Clinical And Laboratory Standards Institute Guidelines Research Articles

Observational Study on Adherence to modified Clinical and Laboratory Standard Institute H3-A6 Guidelines on Blood Sampling Procedures

Background: The accuracy of clinical laboratory results is paramount for effective patient diagnosis and treatment. However, errors in the pre-analytical phase, particularly during phlebotomy, significantly impact the reliability of laboratory data. With the Clinical and Laboratory Standards Institute (CLSI) guidelines serving as a benchmark for best practices in blood collection, adherence to these standards is crucial for minimizing errors. Objective: This study aimed to assess compliance with modified CLSI guidelines in phlebotomy procedures within a healthcare setting and identify the most prevalent errors in the pre-analytical phase that could compromise patient safety and the integrity of laboratory results. Methods: Conducted at the Combined Military Hospital Lahore from January to March 2023, this observational study scrutinized phlebotomy practices across three distinct healthcare settings: emergency department, outpatient department, and clinical wards. A structured checklist, adapted from the European Federation for Clinical Chemistry and Laboratory Medicine (EFLM) guidelines and aligned with local modifications, was employed to evaluate compliance in 20 specific areas of phlebotomy practice. A total of 285 phlebotomies were observed by specialized staff, with data analyzed using SPSS version 25 to compare error frequencies and compliance rates across different settings and among various healthcare professionals. Results: The study revealed an overall compliance rate of 88% for identified request forms, 76% for patient identification according to local guidelines, and 66% for proper hand sanitization. However, significant errors were identified in checking for potential complications of venipuncture (34% compliance), with tubes being clearly under or overfilled (46% compliance), and in verifying that patients were prepared for phlebotomy (46% compliance). The error frequency varied across settings, with the highest discrepancies observed in the emergency department. Differences in compliance rates between laboratory staff and nurses were statistically significant in key areas, including patient identification (p<0.001) and tube labeling (p=0.081). Conclusion: The study highlights a critical need for improving adherence to CLSI guidelines within phlebotomy practices to enhance patient safety and the accuracy of laboratory results. Targeted educational and training interventions are essential for addressing the identified gaps in compliance, particularly in patient identification, sample volume control, and verification of patient preparation for phlebotomy.

In vitro susceptibility to fosfomycin in clinical and environmental extended-spectrum beta-lactamase producing and/or ciprofloxacin-non-susceptible Escherichia coli isolates.

Extended-spectrum beta-lactamase producing and ciprofloxacin-non-susceptible Escherichia coli are clinical and environmental issues. We evaluated the susceptibility profile of fosfomycin in non-susceptible E. coli isolated from urine and the environment. We measured the activity of fosfomycin against 319 and 36 E. coli strains from urine and environmental isolates, respectively, collected from rivers. Fosfomycin resistance profiles were investigated using the minimal inhibitory concentration (MIC), according to the Clinical and Laboratory Standards Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST) guidelines. Antibiotic susceptibility testing revealed that 5% and 6.6% of urine samples were non-susceptible to fosfomycin according to CLSI and EUCAST guidelines, respectively. The fosfomycin MIC50/90 was 0.5/4 mg/L. Of the 36 E. coli isolates from river water, 11.1% and 13,8% were non-susceptible to fosfomycin according to CLSI and EUCAST, respectively (range ≤0.25 ≥512 mg/L). All the isolates with MIC ≥512 mg/L for fosfomycin showed the fosA3 gene. Fosfomycin resistance was more frequent in the environment than in clinical samples.

Comparison of Sample Size and Rejection Limits for Lot-to-Lot Verification Using Two Different Protocols: A Cross-sectional Study

Introduction: Good laboratory practice necessitates verifying each new lot of reagents before it is put into service to ensure that the results of patient samples are consistent across different lots of reagents. Current laboratory protocols use uniform criteria such as a 10% difference, measurement uncertainty, etc., for the acceptance of Lot-to-Lot Verification (LTLV) across all parameters. Although a detailed guideline was introduced by the Clinical and Laboratory Standards Institute (CLSI), there is limited literature on laboratories using this. Aim: To compare the sample size and rejection limits for LTLV for common chemistry analytes using two different protocols. Materials and Methods: The present cross-sectional analytical study was carried out in the Department of Biochemistry, St. John’s Medical College, Bengaluru, Karnataka, India, from January 2017 to January 2019. The LTLV for reagents was done using patient and Internal Quality Control (IQC) data for common analytes such as glucose, creatinine, albumin and calcium on Siemens Dimension EXL 200 chemistry analyser. Sample size and rejection limits obtained using CLSI EP26-A guidelines were compared with one of the current protocols, which uses one sample at each concentration, usually a maximum of two at two different concentrations, and defines less than a 10% difference in value between the two lots as acceptable criteria. The quality control data was analysed, and descriptive statistics such as the coefficient of variation were used to arrive at precision. Rejection limit and sample size were directly read from the EP26-A guideline using the tables given for the same, based on the two ratios at 90% power. Results: The sample size needed was found to be the same for creatinine and albumin using both protocols, while it was higher for glucose and calcium based on EP26-A guideline. The rejection limit obtained using EP26-A guideline was different for each parameter between the two protocols. The rejection limit obtained using EP26-A was lower for all analytes as compared to the first protocol. Conclusion: Lot-to-Lot Verification (LTLV) using the CLSI guideline reinforces the fact that the sample size needed and the rejection limit for each parameter varies based on its performance and the critical difference to be detected. Hence, the practice of using a fixed sample size and fixed criteria, such as a 10% acceptable difference across all parameters, may not be appropriate.

