CLSI Methods Research Articles

P-1101. A Comparative Analysis of Resistance Rates Among Escherichia coli and Klebsiella pneumoniae Isolates Collected from the Gepotidacin Uropathogen Global Surveillance Studies and Phase 3 Uncomplicated Urinary Tract Infection Clinical Studies

Abstract Background Gepotidacin (GEP) is a novel oral antibacterial under development for the treatment of uncomplicated urinary tract infection (uUTI) and gonorrhea. This study reports drug resistance rates for large collections of Escherichia coli (EC) and Klebsiella pneumoniae (KPN) isolates from the GEP uropathogen global surveillance studies vs isolates from 2 global GEP Phase 3 (Ph3) uUTI trials. Methods A total of (G [global]/US [a subset of global isolates from the United States]) 4481/2763 EC and 1769/1296 KPN isolates were collected from the GEP uropathogen global surveillance studies (2019–2022). From an unbiased collection of isolates from all enrolled participants with uUTI, a total of (G/US) 1159/609 EC and 114/80 KPN isolates were from the microbiological intent-to-treat population in the GEP uUTI Ph3 clinical studies. All isolates in both studies were cultured from urine collected from patients in outpatient settings. Isolate identification was confirmed by MALDI-TOF and susceptibility testing by CLSI methods was conducted at a central laboratory (JMI Laboratories [surveillance] and PPD GCL [clinical studies]). MICs for oral antibiotics used for the treatment of uUTI and drug-resistant subsets were interpreted per CLSI guidelines. Results EC: FQ-R rates were 18% and 31% for the US and 21% and 28% for G isolates from the surveillance and Ph3 studies respectively. Resistance rates for SXT and AMP were ≥ 27%; rates for AMC, NIT, FOS, and MEC were ≤ 4% for both studies. ESBL+ % range was 12–15% in both studies (Figure 1). KPN: Rates of FQ-R were similar for US (8%, 9%) and G isolates (14%,16%) from the surveillance and Ph3 studies respectively. Resistance rates for other antibacterials were 19–42% (NIT-R), 13–26% (SXT-R), and 8–16% (ESBL+ %) (Figure 2). Conclusion Drug resistance rates to many uUTI antibacterials tested were high in 1 or both studies for EC and KPN. GEP MIC90s were generally the same or 1 dilution higher than all isolates for resistant subsets of EC and KPN among both surveillance and Ph3 isolates (Table). Funding:Surveillance studies and EAGLE-2 were funded in part by GSK and in part with Federal funds from the US Office of the Assistant Secretary for Preparedness and Response, Biomedical Advanced Research and Development Authority (HHSO100201300011C). EAGLE-3 was funded by GSK. Disclosures Renuka Kapoor, PhD, GSK: Employee|GSK: Stocks/Bonds (Public Company) Deborah Butler, PharmD, GSK: Employee|GSK: Stocks/Bonds (Public Company) John Breton, MCM, GSK: Employee|GSK: Stocks/Bonds (Public Company) Cara Kasapidis, BS, GSK: Employee|GSK: Stocks/Bonds (Public Company) Derrek Brown, BS, GSK: A former agency worker at GSK Amanda Sheets, PhD, GSK: Employee|GSK: Stocks/Bonds (Public Company) Didem Torumkuney, PhD, GSK: Employee|GSK: Stocks/Bonds (Public Company) S.J. Ryan Arends, PhD, JMI: SJRA is an employee of JMI. JMI was contracted by and received financial support from GSK to conduct gepotid Rodrigo E. Mendes, PhD, JMI: RM is an employee of JMI. JMI was contracted by and received financial support from GSK to conduct gepotidac|Paratek Pharmaceuticals: Advisor/Consultant|Paratek Pharmaceuticals: Grant/Research Support Nicole E. Scangarella-Oman, MS, GSK: Employee|GSK: Stocks/Bonds (Public Company)

