? METHODS: This study was a prospective diagnostic trial. In all, 120 patients who were scheduled for revision arthroplasty and had signs of infection underwent joint aspiration in the outpatient operating room between July 2018 and July 2019 and were enrolled in this single-center study. For inclusion, patients must have had a diagnosis of PJI or nonPJI, valid synovial fluid samples, and must not have received antibiotics within 2 weeks before arthrocentesis. As such, 36 patients were excluded; 84 patients were included for analysis, and all 84 patients agreed to participate. The 2018 International Consensus Meeting Criteria (ICM 2018) was used for the classification of 49 patients with PJI (score ≥ 6) and 35 without PJI (score ≤ 2). The classification was used as the standard against which the different timings for reading LE strips were compared. All patients without PJI were followed for more than 1 year, during which they did not report the occurrence of PJI. All patients were graded against the diagnostic criteria regardless of their LE strip test results. In 83 patients, one drop of synovial fluid (50 µL) was applied to LE strips before and after centrifugation, and in one patient (without PJI), the sample was not centrifuged because the sample volume was less than 1.5 mL. The results of the strip test were read on an automated colorimeter. Starting from 1 minute after centrifugation, these strips were automatically read once every minute, 15 times (over a period of 16 minutes), and the results were independently recorded by two observers. Results were rated as negative, ±, 1+, and 2+ upon the machine reading. Grade 2+ (dark purple) was used as the threshold for a positive result. An investigator who was blinded to the study performed the statistics. Optimal timing for reading the LE strip before and after centrifugation was determined by using receiver operative characteristic (ROC) analysis. The specificity, sensitivity, and positive predictive and negative predictive values were calculated for key timepoints. RESULTS: Before centrifugation, the area under the curve was the highest when the results were read at 5 minutes (0.90 [95% CI 0.83 to 0.98]; sensitivity 0.88 [95% CI 0.75 to 0.95]; specificity 0.89 [95% CI 0.72 to 0.96]). After centrifugation, the area under the curve was the highest when the results were read at 10 minutes (0.92 [95% CI 0.86 to 0.98]; sensitivity 0.65 [95% CI 0.50 to 0.78]; specificity 0.97 [95% CI 0.83 to 1.00]). CONCLUSION: The LE strip test results are affected by time and centrifugation. For samples without centrifugation, we found that 5 minutes after application was the best time to read LE strips. We cannot deny the use of centrifuges because this is an effective way to solve the sample-mingling problem at present. We recommend 10 minutes postapplication as the most appropriate time to read LE strips after centrifugation. Multicenter and large-sample size studies are warranted to further verify our conclusion. LEVEL OF EVIDENCE: Level II, diagnostic study.

Infection Screening in Biofluids with Glucose Test Strips.

The four glucosyl esters were synthesized and tested for the determination of infection enzyme leukocyte esterase (LE) in human synovial (joint) fluid and urine. The esters acted as LE substrates releasing glucose in a direct proportion to the activity of LE in a sample. The freed glucose was then detected by a coupled-enzyme assay at either a nitrogen-doped carbon nanotube (N-CNT) electrode or a commercial glucose test strip. The assays at the N-CNT electrode detected LE down to 0.81 nM (25 µg L-1) and showed the fastest kinetics (2.1 × 105 M-1 s-1) for esters with the least crowded space around their carbonyl group. When used with glucose strips, the esters discerned clinically relevant levels of LE up to at least 26 nM (800 µg L-1) in the microliter-sized samples of bodily fluids. The reading of glucose strips with a potentiostat, instead of a personal glucose meter (blood glucometer), shortened the time of required sample incubation from 3 h to 5 min. Correcting the signal of incubated sample for that of original sample eliminated matrix effects and accounted for the presence of native glucose. The new esters have a potential to extend the use of glucose strips (already used by millions for diabetes monitoring) to the quantification of the severity of urinary tract and periprosthetic joint infections.

Determination of diagnostic value (validity) leukocyte esterase (urine dipstick strip) in differentiating inflammatory arthritis from bacterial arthritis.