Assessment of cefiderocol disk diffusion versus broth microdilution results when tested against Acinetobacter baumannii complex clinical isolates.

The aim of this study was to evaluate the correlation between inhibitory zones and minimum inhibitory concentrations (MICs) when testing cefiderocol against Acinetobacter baumannii complex using disk diffusion and the broth microdilution (BMD) method according to the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. Four-hundred and sixty-eight non-duplicated A. baumannii complex clinical isolates were collected from 56 hospitals of the China Antimicrobial Surveillance Network from 2019 to 2021. BMD using iron-depleted cation-adjusted Mueller-Hinton broth (ID-CAMHB) and standard disk diffusion methods were performed according to CLSI guidelines. Results were interpreted according to the CLSI and EUCAST breakpoints. Categorical agreement (CA), minor error (mE), major error (ME), and very major error (VME) were calculated for disk diffusion methods. The susceptibilities of all A. baumannii complex isolates by BMD were 98.7% (CLSI) and 97.6% (EUCAST). For all A. baumannii complex isolates, CA was 98.1% (CLSI) and 97.0% (EUCAST), with 0.9% (CLSI) and 1.9% (EUCAST) of VME, respectively. For the carbapenem-susceptible A. baumannii complex, the CA was 100%, with no mE or VME using both CLSI and EUCAST breakpoints. For carbapenem-resistant A. baumannii complex, CA was 97.5% (CLSI) and 96.2% (EUCAST), with 1.1% (CLSI) and 2.5% (EUCAST) of VME, respectively. Regarding the difficult-to-treat resistance A. baumannii complex isolates, CA was 97.6% (CLSI) and 95.7% (EUCAST), with 1.2% (CLSI) and 3.1% (EUCAST) of VME, respectively. Cefiderocol disk diffusion was difficult to assess in this study. Very few isolates were resistant to cefiderocol by BMD using CLSI breakpoint, and these were categorized as susceptible with the disk diffusion test. This study did, however, show that the main proportion of A. baumannii isolates were susceptible to cefiderocol by BMD, including carbapenem-resistant A. baumannii. IMPORTANCE Carbapenem-resistant Acinetobacter baumannii is a major global health concern due to its high prevalence and limited treatment options. Cefiderocol is the only novel Food and Drug Administration (FDA)-approved β-lactam agent for the salvage treatment of carbapenem-resistant A. baumannii infection. Currently, a commercial automated susceptibility testing panel of cefiderocol is unavailable. Both the preparation of iron-depleted cation-adjusted Mueller-Hinton broth and the performance of broth microdilution are cumbersome in routine microbiology laboratories. A disk diffusion method is convenient for cefiderocol antimicrobial susceptibility testing, but limited data are available specifically for A. baumannii clinical isolates. Moreover, the Clinical and Laboratory Standards Institute published revisions to the A. baumannii cefiderocol disk diffusion breakpoints in 2022. Hence, we evaluated the performance of cefiderocol disk diffusion compared with the reference BMD against A. baumannii clinical isolates, especially those with cefiderocol zone diameters ≤ 14 mm.

B-167 Development and Validation of Liquid Chromatography-Tandem Mass Spectrometry Method for Therapeutic Drug Monitoring of Treosulfan and Busulfan

Abstract Background Treosulfan and busulfan are alkylating agents administered as the conditioning regimen before hematopoietic stem cell transplantation. High busulfan exposure is associated with drug toxicity and low busulfan exposure with rejection or disease recurrence. There is also an association between treosulfan exposure and early toxicity, such as skin toxicity and mucositis. As both busulfan and treosulfan have narrow therapeutic ranges, therapeutic drug monitoring (TDM) is essential. Since the clinical laboratory tests for both drugs are few, a method that can simultaneously measure both drug concentrations under the same conditions would be helpful in practice. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous measurement of treosulfan and busulfan was developed and validated. Methods For preparing calibrators and quality control materials, commercialized reference standards were used for treosulfan, an active metabolite of treosulfan, S,S-EBDM, and busulfan. Treosulfan-D4 and busulfan-D8 isotopes were used as internal standards (ISs). The analytes were separated from 25 µL of plasma after protein precipitation with acetonitrile containing ISs. After centrifugation, the supernatant was diluted with 3% formic acid and then injected into the liquid chromatography system followed by tandem mass spectrometry. Chromatographic separation was performed using ACQUITY Ι-Class plus ultraperformance liquid chromatography (UPLC) system coupled with a XEVO TQ-XS mass spectrometer operated in positive ion electrospray ionization mode. The analytes and ISs were detected in the multiple reaction monitoring (MRM) mode. The limit of detection (LoD), limit of quantification (LoQ), selectivity, linearity, accuracy, precision, carryover, matrix effect, and sample stability were validated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Results Retention times for treosulfan, S,S-EBDM and IS for both analytes were 0.44 min, 0.43 min, and 0.44 min, respectively. Retention times for busulfan and IS for busulfan were 1.20 min and 1.18 min, respectively. The mass-to-charge (m/z) transitions used for quantification in the MRM mode were as follows: treosulfan 296.2 > 87.1; S,S-EBDM 200.2 > 87.1; and busulfan 264.1 > 55.1. The total run time was 4 min. The LoDs of treosulfan, S,S-EBDM, and busulfan were 0.21, 0.23, and 0.001 µg/mL, respectively. The linear range of the calibration curves of treosulfan, S,S-EBDM, and busulfan spanned concentrations of 3.13–100, 0.63–5.00, and 0.16–5.00 µg/mL, respectively. The total precision of the developed method fulfilled the analytical criteria, except for S,S-EBDM. The LC-MS/MS method was adequately selective and accurate and showed no carry-over. It provided an acceptable matrix effect for treosulfan and busulfan; however, the matrix effect could not be excluded for S,S-EBDM. Busulfan and treosulfan were stable in plasma for 2 h at room temperature. When the samples were stored at 10 °C, busulfan was stable for up to a week and treosulfan was stable for up to 3 days, and both were stable for up to a week when stored frozen at −20 °C. Conclusion We developed the LC-MS/MS method to simultaneously measure treosulfan and busulfan. The method met the validation requirements of the CLSI guidelines and showed good performance. Therefore, this method is expected to be helpful in TDM after treosulfan or busulfan treatment in clinical laboratories.

