Clinical And Laboratory Standards Institute Research Articles

Microwave-assisted synthesis of novel Ti/BTB-MOFs as porous anticancer and antibacterial agents

Nano compounds, especially metal-organic frameworks (MOFs), have significant properties. Among the most important properties of these compounds, which depend on their specific surface area and porosity, are biological properties, such as anticancer and antibacterial properties. In this study, a new titanium/BTB metal-organic framework (Ti/BTB-MOF) was synthesized by using titanium nitrate and 1,3,5-Tris(4-carboxyphenyl)benzene (BTB) under microwave radiation. The structure of the synthesized Ti/BTB-MOF was characterized and confirmed using X-ray diffraction (XRD) patterns, X-ray photoelectron spectroscopy (XPS) analysis, Fourier transform infrared (FT-IR) spectra, energy-dispersive X-ray (EDAX) analysis mapping, scanning electron microscope (SEM) images, thermogravimetric analysis (TGA) curves, and Brunauer–Emmett–Teller (BET) analysis. The in vitro anticancer properties of Ti/BTB-MOF were evaluated using the MTT method against MG-63/bone cancer cells and A-431/skin cancer cells. The in vitro antibacterial activity was tested using the Clinical and Laboratory Standards Institute (CLSI) guidelines. In the anticancer activity, IC50 (half-maximal inhibitory concentration) values of 152 μg/mL and 201 μg/mL for MG-63/bone cancer cells and A-431/skin cancer cells, respectively, were observed. In the antibacterial activity, minimum inhibitory concentrations (MICs) of 2–64 μg/mL were observed against studied pathogenic strains. The antimicrobial activity of Ti/BTB-MOF was higher than that of penicillin and gentamicin. Therefore, the synthesized Ti/BTB-MOF could be introduced as a suitable bioactive candidate.

Complete Blood Count Reference Intervals for Children Aged Less Than 1 to 12 Years in the Northern Region of Ghana

Reliable laboratory diagnostic results are key for evaluating and improving children’s health. To interpret these results, child-specific reference intervals (RIs), which account for constant biological changes and physiological development with sex and age, are required, as recommended by the Clinical and Laboratory Standards Institute (CLSI). This study presents age- and sex-specific reference intervals for complete blood count (CBC) parameters in children (<1–12 years old) in the Northern Region of Ghana. In this cross-sectional study, 600 healthy children from randomly sampled schools in Tamale (the Northern Region) were recruited and screened. Data from 388 eligible children were used to nonparametrically determine the reference intervals of CBC parameters at the 2.5th and 97.5th percentiles. The CBC reference intervals were compared for variations in sex and age groups using the Wilcoxon rank-sum test. There were no statistically significant differences in most CBC parameters by sex (RBC, Hb, HCT, MCH, RDW (CV/SD), WBC, LYM#, MON#(%) NEU#(%), EOS#(%), and BAS#(%); p>0.05) and age group (RBC, MCV, RDW (CV/SD), WBC, LYM#, MON#(%) NEU#(%), EOS#(%), and BAS%; p>0.05). However, there were observable differences between this locally established CBC reference interval and that used for children at Tamale Teaching Hospital (manufacturer’s RIs). This study emphasises the importance of determining reference intervals representative of the local child population and incorporating them into the current reporting system of laboratories in the Northern Region to ensure the provision of effective and efficient healthcare services.

Performance of modified colistin broth disc elution vis-a-vis broth microdilution method for susceptibility testing of Enterobacterales

Objectives: Recently, Clinical and Laboratory Standards Institute (CLSI) has approved colistin broth disc elution (CBDE) to be a supplemental test. This requires multiple discs and tubes to get the desired concentrations of colistin -1, 2, and 4 µg/mL and 10 mL volume of cation-adjusted Mueller–Hinton broth for a single isolate. The present study was aimed to evaluate the performance of CBDE in a microtiter plate format modified (mCBDE) with the reference method broth microdilution (BMD) for detection of colistin resistance in carbapenem-resistant Enterobacterales (CRE) isolates. Materials and Methods: One hundred and sixty non-duplicate clinical CRE isolates (May 2021–April 2022) were simultaneously subjected for BMD and mCBDE. For mCBDE, colistin 10 µg discs and Mueller–Hinton broth no-2 control cations were procured from HiMedia, Mumbai, and drug concentrations were prepared following CLSI-M100Ed31. Results of mCBDE were compared with reference BMD (Minimum inhibitory concentration [MIC] ≤2 µg/mL – intermediate and ≥4 µg/mL – resistant). Statistical Analysis: The performance of mCBDE was compared with BMD and expressed in terms of Categorical, essential agreement (EA), very major error (VME), and major error (ME). The sensitivity and specificity were calculated using Fisher’s contingency Table. Results: Of the 160 CRE isolates, 152 had exactly the same minimal inhibitory concentration (MIC) in both the tests with four isolates having higher and four having lower colistin MIC by mCBDE, giving a major error of 2.1% and VME of 5.5%. Categorical and essential agreement of mCBDE were 97.5% and 98.7%, respectively. Conclusions: mCBDE is an easy, economical, and reliable alternative test for determining colistin susceptibility for CRE isolates. Further, large-scale study is needed to strengthen our observation.