BACKGROUND: The current diagnostic cornerstone for septic arthritis contains gram stains, bacterial culture, and cell count with a differential of aspirated synovial fluid. Recently, a synovial leukocyte esterase (LE) test has been used for diagnosing septic arthritis. Since this test measures the esterase activity of leukocytes, there is always a dilemma for using this test in patients with inflammatory arthritis. METHODS: We collected the synovial fluid specimens as part of the general diagnostic protocol for patients suspected of Juvenile Idiopathic Arthritis (JIA) or Septic Arthritis (SA). Each group included 34 patients. We compared the result of the synovial LE test with the result of the culture of each patient. RESULTS: The mean ages of patients were 64.14 ± 31.27 and 50.88 ± 23.19 months in the JIA group and septic arthritis group, respectively. The LE test results were positive in 30 specimens, trace in 3 and negative in one in the first-time test and were positive in 31 specimens and trace in 3 in the second-time test, while it was negative in all patients with JIA. Hence, the sensitivity of the synovial LE test was 80.8%, the specificity, PPV, and NPV were 78.6, 70.0, 86.8% respectively based on a positive culture. CONCLUSION: The leukocyte esterase strip test can be used as a rapid, bedside method for diagnosing or excluding bacterial infections in different body fluids. The synovial LE test can be used as an accurate test to rapidly rule in or out an acute articular bacterial infection, even in patients with concurrent inflammatory arthritis.

The Utility of Leukocyte Esterase Strip Test in the Diagnosis of Pediatric Septic Arthritis.

BACKGROUND: Most tests used to diagnose pediatric septic arthritis are either not accurate or do not produce rapid results. A leukocyte esterase (LE) strip test has previously been validated for the diagnosis of adult native and periprosthetic joint infections. The purpose of this prospective study was to: (1) evaluate the performance characteristics of the LE strip test in the diagnosis of pediatric septic arthritis and (2) determine the false positive rate of LE strip test on the aseptic synovial fluid (SF). METHODS: Between May 2016 and November 2018, SF was obtained from children who were hospitalized at our tertiary referral center on the basis of suspicion of septic arthritis. All patients underwent arthrocentesis, and the aspirate was tested with LE strip test, leukocyte count, and culture. Twenty-five patients satisfied the inclusion criteria. For the second part of the study, SF from 25 children undergoing surgery for developmental dysplasia of the hip was collected and tested with LE strip test, leukocyte count, and culture. RESULTS: In the first part of this study, 19 joints were classified as septic and 6 as aseptic. Considering a positive LE strip test ("++" and "+++" readings) indicative of septic arthritis yielded a sensitivity of 100%, specificity of 83%, positive predictive value of 95%, and negative predictive value of 100%. In the second part, all 25 patients with an aseptic SF had a negative test result ("-" and "+" readings). CONCLUSIONS: The LE strip test seems to be a valuable additional tool in the diagnosis of pediatric septic arthritis. The LE strip test has the advantages of being inexpensive and simple, providing real-time results and having a perfect negative predictive value to rule out the diagnosis of septic arthritis. LEVEL OF EVIDENCE: Level II-diagnostic.

Leukocyte esterase strip test as a reliable intraoperative PJIs biomarker. Our experience.

ackgroud and aim of work: Prosthetic joint infection (PJI) is the most common cause of total knee replacement failure and the third most common cause of total hip replacement failure, accounting for 16.8% of all knee revisions and 14.8% of the hip revisions; nevertheless, the diagnosis of PJI is often a challenge for the orthopaedic surgeon. The aim of these study was to evaluate the reliability of the LE strip test for diagnosis of PJI. MATERIALS AND METHODS: From December 2016 to January 2019, we enrolled 50 patients with suspected PJI; 32 females and 18 males, the average age at the time of the surgery was 76 years. Twenty-four patients underwent knee revision surgery and twenty-six hip revision surgery. In all patients during the surgery, the synovial fluid was aspirated and used for leukocyte esterase strip test. The result of the tests was compared to periprosthetic tissues culture, histological examination and sonication fluid culture for PJI. RESULTS: Comparing the results obtained from the LE test with the results obtained from the other diagnostic methods, we found that the concordance between the results of the leukocyte-esterase test and those of the culture test with peri-prosthetic tissue or synovial fluid was shown to be 93%, between LE and histological examinations, the concordance was 93% and finally with the culture of the sonicated fluid the concordance was 86% of the cases. CONCLUSIONS: The results of our serie show a good intraoperative diagnostic accuracy of the LE test, especially in its ability to exclude the hypothesis of periprosthetic infection in case of a negative result.