DETECTION OF CARBAPENEMASE-PRODUCING CARBAPENEM-RESISTANT IN BLOOD CULTURE ISOLATES BY MCIM AND ECIM AND ITS SUSCEPTIBILITY TO TIGECYCLINE AND MINOCYCLINE

Objective: In this study, we aimed to detect different Carbapenemase-producing carbapenem-resistant Enterobacterales (CREs) in blood isolates by phenotypic, modified carbapenem inactivation methods (mCIM) and amp; EDTA-carbapenem inactivation methods (eCIM), and also to study the susceptibility of these CREs toward Tigecycline and Minocycline. Methods: This prospective study included 100 non-duplicate Enterobacterales organisms isolated from 250 blood samples positive for Enterobacterales that showed resistance to carbapenem (Imipenem). The isolates were identified by conventional routine biochemical tests. CRE isolates were screened for Carbapenemase production by the Clinical and Laboratory Standards Institute (CLSI)-recommended, mCIM and eCIM for evidence of the production of matello-beta-lactamase. Antimicrobial susceptibility for Tigecycline and Minocycline drugs was tested by the disk diffusion method on Mueller–Hinton agar according to CLSI guidelines, and susceptibility patterns were recorded. Clinical diagnosis data were collected from the requisition forms sent to our laboratory during test procedures. Results: Out of 100 (40%) CRE isolates tested for mCIM, 34 samples showed positive results for the Carbapenemase enzyme. Among the mCIM-positive isolates, Klebsiella spp. showed the highest prevalence of 58.8% (20/34). While among mCIM-positive isolates (22/34), 64.7% were positive for eCIM (Matello beta-lactamase producer). Maximum samples had been received from the NICU ward from patients diagnosed with early-onset sepsis; 41.2% of these were Carbapenemase-producing Enterobacterales. Among CRE isolates, 86 isolates were susceptible to Tigecycline and 24 isolates were susceptible to Minocycline. 70 CRE isolates were susceptible to Tigecycline but resistant to minocycline, and all CRE isolates resistant to Tigecycline were not susceptible to Minocycline.

Tick-tock, beat the clock: comparative analysis of disc diffusion testing with 6-, 10-, and 24-h growth for accelerated antimicrobial susceptibility testing and antimicrobial stewardship.

Disc diffusion testing by Kirby-Bauer technique is the most used method for determining antimicrobial susceptibility in microbiological laboratories. The current guidelines by The Clinical and Laboratory Standards Institute (CLSI) 2022 specify using an 18- to 24-h growth for testing by disc diffusion. We aim to determine if using an early growth (6h and 10h) would produce comparable results, thus ultimately leading to reduced turnaround time. Six-hour, 10-h, and 24-h growths of 20 quality control strains and 6-h and 24-h growths of 48 clinical samples were used to perform disc diffusion testing using a panel of appropriate antimicrobial agents. Disc diffusion zone sizes were interpreted for all and comparative analyses were performed to determine categorical agreement, minor errors (mE), major errors (ME), and very major errors (VME) according to CLSI guidelines. On comparing with the standard 24h of incubation, disc diffusion from 6-h and 10-h growths of quality control strains showed 94.38% categorical agreement, 5.10% mE, 0.69% MEs, and no VMEs. Disc diffusion testing for the additional 40 clinical samples yielded a similarly high level of categorical agreement (98.15%) and mE, ME, and VME of 1.29%, 1.22%, and 0% respectively. Disc diffusion testing using early growth is a simple and accurate method for susceptibility testing that can reduce turnaround time and may prove to be critical for timely patient management.