P-1062. In vitro Activity of Gepotidacin and Comparator Agents against a Collection of K. pneumoniae Urine Isolates from the United States During 2019-2022

Abstract Background Gepotidacin is a novel, bactericidal, first-in-class triazaacenaphthylene antibiotic that inhibits bacterial DNA replication by a unique mechanism of action, a distinct binding site and provides well-balanced inhibition (for most uUTI uropathogens) of two different type II topoisomerase enzymes. This study reports on the in vitro activity of gepotidacin and other oral antibiotics tested against contemporary Klebsiella pneumoniae clinical isolates collected from patients with urinary tract infections (UTI) in the United States as part of a gepotidacin global uropathogen surveillance study. Methods A total of 2,001 K. pneumoniae isolates were collected during 2019-2022 from 73 medical centres located in the United States. All isolates were tested for susceptibility by CLSI methods at a central laboratory (Element Iowa City). MIC results for comparator agents were interpreted per CLSI guidelines. MIC results for oral antibiotics approved for the treatment of uncomplicated UTI. Multidrug-resistant (MDR) and extended-spectrum β-lactamase (ESBL) subsets were interpreted per CLSI criteria. Results Gepotidacin (MIC50/90, 4/16 mg/L) displayed activity against 2,001 K. pneumoniae isolates (Table), with 94.9% of all observed gepotidacin MICs ≤ 16 mg/L. Susceptibility rates of oral comparators tested were ≤ 90.0% for amoxicillin-clavulanic acid (90.0%, MIC50/90, 2/8 mg/L), ciprofloxacin (86.3%, MIC50/90, 0.015/0.5 mg/L), levofloxacin (88.3%, MIC50/90, 0.06/1 mg/L), nitrofurantoin (34.0%, MIC50/90, 64/ > 128 mg/L), and trimethoprim-sulfamethoxazole (84.7%, MIC50/90, ≤ 0.12/ > 4 mg/L) (Table). Gepotidacin remained active against the 11.0% and 6.8% of K. pneumoniae isolates that displayed ESBL and MDR phenotypes, respectively, with observed MIC50/90 values of 8/32 mg/L for both. Conclusion Gepotidacin demonstrated potent in vitro activity against contemporary K. pneumoniae, including ESBL-producing and MDR isolates. Almost all oral comparator agents reported against US K. pneumoniae UTI isolates had susceptibility rates between 80-90% while the observed nitrofurantoin susceptibility rates were 34.0%, respectively. Disclosures Renuka Kapoor, PhD, GSK: Employee|GSK: Stocks/Bonds (Public Company) Didem Torumkuney, PhD, GSK: Employee|GSK: Stocks/Bonds (Public Company) Rodrigo E. Mendes, PhD, GSK: Grant/Research Support

P-1059. In vitro Activity of Gepotidacin against Klebsiella pneumoniae, Including Molecularly Characterized ESBL and Carbapenemase positive Subsets Causing Urinary Tract Infections in the United States (2019-2022)