Microbiological Assessment of Commercial Antibiotic Discs from selected Pharmacies in Bwari Area Council, Abuja, Nigeria

Aims: To assess the potency of two brands of locally prepared antibiotics against two bacterial species. Place and Duration of Study: Department of Microbiology, Veritas University, Abuja, Nigeria, between September 2021 and February 2022. Methodology: We purchased two brands of antibiotics and obtained our test isolates from General hospital in Bwari, Abuja. The test isolates were characterized both routinely and molecularly by Sanqer sequencing method to confirm the species. Agar diffusion method was employed to assess the effectiveness of the antibiotics on the test isolates. Finally, the results were compared with the Clinical and Laboratory Standards Institute (CLSI).  Results: The isolates obtained from the Bwari Genera l Hospital were characterized routinely and molecularly to identify the species. Disc - agar diffusion modified method was employed while the potencies of the discs were compared with approved standards of the Clinical and Laboratory Standards Institute (CLSI). The two brands which are locally made and available in multidisc panels, demonstrated performances against Staphylococcus sciuri and Proteus mirabilis but showed significant variations in concentrations and in their inhibitory zone diameters. Both brands containing Tarivid (10 µg) produced only a small zone against Proteus mirabilis, while Streptomycin (30 µg) and Gentamycin (10 µg) exhibited unreadable zones against Staphylococcus sciuri. Unreadable zones of inhibition, which implies large zones that merged together and exceeded plate diameters at the time of reading; were common with both brands and can lead to mis-information, increase in the susceptibility of resistant organisms and eventually, drug abuse. Conclusion: The result of this assessment shows, that there is gross variation in these commercially available but locally prepared antibiotic discs. The disparity in the types of antimicrobial agents and their concentrations will pose a delicate problem to clinical microbiologists and subsequently in the diagnosis, administration of antibiotics and the likelihood of drug resistance.

New plasma LC-MS/MS assays for the quantitation of beta-amyloid peptides and identification of apolipoprotein E proteoforms for Alzheimer's disease risk assessment.

Early detection of Alzheimer's disease (AD) represents an unmet clinical need. Beta-amyloid (Aβ) plays an important role in AD pathology, and the Aβ42/40 peptide ratio is a good indicator for amyloid deposition. In addition, variants of the apolipoprotein E (APOE) gene are associated with variable AD risk. Here, we describe the development and validation of high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for plasma Aβ40 and Aβ42 quantitation, as well as apolipoprotein E (ApoE) proteotype determination as a surrogate for APOE genotype. Aβ40 and Aβ42 were simultaneously immunoprecipitated from plasma, proteolytically digested, and quantitated by LC-MS/MS. ApoE proteotype status was qualitatively assessed by targeting tryptic peptides from the ApoE2, ApoE3, and ApoE4 proteoforms. Both assays were validated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Within-run precision was 1.8%-4.2% (Aβ40), 1.9%-7.2% (Aβ42), and 2.6%-8.3% (Aβ42/40 ratio). Between-run precision was 3.5%-5.9% (Aβ40), 3.8%-8.0% (Aβ42), and 3.3%-8.7% (Aβ42/40 ratio). Both Aβ40 and Aβ42 were linear from 10 to 2500 pg/mL. Identified ApoE proteotypes had 100% concordance with APOE genotypes. We have developed a precise, accurate, and sensitive high-throughput LC-MS/MS assay for plasma Aβ40, Aβ42, and proteoforms of ApoE.