Emerging Patterns in Antimicrobial Susceptibility Profiles of Pseudomonas SPP- Hospital Based Study

Background: There is an emerging trend of pseudomonas infections in immune-compromised patients, specifically in hospital settings. The flagship member of this family is Pseudomonas aeruginosa, which is a major infectious agent. This study assessed the distribution and susceptibility patterns of Pseudomonas species isolated from various specimens as a part of surveillance program, in order to devise antibiograms. Aim: To determine frequency & antimicrobial sensitivities of Pseudomonas species in a tertiary care hospital from Lahore. Study design: Retrospective, descriptive, cross sectional study Place & duration of study: Conducted in a tertiary care hospital in Lahore, from January 2021 to January 2022. Methods: Thirty two isolates of Pseudomonas species from different clinical specimens were isolated in microbiology section of a tertiary care hospital during a period of 13 months. MacConkey and blood agar were utilized for culturing of organisms. Gram staining, oxidase, and catalase testwere utilized for phenotypic characterization. Antibiotic susceptibility testing against anti-pseudomonal drugs was performed according to the Clinical and Laboratory Standards Institute guidelines (CLSI) 2021. Results: The inference drawn at the end of study was that, out of thirty two isolates of Pseudomonas species received and pus was the most common specimen (46.8%), followed by 18.75% from urine specimens. 62.5% of the pseudomonas species were obtained from male patients. The most affected age group was 40-60 years, followed by 1-20 years. The most sensitive options turned out to be Imipenem (65.6%) and Pipericillintazobactam (62.5%), followed by 59.3% sensitivity in Amikacin. The least sensitive options in the study isolates were Aztreonam (15.6%) and Ticarcillin clavulanic acid (25%). In 18% of pseudomonas species isolated from urine cultures, fosfomycin was 83.3%, whereas nitrofurantoin turned out to be 50% sensitive. Conclusions: The steadily rising resistance in Pseudomonas species againstavailable antibiotics optionsnecessitates their use inlife-threatening and hospital acquiredinfections. Keywords: Peudomonas, Flouroquinolones, Carbapenem

Temporal Changes in Extended Spectrum β-Lactamase Producing Organisms in Acute Care Surgery.

Extended-spectrum β-lactamase (ESBL) producing organisms are resistant to a wide range of broad-spectrum antibiotics, and their emergence is a significant driving force of antibiotic resistance. Most South-Asian countries have become hotspots for antibiotic resistance, so specifics of ESBL data are critical to tackling antibiotic resistance. We present the temporal changes in ESBL-producing organisms cultured in our tertiary care referral centre. Over a year, a historical cohort analysis was carried out at our tertiary care referral centre in Southeast Asia. Samples from patients presenting with acute surgical conditions were sent for culture and sensitivity. The phenotype of all specimens was checked using the combination disc method. Antimicrobial susceptibilities to various antibiotics were also checked as per CLSI (Clinical and Laboratory Standard Institute) guidelines. Specimens from 170 patients were analysed. The mean age was 44.73±19.89 years, and there was a female predominance of 62%. The most common organisms were Escherichia coli (70%), Klebsiella pneumoniae (18%), and Pseudomonas aeruginosa (16%). The percentage of ESBL-producing organisms was 54.7%, which is significantly higher than in previous reports. Widespread resistance was found against commonly used antibiotics, including co-amoxiclav (81.9%), ceftriaxone (75%), ciprofloxacin (47%), and levofloxacin (35.7%). Sensitivities to combination antibiotics like piperacillin-tazobactam (79.2% sensitive), cefoperazone-sulbactam (84.3% sensitive), and imipenem-cilastatin (91.1% sensitive) were also noted to be falling. The incidence of ESBL-producing organisms continues to increase at an alarming rate, which mandates strict antibiotic stewardship and amendments to local guidelines.

To Be or Not to Be Resistant: CLSI Versus EUCAST Guidelines for Staphylococcus lugdunensis

Abstract Introduction/Objective Staphylococcus lugdunensis is a coagulase-negative Staphyloccocus, but infections can be as severe as S. aureus. Performing antimicrobial susceptibility testing (AST) is paramount to appropriate treatment. Recently, the College of American Pathologists is requiring clinical labs to implement updated guidelines. We evaluated how the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines would impact our hospital’s resistance rates for S. lugdunensis. Methods/Case Report Cultures with S. lugdunensis isolated between 04/2018 to 02/2022 were included. Minimal inhibitory concentrations (MICs) (Vitek2 and Microscan Walkaway) reported (clindamycin, doxycycline, daptomycin, linezolid, and vancomycin) were interpreted using CLSI (M100Ed32) and EUCAST (v12.0) guidelines. The range of MICs (95% Confidence Interval) was calculated. Results (if a Case Study enter NA) A total of 125 isolates from 120 patients from specimen types including wound (49%), tissue (42%), blood (6%), and body fluid (3%) were identified. Five patients had multiple cultures of S. lugdunensis, four from two wound/tissue sites and one with blood and biopsy positive. For clindamycin (0.7-1.3 ug/mL), EUCAST guidelines determined two more isolates (from blood and wound, MIC=0.5 ug/mL) as resistant. Most drastically, the rate of resistance for doxycycline (0.6-1.0 ug/mL) was 11% higher per EUCAST guidelines (susceptible: 89% EUCAST vs 100% CLSI). The difference was seen in isolates > 2 ug/mL, all isolated from wound and tissue. Regardless of the guidelines used, 100% of the isolates were susceptible to daptomycin (0.4-0.5 ug/mL), linezolid (1.2-1.4 ug/mL), and vancomycin (0.6-0.7 ug/mL); 23% were resistant to oxacillin (1.7-2.0 ug/mL). Conclusion EUCAST guidelines would have reported more resistant doxycycline and clindamycin isolates at our institution. To ensure that clinical teams are utilizing AST results to provide appropriate care, a team effort consisting of the microbiology lab, infectious diseases physicians, and antibiotic stewardship committee is needed to determine which guidelines should be followed.