Abstract Background Gepotidacin (GEP) is a novel, bactericidal, first-in-class triazaacenaphthylene antibacterial that inhibits bacterial DNA replication by a unique mechanism of action, distinct binding site, and provides well-balanced inhibition (for most uncomplicated urinary tract infection [uUTI] uropathogens and Neisseria gonorrhoeae) of two different type II topoisomerase enzymes. GEP recently completed two phase 3 trials for the treatment of uUTI. This study reports the activity of GEP and other oral antibacterials against K. pneumoniae, including molecularly characterized ESBL and carbapenemase producing isolates collected from UTI patients in the US. Methods A total of 2,001 K. pneumoniae were collected from 73 sites. CLSI methods were used for both susceptibility testing and MIC interpretation. Isolates with aztreonam, ceftazidime, ceftriaxone or meropenem MIC of ≥2 mg/L were subjected to genome sequencing, and screening of plasmid-mediated AmpC (pAmpC), ESBL, and carbapenemase genes. Results A total of 11.0% (220/2,001) K. pneumoniae were screened for β-lactamase genes (Table). Majority of these isolates carried blaCTX-M alone (78.2%; 172/220), and a small number carried combinations of blaCTX-M and ESBL variants of blaSHV (2.7%; 6/220), blaSHV alone (2.7%; 6/220), or pAmpC alone (2.3%; 5/220). Among screened isolates, 9.1% (20/220) carried carbapenemases. GEP had MIC50 and MIC90 of 4 mg/L and 8 mg/L, respectively, against isolates that did not meet the MIC criteria for screening of β-lactamases. Other oral agents showed susceptibilities of 92.8–99.5% against this group, except for nitrofurantoin (35.7%). GEP had an MIC90 of 32 mg/L against screened strains carrying ESBL and pAMPC, and an MIC90 of 16 mg/L against carbapenemase positive isolates. Other oral agents had limited activity (< 42%) against strains carrying ESBL and/or pAMPC. Conclusion GEP showed activity against UTI-causing K. pneumoniae in US medical centers, including against isolates carrying ESBL, pAmpC and/or carbapenemases. These data support further clinical development of GEP as a potential treatment option for uUTI caused by K. pneumoniae including when other oral treatment options have limited activity due to drug resistance. Disclosures Rodrigo E. Mendes, PhD, GSK: Grant/Research Support Renuka Kapoor, PhD, GSK: Employee|GSK: Stocks/Bonds (Public Company) Didem Torumkuney, PhD, GSK: Employee|GSK: Stocks/Bonds (Public Company)

P-1086. COT-832, a Novel Polyene Macrolide Antifungal (I): In Vitro Antifungal Activity against Candida, Aspergillus, and Mucor, etc., Hemolytic Activity, and Cytotoxicity

Abstract Background A novel polyene macrolide antifungal, COT-832 was created by SHIONOGI through structurally modifying Amphotericin B(AmB). Invasive fungal infections are commonly treated with antifungal drugs such as azoles, polyenes, and echinocandins; however, their efficacy is limited and mortality rates remain high. Therefore, the development of more effective and safer antifungal drugs is needed. AmB is available in formulations such as Fungizone (FGZ), which utilizes a deoxycholic acid formulation, and AmBisome (L-AMB), which employs a liposome formulation. While these polyene antifungals exhibit activity against a wide range of fungal species, their usage is often discontinued due to the nephrotoxicity associated with AmB itself. This study aimed to evaluate the antifungal activity, hemolytic activity, and cytotoxicity of COT-832 in vitro. Methods Antifungal activity was assessed using the CLSI method on clinical isolates from Japan (2012-2016). Hemolysis activity was measured after applying compounds to 4% sheep red blood cells for 24 hours. Cytotoxicity was evaluated by applying compounds to HepG2 cells for 2 hours and measuring lactate dehydrogenase (LDH) levels in the supernatant with Cytotox96 assay for calculating cell survival rate. Results Antifungal activity: COT-832 exhibited MIC90 of 0.5-2 µg/mL against Aspergillus and Candida, matching AmB's MIC. It also showed efficacy against azole-resistant A. fumigatus and echinocandin-resistant C. glabrata. COT-832's MIC90 for Cryptococcus was 0.5 µg/mL, surpassing AmB. Similar MICs were observed for rare molds. Hemolytic activity: AmB caused hemolysis from 0.625 µg/mL, reaching 80% at 2.5 µg/mL. COT-832 displayed an 8% hemolysis rate at 2.5 µg/mL. Cytotoxicity: AmB reduced cell count from 2 µg/mL, while no noticeable decrease occurred with COT-832 up to 72 µg/mL. Conclusion COT-832 exhibits comparable antifungal activity to AmB while demonstrating reduced toxicity against various cells, up to a tenfold decrease compared to AmB. These findings suggest that COT-832 has potential for development as a safer polyene macrolide antifungal drug, offering potent antifungal activity across a wide range of fungal species. Disclosures Hideki Maki, PhD, Shionogi & Co., Ltd.: employee|Shionogi & Co., Ltd.: employee Yuri Nishiyama, MS, Shionogi & Co., Ltd.: employee Takafumi Ohara, n/a, Shionogi & Co., Ltd.: employee Hideki Sugimoto, n/a, Shionogi & Co., Ltd.: employee