Evaluation of Antibiotics by Disk Diffusion and Minimum Inhibitory Concentration Breakpoints in Urinary Tract Infections

Antibiotic resistance (ABR) has made it more challenging to treat uropathogenic organisms. It is impossible to compromise antimicrobial susceptibility testing (AST), which is essential and has a significant impact on infection treatment strategies. Although labor-intensive and technically challenging for everyday laboratory use, the agar dilution technique is appropriate for monitoring and assessing novel antimicrobials. Objective: To determine the minimum inhibitory concentration (MIC), Agar dilution technique and disk diffusion as susceptibility test methodologies. Methods: This study was carried out at Khyber Girls Medical College (KGMC) Peshawar. Keeping in view the Clinical and Laboratory Standards Institute (CLSI) guidelines AST was executed. BIOMÉRIEUX® API® kits and gram staining were utilized for identification of bacteria. The disk diffusion was performed using Thermo ScientificTM OxoidTM antibiotic discs of Co-trimoxazole, Levofloxacin, Nitrofurantoin and Fosfomycin. The MIC and zone of inhibitions for disk diffusion were noted according to the CLSI protocol. Results: 158 culture positive samples were isolated out of 680 total received. Esherechia Coli (E. coli) (74.1%) being the most isolated organism. In comparison of disk diffusion and agar dilution, categorical agreement for Levofloxacin, Cotrimoxazole, Nitrofurantoin and Fosfomycin were (82.28%, 72.15 %, 87.97% and 82.28%) respectively. Kappa coefficients of (0.64, 0.43, 0.57 and 0.37) (p < 0.0001) were calculated for Levofloxacin, Co-trimoxazole, Nitrofurantoin, and Fosfomycin respectively, revealing considerable level of agreement for these antibiotics. Conclusions: It was concluded that Agar dilution is more precise than disk diffusion but being more labor intensive and technical. Disk diffusion can still produce significantly accurate results with less resource consumption.

Antiphospholipid Syndrome and Lupus Anticoagulant: Where do we stand today ?

Abstract Lupus Anticoagulant (LAC) is a group of auto antiantibodies that interfere with phospholipid dependent tests like Activated Partial Thromboplastin time (APTT) and dilute Russell Viper venom time (dRVVT) on in vitro basis. LA, anti cardiolipin antibodies (aCL) IgG/IgM, and anti-β2 glycoprotein I antibodies [aβ2GPI]IgG/IgM are three diagnostic criteria for laboratory diagnosis of anti-phospholipid antibodies (aPL). The circulating aPLs in the clinical setting of hypercoagulability state or adverse pregnancy outcomes is termed as anti-phospholipid syndrome (APS). The laboratory diagnosis of APS is complicated, it includes constellation of tests like solid phase immunoassays for measurement of aCL and a β 2GPI by and coagulation based assays for detection of LAC. LAC diagnosis is especially challenging in the setting of anti-coagulation therapy, numerous modifications to circumvent this interference have rendered success. The Thrombin generation assays [TGA] for LAC detection and estimation of LAC pathogenicity are available but are yet to be accepted as routine laboratory tests. The medley of assays like Enzyme-linked immunosorbent and chemiluminescent assays are available for detection of aCL and aβ2GPI but due to the non-availability of universal calibrators and standards there is lack of harmonization between various solid phase assays. In this article we intend to highlight new recommendations of laboratory diagnosis of LAC with special emphasis on diagnosis in the setting of pregnancy and anti-coagulation. For risk assessment in APS other non-criteria aPL like as anti-domain β2 glycoprotein I and anti-phosphatidyl serine/prothrombin antibodies are under evaluation. There is an ongoing quest towards harmonization of detection of LAC, thus there has been succession of guidelines to meet the challenges and incorporate newly found knowledge every time, namely International Society for Thrombosis and Hematology- Scientific Standardization Committee (ISTH-SSC) guidelines, followed by British Committee for Standards in Hematology (BCSH), Clinical and Laboratory Standards Institute (CLSI) guidelines 2014 and finally guidelines/updates issued by ISTH- SSC in 2018.