Incidence, Clinical Evaluation and Antibiogram of Bacterial Isolates Obtained from Burn Patients

Background: Microbiological contamination plays salient role in governing the outcome and time span in the hospital for burn victims in burn unit. Therefore, regular supervision of microbes and its resistance pattern is mandatory. The emergence of Multi Drug Resistant (MDR) bacteria provoked researchers to develop new plans to combat against the threat. Objective: To assess the infectious organisms, to know the drug resistance of isolates and ascertain the effectiveness of antibiotics against microbes that are found in burn patients. Methodology: Descriptive case series study performed and Quantitative data was collected in Jan 2019 to April 2019. Two hundred sample from burn patients admitted in Pak Italian Burn Unit Multan was collected, processed for culture, bacterial identifications and to test the antibiotics sensitivity in accordance with CLSI (clinical and laboratory standards institute) guidelines. Results: Gram negative bacteria were 189 (94.5%) and gram positive were 11 (5.5%).Organisms isolated were Proteus vulgaris 53 (26.5%) followed by Escherichia coli 47 (23.5%), Pseudomonas aeruginosa 45 (22.5%), Enterobacter 44 (22%) and Staphylococcus aureus 11 (5.5%). Imipenem 156 (78%), Meropenem150 (75%), Moxifloxacin 17(8.5%), Levofloxacin 67 (33.5%), Piperacillin/Tazobactam 123 (61.5%), Oxifloxacin 22 (11%) and Amikacin 16 (8%) were used for antibiotic sensitivity. Practical implication: Our study results can helpful for all health care personals to select new regime of antibiotics which significantly declined in all types of burn patients and also we are able to decrease the mortality rate in such patients. Assertive contagion limitations can helpful in confining the disease and proliferation of MDR pathogens. Conclusion: This study proved to be successful because antibiotic associated complications were decreased significantly as a result mortality rate declination was also noticed in all the types of burn patients. Keywords: Antibiogram, Burn, Clinical evaluations, Infection, Multi drug resistance (MDR), Mortality.

Analytical and clinical performance of a rapid magnetic immunochromatographic assay for N-terminal pro-B-type natriuretic peptide detection

ObjectiveGoldMag® immunochromatography is a novel point-of-care immunoassay platform utilizing quantitative determination of magnetic signals. A N-terminal pro-B-type natriuretic peptide (NT-proBNP) assay has been developed on this system. The objective of this study was to determine whether GoldMag® immunochromatography for NT-proBNP test fulfils the basic requirements of analytical and clinical performance so as to be used as a point-of-care test. MethodsAnalytical performance was performed for the linearity, sensitivity, precision, interference and recovery by Clinical and Laboratory Standards Institute guidelines (CLSI). Clinical evaluation was performed by testing 206 residual serum samples and comparing with Elecsys® proBNP assay (Roche Diagnostics GmbH, Mannheim, Germany). ResultsGoldMag® immunochromatography was able to differentiate concentrations of NT-proBNP from 0.000 to 8,000 pg/mL with a correlation coefficient R2 = 0.9914 and a limit of detection (LOD) of 43 pg/mL. According to statistics, this system exhibited high precision, high recovery, and no interference with lipemia, bilirubinemia, and hemolysis. Correlation analysis showed that the two assays yielded a good correlation with R2 of 0.9860. Bland-Altmananalysis showed a bias offset of 28.98 pg/mL with 95% confidence interval from 6.056 to 72.74 pg/mL for NT-proBNP. ConclusionGoldMag® immunochromatography system for NT-proBNP detection provides high accuracy, reliability, and easy operation. It can be used as a point-of-care test with a great application value in clinical management of chronic and acute heart failure patients.

P089 Candidemia: prevalence, species characterization ,and the antibiotic susceptibility profile from a tertiary care hospital in north india

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectivesThe aim of this study was to characterize the Candida spp. isolated from blood cultures and determine the antifungal susceptibility pattern of the Candida species prevalent in a tertiary care hospital in North India.MethodsThis retrospective study was conducted in Department of Microbiology of a tertiary care hospital in North India from April 2020 to March 2022. All blood cultures received in the department during this period were included in the study. Candida species isolated were identified and antifungal susceptibility testing was performed by VITEK as per standard protocol, The susceptibility pattern of 50 isolates was also performed by the broth microdilution method as per Clinical and Laboratory Standards Institute guidelines (CLSI) and the results were compared with VITEK results.ResultsOut of 21 804 blood cultures received during this period, 177 grew Candida species. Therefore, the overall prevalence of Candida species was 0.81% in our study. The incidence of bloodstream infection caused by non-albicans Candida species (80%) was higher than C. albicans (20%). Among NAC species, C. tropicalis (45%) was the most common, followed by C. pelliculosa (15%).Candidemia was predominantly observed in ICU patients. Resistance was seen in 14.1% isolates to voriconazole and fluconazole, 4.2% to flucytosine and 3.9% to caspofungin and amphotericin-B. No resistance was seen to micafungin. A total of 15% of the isolates were resistant to more than one drug.ConclusionThere was a predominance of non-albicans Candida over C. albicans. Maximum resistance was seen to voriconazole followed by fluconazole. Continuous surveillance is necessary to follow trends and monitor changes in epidemiological and resistance patterns in different geographical regions, especially in critically ill patients.