P-1060. In vitro Activity of Gepotidacin Tested Against Molecularly Characterized Escherichia coli Isolates Responsible for Urinary Tract Infections in the US (2019-2022)

Abstract Background Gepotidacin (GEP) is a novel, bactericidal, first-in-class triazaacenaphthylene antibacterial that inhibits bacterial DNA replication by a unique mechanism of action, distinct binding site and provides well-balanced inhibition (for most uncomplicated urinary tract infections [uUTI] uropathogens and Neisseria gonorrhoeae) of two different type II topoisomerase enzymes GEP completed two phase 3 trials for treatment of uUTI This study reports the activity of GEP and other oral antibacterials against E. coli (EC), including molecularly characterized isolates carrying ESBL and carbapenemase genes collected from UTI patients in the US. Methods 3,995 EC collected from 52 US sites as part of the gepotidacin uropathogen global surveillance study were included (2019-2022). CLSI methods were used for susceptibility (S) testing and MIC interpretations. Isolates with aztreonam, ceftazidime, ceftriaxone or meropenem MIC of ≥2 mg/L were subjected to genome sequencing, and screening of β-lactamase genes and epidemiology typing (MLST, O:H, and fimH). Results A total of 86.2% (3,444/3,995) of EC did not meet the MIC criteria for molecular characterization (Table) and had GEP MIC50 and MIC90 values of 2 mg/L and 4 mg/L, respectively. Cefazolin (97.0%S), fosfomycin (FOS) (99.7%S), and nitrofurantoin (NIT) (98.7%S) had activity among oral comparator agents tested against this group. A total of 13.8% (551/3,995) of EC were screened for β-lactamase genes, and this group had GEP MIC50 and MIC90 values of 2 mg/L and 4 mg/L respectively. Other oral agents had limited activity, except for FOS (98.0%S) and NIT (92.7%S). In general, GEP retained activity with MIC50 and MIC90 values of 2 mg/L and 4 mg/L, respectively, against isolates carrying ESBL, AmpC and carbapenemase genes, and against clonal complex (CC) 131 and the resistant subset O25b:H4. Conclusion GEP showed generally consistent activity against UTI-causing EC in the US, including against isolates carrying ESBL, AmpC and/or carbapenemase genes. In addition, GEP had activity against the CC131 high-risk EC clone. These data support further clinical development of GEP as a potential treatment option for uUTI caused by EC including when other oral treatment options are limited due to drug resistance. Disclosures Rodrigo E. Mendes, PhD, GSK: Grant/Research Support Renuka Kapoor, PhD, GSK: Employee|GSK: Stocks/Bonds (Public Company) Didem Torumkuney, PhD, GSK: Employee|GSK: Stocks/Bonds (Public Company)

P-1061. In vitro Activity of Gepotidacin against Klebsiella pneumoniae, Including Molecularly Characterized Fluoroquinolone not Susceptible Subsets Causing Urinary Tract Infections in the United States (2019-2022)