Investigation of the Resistance of Some Disinfectant Active Substances in ESBL-Producing Enterobacteriaceae

In this study, a total of 200 samples, including 100 neck skin and 100 cecum samples, were collected and analyzed from various poultry slaughterhouses on different sampling days. ESBL-producing Enterobacteriaceae were isolated and ESBL production was confirmed phenotypically by combined disk diffusion and E-test gradient strips. While ESBL production was confirmed in 10 (10%) of 100 neck skin samples, no significant ESBL production could be confirmed in 100 cecal samples. The broth microdilution method of Clinical and Laboratory Standards Institute (CLSI) was used to determine the resistance profiles against benzalkonium chloride (BC), cetylpyridinium chloride (CPC), N-alkyl dimethyl benzyl ammonium chloride (ADBAC) and potassium peroxymonosulfate (PPMS) disinfectants in 10 neck skin isolates with confirmed ESBL production. In the study, it has been determined that MIC50 and MIC90 values were respectively ADBAC (8 and 16 mg/L), BC (16 and 32 mg/L), CPC (16, and 32 mg/L), PPMS (≥ 1024 mg/L). The impacts of Enterobacteriaceae strains on food safety and public health are significant; Disinfectant resistance can lead to increased transmission of antibiotic-resistant bacteria, leading to serious infections in humans that are difficult to treat. For that reason, it is of great importance to develop effective control methods, including appropriate disinfectant use, hand hygiene and appropriate isolation measures, to prevent the spread of disinfectant resistant Enterobacteriaceae strains in food production systems.

Using the CLSI rAST breakpoints of Enterobacterales in positive blood cultures

ObjectivesThe objective of this study was to provide the clinic with rapid and accurate results of antimicrobial susceptibility testing for the treatment of patients with bloodstream infections. To achieve this, we applied the Clinical and Laboratory Standards Institute (CLSI) blood culture direct rapid antimicrobial susceptibility test (rAST) to assess the susceptibility of the most common Enterobacterales found in blood cultures. MethodsIn this study, we utilized the CLSI blood culture direct rapid antimicrobial susceptibility test to assess the susceptibility (rAST) of the most common Enterobacterales present in blood cultures. We chose this method for its simplicity in analysis, and our aim was to predict minimum inhibitory concentrations (MICs) using the rAST. As a benchmark, we assumed that Broth Macrodilution method (BMD) results were 100% accurate. For data evaluation, we employed the terms categorical agreement (CA), very major errors (VME), and major errors (ME). ResultsOur findings demonstrate that the CLSI rAST method is reliable for rapidly determining the in vitro susceptibility of Enterobacterales to common antimicrobial drugs in bloodstream infections. We achieved a concordance rate of 90% in classification within a 10-hour timeframe. We identified a total of 112 carbapenem-resistant Enterobacterales (CRE) strains, and there was no significant difference in the detection rate of CRE at 6, 10, and 16 hours. This suggests that CRE can be identified as early as 6 hours. ConclusionThe CLSI rAST is a valuable tool that can be utilized in clinical practice to quickly determine the susceptibility of Enterobacterales to antimicrobial drugs within 10 hours. This capability can greatly assist in the clinical management of patients with bloodstream infections.

Amoxicillin-clavulanate Breakpoints Against Enterobacterales: Rationale for Revision by the Clinical and Laboratory Standards Institute.

Amoxicillin-clavulanate (AMC) is among the most frequently prescribed antibiotics globally. It has broad antibacterial activity against gram-positive, gram-negative, and anaerobic bacteria, and has been utilized to treat infections caused by a broad range of pathogens. AMC breakpoints against Enterobacterales were initially set in the 1980s but since then increases in antibiotic resistance, advances in pharmacokinetic (PK)/pharmacodynamic (PD) analyses, and publication of additional clinical data prompted a reassessment by the Clinical and Laboratory Standards Institute (CLSI) Subcommittee on Antimicrobial Susceptibility Testing. Based on this contemporary reappraisal, the CLSI retained the Enterobacterales breakpoints but revised comments regarding dosing associated with use of the AMC breakpoints in the 2022 supplement of M100. This viewpoint provides insight into the CLSI breakpoint reevaluation process and summarizes the data and rationale used to support these revisions to the AMC Enterobacterales breakpoint.