MIC Discrepancies between Parenteral and Oral Anti-Staphylococcal Beta-Lactams among MSSA

Introduction: Recent evidence has shown that oral antibiotic therapy is not inferior to IV antibiotic therapy in the treatment of complicated Staphylococcus aureus infections. Therefore, oral antibiotic therapy is now frequently prescribed in clinical practice due to cost benefit, ease of administration, decreased complication rate, and lack of need for IV access. In vitro susceptibility testing for β-lactam oral antibiotics is not routinely performed as the guidelines provided by the Clinical and Laboratory Standards Institute (CLSI) recommend using oxacillin and cefoxitin as surrogate markers. Hence, oral antibiotic susceptibilities for cephalexin and dicloxacillin are not reported and implied based on oxacillin and cefoxitin. The objective of the current study was to determine whether susceptibilities among S. aureus isolates are predictable when comparing commonly used IV and oral beta-lactams. Methods: Cefazolin, cephalexin, dicloxacillin, and oxacillin broth microdilution minimum inhibitory concentrations (MICs) were determined for 100 clinical isolates of methicillin-sensitive S. aureus by broth microdilution following CLSI guidelines. Results: Among these isolates, median MICs for cephalexin were eight-fold higher than cefazolin MICs and median MICs for dicloxacillin were four-fold less than oxacillin MICs. Ten percent of more strains studied had a major or very major error in its susceptibility reporting when cephalexin was compared to its surrogate marker oxacillin. Discussions/Conclusions: The variations in MICs observed compounded with the dosing and pharmacokinetic differences of oral versus IV β-lactam suggests that establishing breakpoints for oral β-lactam antibiotics is necessary to ensure adequate therapy is selected for the treatment of complex S. aureus infections.

Prevalence of ESBL genes in Klebsiella pneumoniae from individuals with community-acquired urinary tract infection in rural communities of Enugu State, Nigeria

Background: The emergence and spread of extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae is now a major challenge in treating urinary tract infections. This study was conducted to determine the Prevalence of ESBL genes in Klebsiella pneumoniae and the influence of these genes on the antimicrobial susceptibility patterns of the isolates, obtained from community acquired urinary tract infection in Enugu state rural communities. Method: A total of 735 clean catch mid-stream urine samples were collected from February, 2021 to June, 2021. The urine samples were cultured and Klebsiella pneumoniae were identified morphologically, biochemically, and then typed down, using PCR, Gel electrophoresis, and Sanger Sequencing by Genewiz. Antibiotic susceptibility testing was done by modified Kirby-Bauer disc diffusion method and interpretation was done following Clinical and Laboratory Standard Institute (CLSI) guidelines. ESBL screening was done by the phenotypic method, using the standard disk diffusion method, whereas the phenotypic confirmation of ESBL producers was done by the double-disk synergy method, and interpreted according to CLSI guidelines. Multiplex PCR was used to detect the genes for SHV and CTX-M while conventional linear PCR was done for TEM and blas GES type ESBL genes. Results: A total of 77 isolates were identified as Klebsiella pneumoniae, and the prevalence of positive ESBL in them were 10(12.9%). Of the 77 isolates that were tested for antibiogram, 29 isolates were multidrug-resistance (MDR). Out of the MDR isolates, 10 were ESBL positive, whereas 19 were not. Of this 29 MDR isolates, 6 were extensively-drug resistant (XDR). Of the 6 XDR isolates, 3 do not possess ESBL enzymes, whereas 3 of them do. Age range of 31-40 contributed the highest prevalence of ESBL genes (60%), whereas age range of 10-20, 21-30, and 51-70 did not produce any ESBL gene. bla TEM gene was the most prevalent ESBL resistant genes with a prevalence of 10 (100%), followed by SHV 9(90%), whereas bla GES gene and bla CTX-M have the least prevalence of 4 (40%) each. Conclusion: Higher prevalence of MDR, XDR, and ESBL-producing Klebsiella pneumoniae were observed, thus the need for public health intervention for effective prevention and control of antimicrobial resistance, and proper treatment of UTIs.

Characterisation of blaKPC-2–harbouring plasmids recovered from Pseudomonas aeruginosa ST654 and ST235 high-risk clones