Reliability of synovial fluid alpha-defensin and leukocyte esterase in diagnosing periprosthetic joint infection (PJI): a systematic review and meta-analysis.

BACKGROUND: Synovial fluid proteins had been applied as diagnostic biomarkers for periprosthetic joint infection (PJI) in recent research papers. Thus, this meta-analysis aimed to estimate the diagnostic efficiency of synovial fluid α-defensin and leukocyte esterase (LE) for PJI. METHODS: We conducted our systematic review by searching the keywords in online databases such as PubMed, Embase, Cochrane, Elsevier, Springer, and Web of Science from the time of database inception to October 2018. Inclusion criteria were as follows: patients who have undergone knee, hip, or shoulder joint replacements; α-defensin or leukocyte esterase (LE strip) of synovial fluid was detected as the biomarker for PJI diagnosis; and Musculoskeletal Infection Society (MSIS) or utilizing a combination of clinical data was considered as the gold standard. Diagnostic parameters including sensitivity, specificity, diagnostic odds ratio (DOR), and area under the summary of receiver operating characteristics curve (AUSROC) were calculated for the included studies to evaluate the synovial fluid α-defensin and LE for PJI diagnosis. RESULTS: After full-text review, 28 studies were qualified for this systematic review, 16 studies used α-defensin and the other 12 were conducted using LE strip. The pooled sensitivity, specificity, and DOR of LE strip were 87% (95% CI 84-90%), 96% (95% CI 95-97%), and 170.09 (95% CI 97.63-296.32), respectively, while the pooled sensitivity, specificity, and DOR of α-defensin were 87% (95% CI 83-90%), 97% (95% CI 96-98%), and 158.18 (95% CI 74.26-336.91), respectively. The AUSROC for LE strip and α-defensin were 0.9818 and 0.9685, respectively. CONCLUSION: Both LE strip and α-defensin of synovial fluid provide rapid and convenient diagnosis for PJI. Sensitivity of α-defensin and LE strip are the same, while both these two methods have high specificity in clinical practice.

The Quality of Diagnostic Studies in Periprosthetic Joint Infections: Can We Do Better?

BACKGROUND: The diagnosis of periprosthetic joint infections (PJIs) continues to be a subject of extensive debate. This is in part due to the lack of a single "gold standard" test, and the marked heterogeneity in the design of studies evaluating the accuracy of different diagnostic modalities. The goal of this review is to critically analyze the evidence cited by the proceedings of the 2013 International Consensus Meeting (ICM) on PJI with regards to the diagnosis of PJI. METHODS: References from the Proceedings of the ICM on PJI related to PJI minor criteria were retrieved and manually reviewed. A total of 25 studies were analyzed using a Validated Quality Assessment of Diagnostic Accuracy Studies tool. RESULTS: A large number of studies were determined to have a high risk of bias for flow and timing domains due to the large numbers of exclusions. Studies of synovial white blood cells count and polymorphonuclear neutrophils percentage suffered from threshold optimization and lack of internal validity. Furthermore, due to the lack of homogeneity across studies, index test and reference standard domains showed high risk of bias for white blood cell/polymorphonuclear neutrophil percentage and the utility histological analysis, respectively. Leukocyte esterase testing lacked standardization with regard to the strip reagent used, and the exclusion of bloody samples limited sample sizes. CONCLUSION: The 2013 ICM minor criteria were based on studies with a low quality of evidence. As the committee continues to adjust these guidelines, they should encourage future studies with sound clinical design, patient selection, and testing procedures.