Comparison of the sigma metrics using the total error allowable algorithm with variation of bias source

Sigma Metrics, as a quality indicator, have been widely applied in clinical laboratories to assess the performance of analytical methods. Described in the document Clinical and Laboratory Standards Institute (CLSI) EP15- A3, the use of target values can be sourced from certified reference standards, survey materials from the Proficiency Testing (PT)/External Quality Assessment (EQA), materials used in inter-laboratory quality control programs and internal quality control materials with predetermined targets. This research aims to determine whether there is a difference in the sigma metrics between the bias derived from the manufacturer's target value and those from the peer group source in the External Quality Assurance Services (EQAS) program. The research methodology employed is descriptive comparative analysis, utilizing the results of material inspection data for 15 internal quality control parameters of Clinical Chemistry over a span of 2 years at the Pramita Laboratory in Bandung. The calculation of the sigma metrics commences with computing the coefficient of variation (CV), and the appropriate Total Error aalowable (Tea) sources for each parameter are determined beforehand using the TEa algorithm. The research findings indicate a difference between the sigma metrics derived from the manufacturer's target value and those from the EQAS-peer group target value, accounting for 33% or 10 parameters out of the total parameters with 2 levels of inspection are calculated on the sigma scale. However, in 67% or 20 parameters out of the total parameters, no such difference is observed. Bias associated with the target value from the manufacturer and the EQAS peer group shows no significant difference, suggesting that the laboratory can utilize pre-existing target values confidently.

Antimicrobial Resistance Profile of Uropathogenic Escherichia coli Isolated in Garaku, Keffi and Mararaban-Gurku Healthcentres, Nasarawa-West Senatorial District, Nasarawa State, Nigeria

Aims: This Study is aimed at studying the Antimicrobial Resistance profile of Uropathogenic Escherichia coli isolated in Garaku, Keffi and Mararaban-gurku Healthcentres, Nasarawa-West Senatorial District, Nasarawa State, Nigeria. Study Design: is a survey research. Place and Duration of Study: Department of Microbiology, Nasarawa State University, Keffi, between June, 2021 and January,2024. Methodology: 384 urine samples of patients with suspected cases of UTIs were collected and E. coli was isolated and identified using standard microbiological methods. Antimicrobial Susceptibility Testing of the isolates was carried out in accordance with the Clinical and Laboratory Standards Institute (CLSI) method. Results: The occurrence of E. coli was 92(24%). The isolates were generally more resistant to Ampicillin (AMP) with 75.0%.  Streptomycin (S) 70.7% Cefotaxime (CTX) 68.5% but less resistant to Ciprofloxacin (9.8%) followed by Gentamicin 17.4%. The commonest resistance phenotype was AMC-AMP-CAZ-CTX-S-SXT (5.4%). Most of the isolates showed multiple antibiotic resistance (MAR) Index of above 0.2 with the commonest MAR Index at GHK, GHG and MCM-g of 0.7(21.4%), 0.4(28.0%) and 0.3(23.1%) respectively. The antibiotics resistant E. coli isolates were grouped into different categories of antibiotic resistance namely: Non Multiple Drug Resistance (NMDR); Excessive Drug Resistance (XDR); Multiple Drug Resistance (MDR) and Pan Drug Resistance (PDR). The occurrence of classes of antibiotic resistance was of the orderMDR75(81.5%) ˃ NMDR9(9.8%) ˃XDR7(7.6%) > PDR 1(1.1%). Conclusion: with the results from this research, E. coli being non susceptible to many antimicrobials is a clear indication that, prescription of these antimicrobials by physicians and usage by patients must be done with diligence and utmost care and vigorous campaign by stakeholders must be step up to bring to the barest minimum incidences of Antimicrobial resistance.

Comparison of broth microdilution and E-test for susceptibility of MRSA to vancomycin

Introduction: Healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) developed soon after the introduction of methicillin. These bacteria have shown resistance to multiple drugs and therefore vancomycin became the antibiotic of choice for treatment of MRSA infections. Vancomycin is a bactericidal antibiotic that acts by inhibiting bacterial cell wall synthesis. This study aimed to compare broth microdilution (BMD) and E-test in determining vancomycin minimum inhibitory concentration (MIC) against MRSA.Methods: A total of 30 clinical isolates of MRSA were acquired from Colombo South Teaching Hospital, Sri Lanka. These MRSA strains were identified by cefoxitin disk diffusion and the vancomycin MIC was determined through BMD and E-test. The Kruskal-Wallis test was applied to test if the samples originated from the same distribution with post hoc determination of the correlation between methods using the Mann-Whitney U test.Results: All 30 MRSA isolates were 100% vancomycin susceptible (≤2 μg/mL) irrespective of methodology, according to the Clinical and Laboratory Standards Institute (CLSI) established breakpoints. However, the E-test MIC values were 1 to 2 dilutions higher than those of BMD. A statistically significant difference between vancomycin MICs of BMD and E-test (p = <0.00001) was calculated which indicated a difference in accuracy.Conclusion: Due to cost, convenience, and the ability to detect vancomycin intermediate Staphylococcus aureus (VISA), exploring the possibility of using E-test as an alternative to BMD is worthwhile.