The main objectives were to describe two blaKPC-2 plasmids recovered from Pseudomonas aeruginosa isolates belonging to the ST654 and ST235 high-risk clones, and to compare with complete sequences of blaKPC-2 harbouring plasmids available in public databases. Antimicrobial susceptibility was determined according to CLSI (Clinical and Laboratory Standards Institute) guidelines. Genomes were sequenced using an Illumina MiSeq platform, and blaKPC-2 plasmid sequences were achieved using MinION platform. Sequences were analysed using Unicycler and RAST. In silico predictions of the isolates sequence type (ST), antimicrobial resistance genes, plasmid replicon typing and MOB relaxases were fulfilled using bioinformatics tools. PA_2047 and PA_HdC isolates corresponded to the high-risk clones ST654 and ST235, respectively. The carbapenem resistance was mediated by KPC-2. Both blaKPC-2 harbouring plasmids, pPA_2047 and pPA_HdC, were different among them, non-conjugative and untypable by PlasmidFinder. pPA_2047 presented high identity with a Pae-13 plasmid, and these both located blaKPC-2 in Tn4401b isoform. pPA_HdC displayed a novel architecture, and the genetic context of blaKPC-2 was original. Besides the blaKPC-2 gene, resistance genes to aminoglycosides and quinolones were detected, including the novel phosphotransferase CrpP in PA_HdC. This study expands the limited knowledge about the molecular epidemiology of blaKPC-2 in P. aeruginosa from Latin America. Two novel plasmids harbouring blaKPC-2 were described that were untypable by their incompatibility group. The plasmid recovered from P. aeruginosa PA_HdC (ST235) displayed a novel architecture and an original context for blaKPC-2. On the other hand, the genetic platform carrying blaKPC-2 in P. aeruginosa PA_2047 (ST654) seems to a be a classical one.

Increase in Penicillin and multidrug resistance in Streptococcus pneumoniae (1993-2016): Report from a tertiary care hospital laboratory, Pakistan.

To determine the trend of resistance to antimicrobials in Streptococcus pneumoniae infections, and the impact of new Clinical and Laboratory Standards Institute guidelines on 1211 among meningeal isolates. The descriptive observational retrospective study was conducted at the Aga Khan University Hospital laboratory in Karachi, and comprised Streptococcus pneumoniae isolation and antimicrobial susceptibility data over a period of 24 years, from 1993 to 2016, which was compared in terms of pre-2008 and post-2008 data, which was analysed using SPSS 19. Of the 7415 non-duplicate isolates identified, 4700(63.4%) were from male patients and 2,715(36.6%) were from female patients. The overall mean age of the patients was 38±27 years. Penicillin resistance in non-meningeal isolates during the two periods was not significantly different (p>0.05), but a significant rise in penicillin resistance in meningeal isolates was observed in the second period (p<0.05). High resistance rates were observed for co-trimoxazole, tetracycline and erythromycin, and an increased trend of multi-drug resistant strains was also noted from 1999 {n=35/317(11%)} to 2016 {n=110/314 (36%)}. The emergence of multi-drug resistant strains was evident. The spike in penicillin-resistant Streptococcus pneumoniae in meningeal isolates may have been due to the revised guidelines by the Clinical and Laboratory Standards Institute.

Effect of the Different Plasmid-Mediated AmpC Beta-Lactamase Genotypes on the Phenotypic Detection of ESBL in Enterobacteriaceae Isolates.

Increasingly, Enterobacteriaceae isolates that are positive for the extended-spectrum-beta-lactamase (ESBL) by phenotypic screening tests yield a negative result when tested with the reference Clinical and Laboratory Standards Institute (CLSI) ESBL confirmatory test. The aim is to determine to what extent the different plasmid AmpC (pAmpC) genotypes could affect the CLSI ESBL confirmatory test for detection of the ESBL phenotype in ESBL/pAmpC co-producers. A total of 253 Enterobacteriaceae isolates were screened for ESBL and AmpC production according to CLSI guidelines. Out of 186 ESBL and AmpC-screen-positive isolates, 96 isolates were selected for ESBL confirmation by the combined disc diffusion test (CDDT) as well as for detection of the most common ESBL and pAmpC encoding-genes by multiplex PCR. Out of the 96 ESBL/AmpC-screen-positive isolates, all (100%) were positive for at least one of the investigated ESBL genes, and 88 (91.7%) were positive for any of the investigated pAmpC genes. CDDT correctly identified ESBL phenotype more frequently in non-pAmpC carriers than pAmpC carriers (75% vs. 52.3%). CIT alone-containing isolates were associated more with non-confirmed ESBL phenotype rather than confirmed ESBL phenotype (76.2% vs. 30.4%, p < 0.001), especially when co-harbored blaCTXM alone (76.9% vs. 33.3%, p < 0.001) or both blaCTXM/blaSHV genes (100% vs. 0%). On the other hand, DHA-carrying isolates were more associated with confirmed ESBL phenotype than with non-confirmed ESBL phenotype when co-harboring either blaCTXM alone (47.6% vs. 0%, p < 0.001) or blaCTXM/blaSHV genes (100% vs. 0%, p = 0.022) or blaCTX-M/blaTEM genes (100% vs. 0%, p = 0.03). For ESBL/pAmpC co-producers of Enterobacteriaceae, CDDT results vary with the type of pAmpC genes and with different ESBL/pAmpC genotype combinations. Therefore, the ESBL-screening test is more sensitive than the CDDT in detecting ESBL phenotype among ESBL/pAmpC coproducers of Enterobacteriaceae.