Abstract Background Gepotidacin (GEP) is a novel, bactericidal, first-in-class triazaacenaphthylene antibiotic that inhibits bacterial DNA replication by a unique mechanism of action, distinct binding site and provides well-balanced inhibition (for most uUTI uropathogens) of two different type II topoisomerase enzymes. This study reports the activity of GEP and other oral antibiotics against K. pneumoniae, including molecularly characterized fluoroquinolone (FQ) not susceptible (NS) isolates collected from urinary tract infection (UTI) patients in the US. Methods A total of 2,001 K. pneumoniae were collected from 73 sites. CLSI methods were used for both susceptibility (S) testing and MIC interpretation. Isolates with MIC≥0.5 mg/L for ciprofloxacin (NS) and/or ≥1 mg/L for levofloxacin (NS) were screened for FQ-resistance (R) mechanisms (plasmid-mediated quinolone resistance [PMQR]), QRDR mutations, and expression of acrAB and oqxAB by qRT-PCR. Results GEP inhibited 94.9% of all these isolates at ≤16 mg/L (MIC50/90 = 4/16mg/L). GEP MIC50/90 against FQ-S isolates was 4/8 mg/L, and other agents showed activity against ≥90% of isolates, except nitrofurantoin (37.4%S). 14.5% (291/2,001) of isolates were screened for FQ-R mechanisms. GEP MIC50/90 against FQ-NS strains was 16/32 mg/L, whereas other agents had S of 5.8–48.5%. GEP had generally similar activity (MIC50/90, 16-32/32-64 mg/L) against isolates with wildtype QRDR and with or without PMQR genes. Most of the isolates (95.2%) without PMQR genes overexpressed oqxAB and/or acrAB, against which amoxicillin-clavulanate and cefazolin (90.3–95.2%S) were active. GEP had consistent MIC90 of 16 mg/L against isolates with distinct QRDR mutations and absence of PMQR genes, whereas other agents had S of ≤68%. Conclusion GEP showed activity against FQ-S and FQ-NS K. pneumoniae UTI isolates from the US, in particular against FQ-NS isolates carrying QRDR mutations, where standard oral antibiotics showed limited activity. GEP MICs were not substantially affected by QRDR mutations. Highest MICs were seen against FQ-NS isolates with wildtype QRDR and PMQR genes and/or overexpression of efflux-pump genes. These data support the development of GEP for the treatment of uUTI caused by K. pneumoniae. Disclosures Rodrigo E. Mendes, PhD, GSK: Grant/Research Support Renuka Kapoor, PhD, GSK: Employee|GSK: Stocks/Bonds (Public Company) Didem Torumkuney, PhD, GSK: Employee|GSK: Stocks/Bonds (Public Company)

Clonal outbreak of Candida vulturna in a paediatric oncology ward in Maranhão, Brazil

To describe an outbreak due to Candida vulturna, a newly emerging Candida species belonging to the Candida haemulonii species complex in the Metschnikowiaceae family. In this retrospective cohort study we genotyped 14 C. vulturna bloodstream isolates, occurring in a 4-month-period in paediatric cancer patients in a Brazilian hospital. To prove an outbreak, ITS sequence analysis and whole genome sequencing (WGS) was done. Antifungal susceptibility was performed with the reference CLSI method and the commercial Sensititre YeastOne (SYO) YO10 plates. A control C. vulturna isolate from another region in Brazil was included in all analyses. MALDI-TOF-MS identified isolates as C. pseudohaemulonii and C. duobushaemulonii albeit with low scores and therefore molecular methods were required for accurate identification. ITS sequence analyses clearly differentiated C. vulturna from other species in the C. haemulonii species complex. WGS proved the presence of a clonal outbreak with C. vulturna involving 14 paediatric patients. Antifungal susceptibility testing (AFST) with two methods showed the isolates had low MICs of commonly available antifungals. This study describes an outbreak due to the rare yeast C. vulturna, related to C. auris, during a four-month period in patients admitted to a paediatric oncology ward in a Brazilian hospital. In contrast to previous studies the yeast was susceptible to all antifungals and patient outcome was good.