Antimicrobial Resistance Profile and Extended Spectrum Beta-lactamase Resistance Genes in Escherichia coli from Patients in General Hospital, Karshi, Abuja, Nigeria

Aims: This study investigated the antimicrobial resistance profile and extended spectrum beta-lactamase resistance genes in Escherichia coli of from urine of patients sourced from General Hospital, Karshi, Abuja, Nigeria. Study Design: Cross-sectional study. Place and Duration of Study: Department of Microbiology, Nasarawa State University, Keffi, between August 2022 and February 2023. Methodology: A total of 120 samples were collected from patients. Escherichia coli was isolated from the samples using standard microbiological methods. Antibiotic susceptibility testing was evaluated as described by the Clinical and Laboratory Standards Institute (CLSI). The detection of ESBL production in E. coli isolates was carried out using double disc synergy test.  In addition, molecular detection of ESBL genes was carried out using Polymerase Chain Reaction (PCR) method. Results: The prevalence of E. coli was 17.5% (21/120), out of which two (2) of the positive isolates (9.5%) were male, and 19 (90.5%) female. Antibiotic resistances in the isolates in decreasing order were as follows: sulphamethoxazole / trimethoprim (SXT: 81.0%), amoxicillin/clavulanic acid (AMC: 61.9%), ofloxacin (OFX: 66.7%), cefotaxime (CTX: 53.4%), gentamicin (CN: 42.9%), ceftriaxone (CRO: 33.3%), imipenem (IPM: 33.3%), meropenem (MOR: 42.4%), nitrofurantoin (NET: 20.3%) and ciprofloxacin (CIP: 23.8%). The commonest antibiotic resistant resistance phenotype was CIP-OFX-SXT (23.8%). Multiple antibiotic resistance (MAR) was observed in 90.5% (19/21) of the isolates, with the common MAR index being 0.3 (23.8%). Six of the twenty one beta-lactam resistant isolates (28.5%) were confirmed ESBL producers. The 6 ESBL positive isolates carried bla genes as follows: blaTEM (1/6, 16.7%) and blaCTX-M (1/6, 16.7%). blaSHV only was not found in any of the isolates. Conclusion: The E. coli isolates from urine of patients in General Hospital, Karshi, Abuja, Nigeria was less resistant to ciprofloxacin, nitrofurantoin, meropenem and imipenem. This implies that the antibiotics are useful in the treatment of infection caused by E. coli. Also, ESBL-positive E. coli isolates harbored ESBL genes, with blaCTX-M and blaTEM as the most common.

In Vitro Activity of Dalbavancin and Fourteen Other Antimicrobial Agents Against Toxigenic Clostridioides Difficile Clinical Isolates in a Greek Tertiary-Care Hospital.

Clostridioides difficile is a major cause of healthcare-associated diarrhea worldwide. For years, metronidazole and vancomycin were considered the standard treatment for C. difficile infection (CDI). However, they are increasingly being associated with treatment failure and recurrence. In this study we investigated the in vitro activity of dalbavancin and fourteen other antimicrobials against 155 toxigenic C. difficile isolates originating from patients with C. difficile-associated diarrhea. Antimicrobial susceptibility was evaluated by the MIC Test Strip and the results were interpreted using both the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial susceptibility Testing (EUCAST) breakpoints. C. difficile isolates were fully susceptible to metronidazole, vancomycin, amoxicillin/ clavulanate, piperacillin/tazobactam, and tigecycline. All isolates were dalbavancin susceptible by the CLSI breakpoint (≤ 0.25 μg/ml) compared with 97.4% susceptibility by the EUCAST breakpoint (≤ 0.125 μg/ml). Dalbavancin demonstrated significantly lower MIC50 and MIC90 values compared to vancomycin (0.047 vs. 0.38 and 0.125 vs. 0.5, respectively, p < 0.001). Resistance rates to penicillin, ampicilin, cefoxitin, imipenem, meropenem, clindamycin, moxifloxacin, chloramphenicol, and tetracycline were 20%, 14.2% , 100%, 75.5%, 0.6%, 51%, 36.1%, 3.2%, and 14.8%, respectively. Multidrug-resistant (MDR) phenotypes were detected among 41.3% of the isolates. Dalbavancin exhibited potent activity against the isolates tested. As C. difficile is an important healthcare-associated pathogen, continued surveillance is required to monitor for development of resistance.