1220. Is MIC all that matters? MIC Distributions of Ceftazidime and Cefepime in Ceftriaxone-Resistant E. coli and Klebsiella spp

BackgroundThe Clinical and Laboratory Standards Institute (CLSI) lowered MIC breakpoints for many beta-lactam antibiotics to enhance detection of resistance among Enterobacterales. This shift was also meant to eliminate the need for routine testing for extended-spectrum beta-lactamases (ESBLs). The recommended treatment for ESBL-producing Enterobacterales is carbapenems. The IDSA guidelines for MDR-GN organisms recommend using ceftriaxone (CRO) resistance as a proxy for ESBL production and thus carbapenem treatment. Under CLSI guidelines, alternative beta-lactams such as ceftazidime (CAZ) and cefepime (FEP) may still be reported as susceptible and thus used by clinicians even in light of IDSA recommendations. The aim of this project was to characterize the MIC distributions of CAZ and FEP stratified by CRO susceptibility.MethodsClinical E. coli, K. pneumoniae, and K. oxytoca isolates from blood cultures in adult patients from Nov 2016-Dec 2018 that had MICs tested by the Vitek-2 automated susceptibility testing system for CRO, FEP and CAZ were identified. Descriptive statistics were used to compare MIC distributions across the antibiotics of interest (SPSS).Results573 isolates were included, of these, 17.3% were CRO resistant. Most (53%) CRO-R isolates had FEP MICs ≤2 which is considered susceptible per CLSI; 19% had FEP MICs of 4-8 which would be considered S-DD by CLSI (Figure 1A; breakpoints noted by dashed lines). Using the EUCAST breakpoint of ≤1, only 11% of CRO-R isolates would be reported as FEP-S. For CAZ, 40% of CRO-R isolates had CAZ MICs ≤4, which is considered S by CLSI. Using the EUCAST breakpoint of ≤1, only 12% of CRO-R isolates would be reported as CAZ-S (Figure 1B). Cefepime MIC Distribution for Ceftriaxone Resistant Isolates Distribution of MICs for cefepime for ceftriaxone resistant isolates with the breakpoints for EUCAST and CLSI noted with a dashed lineCeftazidime MIC Distribution for Ceftriaxone Resistant Isolates Distribution of MICs for ceftazidime for ceftriaxone resistant isolates with the breakpoints for EUCAST and CLSI noted with a dashed lineConclusionHalf of CRO-R E. coli, K. pneumoniae and K. oxytoca have FEP and CAZ MICs at or below the current CLSI breakpoints. This may lead to their use for serious ESBL infections where a carbapenem is preferred. To prevent unnecessary use, laboratories should consider suppressing FEP and CAZ susceptibilities when CRO-R or adopting more the aggressive EUCAST breakpoints for these agents. Disclosures Emily Heil, PharmD, MS, BCIDP, Nothing to disclose Kimberly C. Claeys, PharmD, GenMark (Speaker’s Bureau)

1256. Clinical Response by Minimum Inhibitory Concentrations in Carbapenem-Resistant Pseudomonas aeruginosa Infections under Cefiderocol Compassionate Use Program

BackgroundCefiderocol (CFDC) has been developed for the treatment of serious infections caused by drug-resistant aerobic Gram-negative pathogens, including carbapenem-resistant (CR) Pseudomonas aeruginosa (CRPA). The current CFDC susceptibility breakpoints for P. aeruginosa differ between US Food and Drug Administration (FDA) and Clinical and Laboratory Standards Institute (CLSI) (Table). Data characterizing the impact of CFDC minimum inhibitory concentrations (MICs) on the clinical responses of patients treated with CFDC for CRPA are sparse.MethodsWe reviewed patients treated with compassionate-use CFDC (2 g, q8h or renally adjusted dosages) for infections caused by CRPA with no alternative treatment options. CFDC minimum inhibitory concentrations (MICs) were evaluated according to CLSI guidelines in iron-depleted cation-adjusted Müller–Hinton broth for available CRPA isolates. We then assessed physician-reported clinical responses to CFDC therapy and stratified results by CFDC MIC. ResultsThere were 71 patients overall with CRPA treated with CFDC. Treatment duration ranged from 1 to 132 days. For the subset of 33 patients for whom CFDC MIC values were available, the most common infection sites were the respiratory tract (n=15), blood (n=12), and urinary tract (n=4). Patients could have had an infection at ≥1 sites and in other locations. CFDC MIC range was ≤0.03– >64 µg/mL. The modal MIC value was 2 µg/mL (n=13; Table). CRPA isolates were susceptible to CFDC in 13/33 patients (39.4%) based on the FDA breakpoint (MIC ≤1 µg/mL) and in 31/33 patients (93.9%) based on the CLSI breakpoint (MIC ≤4 µg/mL). Clinical response was reported for 15/18 patients (83.3%) who had infections with CFDC MICs of 2–4 µg/mL, organisms that are considered susceptible by CLSI but not by FDA breakpoints (Table). Clinical response was reported in 6/13 patients (46.1%) with infections with CFDC MIC ≤1 µg/mL and in 1 of 2 patients (50.0%) with CFDC MIC ≥8 µg/mL (Table). 21 (63.6%) patients survived to Day 28 and there were no trends in mortality by CFDC MIC. ConclusionClinical response rate was high for CRPA infections with CFDC MICs of 2–4 µg/mL, supporting the higher CLSI susceptibility breakpoint.Disclosures Michael J. Satlin, MD, MS, Achaogen (Consultant)Allergan (Research Grant or Support)BioFire Diagnostics (Research Grant or Support)Merck (Research Grant or Support)Shionogi (Consultant) David Fam, PharmD, Shionogi (Employee) Roger Echols, MD, Shionogi (Consultant) Christopher Longshaw, PhD, Shionogi (Employee) Miki Takemura, MS, SHIONOGI & CO., LTD. (Employee) Yoshinori Yamano, PhD, Shionogi (Employee)