Investigating the effect of intracameral cefuroxime on oxidative stress and apoptosis in the rat cornea / Investigando o efeito de cefuroxima intracameral sobre o estresse oxidativo e a apoptose na córnea de ratos

ABSTRACT PURPOSE: We examined the effect of intracameral administration of cefuroxime on oxidative stress and endothelial apoptosis in rat corneal tissue. METHODS: In total, 30 rats were divided into 3 groups of 10 rats each (intracameral administration of cefuroxime 0.1 mg/0.01 mL (cefuroxime group); intracameral administration of balanced salt solution 0.01 mL (control group); or absence of intracameral injection (sham group). Corneal endothelial apoptosis was assessed by immunohistochemical analysis using caspase-3 and caspase-8. Total oxidant status, total antioxidant status, oxidative stress index, and paraoxonase and arylesterase levels were examined in corneal endothelial tissue and serum. RESULTS: Paraoxonase levels in the serum were significantly different between the sham and cefuroxime groups (p=0.027). A significant difference was also observed in total oxidant status levels between the cefuroxime and balanced salt solution groups (p=0.023). In addition, there were significant differences in total antioxidant status levels in corneal tissue between the cefuroxime and sham groups (p<0.001) and between the cefuroxime and balanced salt solution groups (p<0.001). Furthermore, significant differences were also observed in oxidative stress index levels between the cefuroxime and balanced salt solution groups (p=0.001) and between the cefuroxime and sham groups (p=0.026). According to the immunohistochemical staining results, a significant association with caspase-3 activity existed between the cefuroxime and balanced salt solution groups (p=0.007), while no significant difference was found with caspase-8 activity (p=0.541). Caspase-3 activity exhibited a significant relationship between the sham and balanced salt solution groups (p=0.018), but no relationship was found with caspase-8 activity (p=0.623). CONCLUSION: Immunohistochemical examination revealed that intracameral cefuroxime increased apoptosis when compared to the sham and balanced salt solution groups. Moreover, intracameral cefuroxime increased oxidative stress in the cornea and simultaneously induced apoptosis.

RESUMO OBJETIVO: Examinamos o efeito da administração intracameral da cefuroxima sobre o estresse oxidativo e a apoptose endotelial no tecido corneano de ratos. MÉTODOS: No total, 30 ratos foram divididos em 3 grupos de 10 ratos cada (administração intracameral de cefuroxima 0,1 mg/0,01 mL (grupo cefuroxima), administração intracameral de solução salina balanceada 0,01 mL (grupo controle) ou ausência de injeção intracameral (grupo sham)). A apoptose endotelial da córnea foi avaliada por análise imuno-histoquimica usando caspase-3 e -8. O status oxidante total, o status antioxidante total, o índice de estresse oxidativo e os níveis de a paraoxonase e arilesterase foram investigados no tecido endotelial da córnea e no soro. RESULTADOS: Os níveis de paraoxonase no soro foram significativamente diferentes entre os grupos sham e cefuroxima (p=0,027). Foi também observada uma diferença significativa nos níveis de estado oxidante total entre os grupos cefuroxima e solução salina balanceada (p=0,023). Além disso, houve diferenças significativas nos níveis de status antioxidante total no tecido da córnea entre os grupos cefuroxima e sham (p<0,001) e entre os grupos cefuroxima e solução salina balanceada (p<0,001). Diferenças significativas também foram observadas nos níveis do índice de estresse oxidativo entre os grupos cefuroxima e solução salina balanceada (p=0,001) e entre os grupos cefuroxima e sham (p=0,026). De acordo com os resultados de coloração imuno-histoquimica, houve associação significativa com a atividade da caspase-3 entre os grupos cefuroxima e solução salina balanceada (p=0,007), enquanto não houve diferença significativa com a atividade da caspase-8 (p=0,541). A atividade da caspase-3 exibiu uma relação significativa entre os grupos sham e solução salina balanceada (p=0,018), mas nenhuma relação foi encontrada com a atividade da caspase-8 (p=0,623). CONCLUSÃO: O exame imuno-histoquímico revelou que a cefuroxima intracameral aumentou a apoptose quando comparada com os grupos sham e solução salina balanceada. Além disso, a cefuroxima intracameral aumentou o estresse oxidativo na córnea e induziu simultaneamente a apoptose.