Comparative evaluation of MIC values of Trichosporon spp. by MTT assay and CLSI M27-A3 broth microdilution reference methods

Objectives: The objective of this study was to determine and compare the minimum inhibitory concentration (MIC) values of Trichosporon spp. by MTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl- 2H-tetrazoliumbromide) assay, and Clinical and Laboratory Standards Institute M27-3rd edition (CLSI M27-A3) broth microdilution methods. Materials and Methods: Antifungal susceptibility testing was done by CLSI M27-A3 broth microdilution and MTT assay for all the 72 Trichosporon isolates after genus specific and Trichosporon asahii specific polymerase chain reaction (PCR). Candida krusei ATCC 6258 was used as the reference strain. Statistical Analysis: All statistical data were analyzed using the Statistical Package for the Social Sciences, version 17 for Microsoft Windows. The percentage of agreement was calculated using the Type C intraclass correlation coefficient. Results: The MICs by MTT assay strongly correlated with those obtained by CLSI M27-A3 method, by being either the same or within 1 dilution of MIC by CLSI method. Furthermore, the ranges of MICs obtained by MTT and CLSI method were all identical in our study. The overall agreement between the two methods for the Trichosporon isolates was good, that is, 90.8% in our study. Conclusions: MTT assay can be an alternative method that assists reading of MICs visually with a colored end point, making it easier compared to CLSI M27-A3 method. MTT assay can also be standardized for other yeasts and molds so that antifungal susceptibility tests can be done for different fungi.

Increased terbinafine resistance among clinical genotypes of Trichophyton mentagrophytes/T. interdigitale species complex harboring squalene epoxidase gene mutations

Terbinafine resistance has become epidemic as an emerging problem in treatment of dermatohpytosis. This could be attributed in part to a point mutation in the squalene epoxidase (SQLE) gene. In this study, point mutations in the SQLE gene were studied in T. rubrum and T. mentagrophytes/T. interdigitale species complex as two main causative agents of dermatophytosis. Antifungal susceptibility of clinical isolates of T. rubrum (n = 27) and T. mentagrophytes/T. interdigitale (n = 56) was assessed using the M38–3rd edition CLSI method. The SQLE gene and ITS region were sequenced for all the fungal strains, and the mutation sites and genotypes of the terbinafine-resistant strains were characterized. The results demonstrated that, in T. rubrum, the minimum inhibitory concentration of terbinafine (MIC50 and MIC90) was 0.03 μg/ml, and the geometric mean (G mean) concentration was 0.02. For the T. mentagrophytes complex, the MIC50 and MIC90 were 0.03 and 1.0 μg/ml, respectively, and the G mean concentration was 0.04 μg/ml. Four out of the five resistant strains were T. indotineae harboring the F397L and Q408L mutations, while the last one was T. mentagrophytes genotype VII, which harbors the F397L mutation. T. indotineae was the prominent causative agent of terbinafine resistance, with 80 % of the isolates, and T. mentagrophytes genotype VII was introduced as a new genotype in the terbinafine-resistant T. mentagrophytes complex. Our findings further substantiate the importance of antifungal susceptibility testing in selecting the choice of drug for effective treatment of dermatophytosis and highlight the importance of screening dermatophyte species for point mutations responsible for newly developed resistant strains to improve the current knowledge of overcoming infections caused by resistant species.

A cluster of Candida parapsilosis displaying fluconazole-trailing in a neonatal intensive care unit successfully contained by multiple infection-control interventions.