Antibacterial and antibiofilm activity of cinnamic acid against Multidrug-resistant bacteria isolated from Ramadi Teaching hospitals, Iraq

Various bacterial pathotypes remain a significant public health concern due to their pathogenicity and antimicrobial resistance. Moreover, the ability of bacteria to form biofilms can hinder host defense and antimicrobial eradication, leading to additional resistance. This study aimed to estimate the cinnamic acid's anti-biofilm activity against biofilms-producing bacteria. From October to November 2023, various clinical isolates were obtained from the Bacteriology Unit, Ramadi Teaching Hospital, Iraq. All isolates were identified using a conventional and automated VITEK-2 compact system. Based on the Clinical and Laboratory Standards Institute (CLSI) guidelines and the European Committee on Antimicrobial Susceptibility Testing (EUCAST), the Kirby- Bauer disk diffusion and AST were performed for antimicrobial susceptibility. Biofilm formation was estimated using a 96-well Enzyme-linked immunosorbent assay (ELISA) reader. Based on the antimicrobial susceptibility test,  all bacterial isolates showed antibiotic resistance, including Kleibsella  pneumonieae, Psuedomonas aeruginosa, Escherichia coli and Staphylococcus aureus. The data showed that MICs of cinnamic acid against bacteria were 125 μg /ml.The biofilms formed by all isolates exhibited strong strength (OD570 : 0.078-0.099). Cinnamic acid demonstrated significant inhibition of biofilm production in Multidrug resistance (MDR) bacteria (P-value = 0.0236). The results indicated that Cinnamic acid could be a promising anti-infective agent based on its ability to inhibit MDR-bacterial infections through biofilm formation.  

Evaluation of the Vitek 2 system for antifungal susceptibility testing of Candida auris using a representative international panel of clinical isolates: overestimation of amphotericin B resistance and underestimation of fluconazole resistance.

Although the Vitek 2 system is broadly used for antifungal susceptibility testing of Candida spp., its performance against Candida auris has been assessed using limited number of isolates recovered from restricted geographic areas. We therefore compared Vitek 2 system with the reference Clinical and Laboratory Standards Institute (CLSI) broth microdilution method using an international collection of 100 C. auris isolates belonging to different clades. The agreement ±1 twofold dilution between the two methods and the categorical agreement (CA) based on the Centers for Disease Control and Prevention's (CDC's) tentative resistance breakpoints and Vitek 2-specific wild-type upper limit values (WT-ULVs) were determined. The CLSI-Vitek 2 agreement was poor for 5-flucytosine (0%), fluconazole (16%), and amphotericin B (29%), and moderate for voriconazole (61%), micafungin (67%), and caspofungin (81%). Significant interpretation errors were recorded using the CDC breakpoints for amphotericin B (31% CA, 69% major errors; MaEs) and fluconazole (69% CA, 31% very major errors; VmEs), but not for echinocandins (99% CA, 1% MaEs for both micafungin and caspofungin) for which the Vitek 2 allowed correct categorization of echinocandin-resistant FKS1 mutant isolates. Discrepancies were reduced when the Vitek 2 WT-ULV of 16 mg/L for amphotericin B (98% CA, 2% MaEs) and of 4 mg/L for fluconazole (96% CA, 1% MaEs, 3% VmEs) were used. In conclusion, the Vitek 2 system performed well for echinocandin susceptibility testing of C .auris. Resistance to fluconazole was underestimated whereas resistance to amphotericin B was overestimated using the CDC breakpoints of ≥32 and ≥2 mg/L, respectively. Vitek 2 minimun inhibitory concentrations (MICs) >4 mg/L indicated resistance to fluconazole and Vitek 2 MICs ≤16 mg/L indicated non-resistance to amphotericin B.