Clinical usefulness of iQ200/iChem Velocity workstation for screening of urine culture.

BACKGROUND: Clinical microbiology laboratories are asked to process large numbers of urine specimens for culture, but only 20-40% of them are positive. Therefore, a rapid, reliable screening method is necessary to speed up the reporting of a negative result. In this study, we evaluated the iQ200/iChem workstation, which is a combination of digital imaging software and a strip reader to predict negative urine culture. METHOD: A total of 1942 urine specimens were processed through both culture and iQ200/ iChem workstation. We analyzed the performance using two definition of positive urine culture; one or two potential uropathogens at a concentration of ≥105 CFU/ml and ≥ 104 CFU/ml. We assessed combinations of parameters (ASP; all small particles, WBC; leukocyte, BACT; bcteria, LE; leukocyte esterase) applying various cut-offs which can achieve the negative predictive value (NPV) ≥97% and culture reduction rate ≥ 50%. RESULTS: The culture positive rate was 12.8 and 18.4% applying the criteria of ≥105 CFU/ml and ≥ 104 CFU/ml, respectively. The area under the curve (AUC) of each parameter for ≥105 CFU/ml / ≥104 CFU/ml bacteriuria was 795 /0.719 for WBC, 0.722 / 0.701 for ASP and 0.740 /0.704 for bacteria. Therefore, we investigated the combination of the parameters. With the fixed parameter of BACT≥1/HPF and positive LE, the combinations of WBC ≥ 4/HPF and ASP ≥8500/µl or WBC ≥ 6/HPF and ASP≥5500/µl showed good performance for detecting ≥105 CFU/ml uropathogen. The ranges of sensitivity, specificity, negative predictive value and culture reduction rate were 91.5-92.3%, 49.8-52.6%, 97.7-97.9% and 50.4-53.0%, respectively. However, none of the combined setting yielded acceptable range of NPV for detecting ≥104 CFU/ml uropathogen (NPV 92.9-94.9%). Enterococcus spp. was the most common uropathogen causing the false negative results (55.7%), and also the main pathogen among the positive culture of 104-5 CFU/ml bacteriuria (45%). CONCLUSIONS: iQ200/iChem workstation was excellent in detection of ≥105 CFU/ml uropathogen, but unsatisfactory in detection of 104-5 CFU/ml uropathogen and Enterococcus spp. It can be useful for screening of urine specimens to reduce bacterial culture. However, notice from clinician will be necessary for specimens from the patients with high risk for UTI, such as pregnant woman, infant, elderly or immune compromised patients.

Arylacetamide Deacetylase Enzyme: Presence and Interindividual Variability in Human Lungs.

Human arylacetamide deacetylase (AADAC) is a single microsomal serine esterase involved in the hydrolysis of many acetyl-containing drugs. To date, the presence and activity of the AADAC enzyme in human lungs has been scarcely examined. We investigated its gene and protein expression as well as interindividual variations in AADAC activities in a large number of human lungs (n = 25) using phenacetin as a selective substrate. The kinetic parameters K m and V max were determined. Our findings highlighted a high interindividual variability in both AADAC mRNA levels and hydrolysis activities. Furthermore, for the first time we demonstrated the presence of the AADAC protein in various lung samples by means of immunoblot analysis. As a comparison, phenacetin hydrolysis was detected in pooled human liver microsomes. Lung activities were much lower than those found in the liver. However, similar K m values were found, which suggests that this hydrolysis could be due to the same enzyme. Pulmonary phenacetin hydrolysis proved to be positively correlated with AADAC mRNA (*P < 0.05) and protein (*P < 0.05) levels. Moreover, the average values of AADAC activity in smokers was significantly higher than in nonsmoker subjects (*P < 0.05), and this might have an important role in the administration of some drugs. These findings add more information to our knowledge of pulmonary enzymes and could be particularly useful in the design and preclinical development of inhaled drugs. SIGNIFICANCE STATEMENT: This study investigated the presence and activity of the AADAC enzyme in several human lungs. Our results highlight high interindividual variability in both AADAC gene and protein expression as well as in phenacetin hydrolysis activity. These findings add more information to our knowledge of pulmonary enzymes and could be particularly useful in the design and preclinical development of inhaled drugs.