This study aimed to investigate and contain a cluster of invasive candidiasis cases caused by fluconazole-resistant Candida parapsilosis (FRC) in a neonatal intensive care unit. Active surveillance was initiated. Direct observations of hand-hygiene compliance (HHC) among staff were conducted before and after the implementation of hand-hygiene (HH) education. Thirty-five environmental cultures were obtained. Phylogenetic analysis of FRC was performed using Fourier-transform infrared spectroscopy and microsatellite genotyping. A total of 14 patients (mean birth weight = 860 g, gestational age = 25 weeks) infected with FRC were identified using the fully automated analyzer, including 5 with clinical infection (three with catheter-related bloodstream infection, one with cutaneous infection, and one with fatal peritonitis) and 9 with colonization. The HHC rate in nurses before performing a sterile or aseptic procedure significantly improved after the HH education (P < .05). Sinks near the patients were contaminated with FRC. All FRC strains were confirmed to be susceptible to fluconazole using the CLSI method, and the microdilution procedure indicated a trailing effect. Phylogenetic analysis showed that all the fluconazole-trailing isolates from patients were clustered together and had the same genotype. Sinks were successfully decontaminated using accelerated hydrogen peroxide and drainage pipes were replaced. Ultraviolet-C decontamination was applied in the milk preparation room. No new cases were detected after the education and disinfection interventions. Sinks are an important reservoir of C. parapsilosis. Active surveillance, environmental hygiene, and constant staff education on maintaining a high level of HHC are necessary to limit the spread of C. parapsilosis.

Imidazole derivatives as novel and potent antifungal agents: Synthesis, biological evaluation, molecular docking study, molecular dynamic simulation and ADME prediction

Fungal infections have become a serious threat to human health due to their high incidence and mortality. In this study, a new set of imidazole derivatives (7a-f) were synthesized and their chemical structures were characterized using spectroscopic techniques including IR, 1H-NMR, 13C-NMR and Mass. In the following, the antifungal activities of these derivatives were evaluated against several yeasts, aspergillus and dermatophyte species using the CLSI method. In addition, to check the safety of the synthesized compounds, the in vitro cytotoxicity activity of these compounds was investigated towards normal human fibroblasts cell line (MRC-5) using MTT assay. The obtained biological results indicated that these compounds acted as the most potential antifungal agents. In particular, compounds 7c, 7d and 7f exhibited excellent activity against Candida albicans, Candida tropicalis, Candida glabrata, Candida krusei, Candida dubliniensis, Candida parapsilosis, and Cryptococcus neoformans species with MIC50 values in the range of 0.5-16 µg/ml. The cytotoxicity results showed that these compounds have low toxicity against normal human fibroblasts cell line (MRC-5). Additionally, molecular modelling studies and molecular dynamic simulation were carried out to predict the binding energy and possible interaction mode of these compounds in the binding site of lanosterol 14α-demethylase (CYP51) as a conventional target for azole derivatives. In silico ADME study was also performed to investigate the physicochemical features of the synthesized imidazole derivatives. According to the results, these compounds can be considered as promising antifungal candidates for further development.

2159. Analysis of Resistance to Oral Standard-of-Care Antibiotics for Urinary Tract Infections Caused By Escherichia coli and Staphylococcus saprophyticus Collected in the United States in 2022