High prevalence of ST5-SCCmec II-t311 clone of methicillin-resistant Staphylococcus aureus isolated from bloodstream infections in East China

ObjectivesMethicillin-resistant Staphylococcus aureus (MRSA) is a challenging global health threat, resulting in significant morbidity and mortality worldwide. This study aims to determine the molecular characteristics and antimicrobial susceptibility of 263 MRSA isolates in Zhejiang Province, east China.MethodsFrom 2014 to 2019, a total of 263 MRSA isolates from bloodstream infections (BSIs) were collected from 6 hospitals in 4 cities in Zhejiang province, east China. Antimicrobial susceptibility tests were conducted according to the guidelines set forth by the Clinical and Laboratory Standards Institute (CLSI). To characterize and analyze these isolates, multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) typing and virulence genes gene profiles were performed.ResultsThe most predominant clone was ST5-SCCmec II-t311, which accounted for 41.8% (110/263), followed by ST59 (44/263, 16.7%). Compared with non-ST5-II-t311 isolates, ST5-II-t311 isolates were more resistant to erythromycin, tetracycline, levofloxacin, moxifloxacin, and ciprofloxacin, but more susceptible to clindamycin. Moreover, the rates of multidrug resistance were higher in ST5-II-t311 isolates compared to the non-ST5-II-t311 isolates. In comparison to the non-ST5-II-t311 isolates, ST5-II-t311 isolates showed no significant difference in virulence genes detected.ConclusionsMRSA ST5-II-t311 clone has become the most predominant clone in Zhejiang Province, east China and has higher rates of multidrug resistance than other isolates, that should be kept in mind when treating BSI. Moreover, MRSA ST59 clone shows an upward trend and has begun to spread into hospitals. Our findings highlight the importance of epidemiological studies of S. aureus carriage in the eastern region.

Cefazolin as a predictor of urinary cephalosporin activity in indicated Enterobacterales.

Traditionally, cephalothin susceptibility results were used to predict the susceptibility of additional cephalosporins; however, in 2013-2014, the Clinical and Laboratory Standards Institute (CLSI) revisited this practice and determined that cefazolin is a more accurate proxy than cephalothin for uncomplicated urinary tract infections (uUTIs). Therefore, a cefazolin surrogacy breakpoint was established to predict the susceptibility of seven oral cephalosporins for Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis in the context of uUTIs. Clinical microbiology laboratories face several operational challenges when implementing the cefazolin surrogacy breakpoint, which may lead to confusion for the best path forward. Here, we review the historical context and data behind the surrogacy breakpoints, review PK/PD profiles for oral cephalosporins, discuss challenges in deploying the breakpoint, and highlight the limited clinical outcome data in this space.

Novel automated antifungal susceptibility testing system for yeasts based on dual-detection algorithm of turbidimetry and colorimetry.

Introduction. The increasing prevalence and growing resistance of fungi present a significant peril to public health. There are only four classes of antifungal medicines available today, and few candidates are in clinical trials.Hypothesis/Gap Statement. Rapid and sensitive diagnostic techniques are lacking for most fungal pathogens, and those that do exist are expensive or hard to obtain.Aim. This study aimed to evaluate the feasibility of a novel automated antifungal susceptibility testing system, Fungus AST, in comparison to the broth microdilution method (BMD) recommended by the Clinical and Laboratory Standards Institute (CLSI).Methodology. A total of 101 clinical Candida spp. isolates were collected from the Zengcheng Branch of Nanfang Hospital and subjected to antifungal susceptibility testing. Antifungal susceptibility was assessed using the Fungus AST method and the BMD.Results. In this study, we introduce a novel automated antifungal susceptibility testing system, Fungus AST, which detects the turbidity and/or colour intensity of microdilution wells using a four-wavelength detection technology in real time and is designed to match the growth characteristics of strains over time. Based on our analysis, all reportable ranges of Fungus AST were suitable for clinical fungal isolates in PR China. Within ±twofold dilutions, reproducibility was 100 %. Considering the BMD as a referenced method, ten antifungal agents (anidulafungin, caspofungin, micafungin, fluconazole, voriconazole, posaconazole, itraconazole, amphotericin B, 5-flucytosine and nystatin) showed an essential agreement of >95 %. The category agreement of five antifungal agents (anidulafungin, caspofungin, micafungin, fluconazole and voriconazole) was excellent at >90 %. One Candida albicans isolate and voriconazole showed a major error (ME) (1.7 %), and no other ME or very ME agents were found.Conclusion. Given the above, it can be argued that the utilization of Fungus AST is a discretionary automated approach. More improvements are needed in Fungus AST compared to the BMD system for a wider range of clinical isolates, including different types of fungi.