Investigating the effect of intracameral cefuroxime on oxidative stress and apoptosis in the rat cornea.

PURPOSE: We examined the effect of intracameral administration of cefuroxime on oxidative stress and endothelial apoptosis in rat corneal tissue. METHODS: In total, 30 rats were divided into 3 groups of 10 rats each (intracameral administration of cefuroxime 0.1 mg/0.01 mL (cefuroxime group); intracameral administration of balanced salt solution 0.01 mL (control group); or absence of intracameral injection (sham group). Corneal endothelial apoptosis was assessed by immunohistochemical analysis using caspase-3 and caspase-8. Total oxidant status, total antioxidant status, oxidative stress index, and paraoxonase and arylesterase levels were examined in corneal endothelial tissue and serum. RESULTS: Paraoxonase levels in the serum were significantly different between the sham and cefuroxime groups (p=0.027). A significant difference was also observed in total oxidant status levels between the cefuroxime and balanced salt solution groups (p=0.023). In addition, there were significant differences in total antioxidant status levels in corneal tissue between the cefuroxime and sham groups (p<0.001) and between the cefuroxime and balanced salt solution groups (p<0.001). Furthermore, significant differences were also observed in oxidative stress index levels between the cefuroxime and balanced salt solution groups (p=0.001) and between the cefuroxime and sham groups (p=0.026). According to the immunohistochemical staining results, a significant association with caspase-3 activity existed between the cefuroxime and balanced salt solution groups (p=0.007), while no significant difference was found with caspase-8 activity (p=0.541). Caspase-3 activity exhibited a significant relationship between the sham and balanced salt solution groups (p=0.018), but no relationship was found with caspase-8 activity (p=0.623). CONCLUSION: Immunohistochemical examination revealed that intracameral cefuroxime increased apoptosis when compared to the sham and balanced salt solution groups. Moreover, intracameral cefuroxime increased oxidative stress in the cornea and simultaneously induced apoptosis.

The Major Storage Protein in Potato Tuber Is Mobilized by a Mechanism Dependent on Its Phosphorylation Status.

The role of the protein phosphorylation mechanism in the mobilization of vegetative storage proteins (VSPs) is totally unknown. Patatin is the major VSP of the potato (Solanum tuberosum L.) tuber that encompasses multiple differentially phosphorylated isoforms. In this study, temporal changes in the phosphorylation status of patatin isoforms and their involvement in patatin mobilization are investigated using phosphoproteomic methods based on targeted two-dimensional electrophoresis (2-DE). High-resolution 2-DE profiles of patatin isoforms were obtained in four sequential tuber life cycle stages of Kennebec cultivar: endodormancy, bud break, sprouting and plant growth. In-gel multiplex identification of phosphorylated isoforms with Pro-Q Diamond phosphoprotein-specific stain revealed an increase in the number of phosphorylated isoforms after the tuber endodormancy stage. In addition, we found that the phosphorylation status of patatin isoforms significantly changed throughout the tuber life cycle (P < 0.05) using the chemical method of protein dephosphorylation with hydrogen fluoride-pyridine (HF-P) coupled to 2-DE. More specifically, patatin phosphorylation increased by 32% from endodormancy to the tuber sprouting stage and subsequently decreased together with patatin degradation. Patatin isoforms were not randomly mobilized because highly phosphorylated Kuras-isoforms were preferably degraded in comparison to less phosphorylated non-Kuras isoforms. These results lead us to conclude that patatin is mobilized by a mechanism dependent on the phosphorylation status of specific isoforms.