Abstract Background Gepotidacin is a novel, bactericidal, first-in-class triazaacenapthylene antibiotic that inhibits bacterial DNA replication by a distinct mechanism of action and binding site and provides well-balanced inhibition of 2 different Type II topoisomerase enzymes. This study reports on the in vitro activity of gepotidacin and other oral antibiotics when tested against contemporary Escherichia coli and Staphylococcus saprophyticus clinical isolates collected from patients with UTIs for a gepotidacin uUTI global surveillance study. Methods A total of 1,001 E. coli and 92 S. saprophyticus isolates were collected during 2022 from 45 medical centers located within the United States. Most isolates (77%) tested were cultured from urine specimens collected from patients seen in ambulatory, emergency, family practice, and outpatient medical services. Bacterial identifications were confirmed by MALDI-TOF MS. Isolates were tested for susceptibility by CLSI methods at a central laboratory (JMI Laboratories). MIC results for oral antibiotics licensed for the treatment of uUTI and drug-resistant subsets were interpreted per CLSI guidelines. Results Gepotidacin (MIC50/90, 2/4 µg/mL) displayed good activity against 1,001 E. coli isolates as 98.8% of all observed gepotidacin MICs were ≤4 µg/mL (Table). Other oral agents tested against these isolates demonstrated the following rates of susceptibility (S): amoxicillin-clavulanate (85.2% S), ampicillin (55.6% S), ciprofloxacin (78.8%S), fosfomycin (99.3% S), mecillinam (93.5%S), nitrofurantoin (97.5% S), and trimethoprim-sulfamethoxazole (73.3% S). Gepotidacin maintained similar MIC50 values (ranging from 1 – 2 µg/mL) and MIC90 values (ranging from 2 – 4 µg/mL) against these drug-resistant subsets. Against S. saprophyticus isolates, gepotidacin (MIC50/90, 0.06/0.12 µg/mL) inhibited all isolates at ≤0.12 µg/mL. Most oral agents showed &amp;gt;90% S against S. saprophyticus isolates, except for penicillin (7.6% S). Conclusion Gepotidacin demonstrated in vitro activity against contemporary E. coli and S. saprophyticus from US urine isolates. This activity remained unaffected by resistance to other oral standard-of-care antibiotics. Disclosures S J Ryan Arends, PhD, Cipla: Grant/Research Support|GSK: Grant/Research Support Renuka Kapoor, PhD, GSK: Grant/Research Support Didem Torumkuney, PhD, GSK: Grant/Research Support Nicole E. Scangarella-Oman, MS, GSK: Employee and shareholder Rodrigo E. Mendes, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|Cipla: Grant/Research Support|Entasis: Grant/Research Support|GSK: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support

A-020 Evaluation of the Analytical Performance of BNP and NT-proBNP Assays on the Atellica CI 1900 Analyzer

Abstract Background The Atellica® CI 1900 Analyzer is an automated, high-throughput integrated chemistry and immunoassay analyzer employing both Atellica CH and Atellica IM assays. This study was designed to evaluate the analytical performance of the Atellica IM B-type Natriuretic Peptide (BNP) and NT-proBNP (PBNP) assays* on the Atellica CI 1900 Analyzer. Methods The Atellica CI 1900 BNP and PBNP assays use the same reagents and calibrators as the Atellica IM assays. Precision and method comparison were used as performance indicators for the Atellica CI 1900 Analyzer. Precision studies were performed according to CLSI EP05-A3 and method comparison according to CLSI EP09-A3 using native and contrived human plasma (BNP) and serum (PBNP) samples. One aliquot of each sample pool was tested in duplicate in two runs per day &amp;gt;2 h apart on each analyzer for &amp;gt;20 days. BNP and PBNP were evaluated with one reagent lot on two systems. For the method comparison, individual native and contrived human plasma samples (BNP) or native human serum samples (PBNP) were analyzed using the Atellica IM BNP and PBNP assays on both the Atellica IM and Atellica CI 1900 Analyzers. Results Representative precision and MC results for each assay are listed in the table. For the two assays tested, repeatability and within-lab %CVs were &amp;lt;2.5% and &amp;lt;3.0%, respectively. Slopes determined by the Deming linear regression model were approximately equal to 1. Conclusion Evaluation of the Atellica IM BNP and PBNP assays using the Atellica CI 1900 Analyzer demonstrated acceptable precision and equivalent performance compared to the same assays on the Atellica IM Analyzer.

A-023 Performance Evaluation of the Atellica IM High-Sensitivity Troponin I Assay on the Atellica CI 1900 Analyzer

Abstract Background The Atellica® CI 1900 Analyzer is an automated, high-throughput, integrated chemistry and immunoassay analyzer employing both Atellica CH and Atellica IM assays. This study was designed to evaluate the analytical performance of the Atellica IM High Sensitivity Troponin I (TnIH) assay* on the Atellica CI 1900 Analyzer*. Methods Precision and method comparison were used as performance indicators for the Atellica CI 1900 Analyzer. Precision studies were performed according to CLSI EP05-A3 and method comparison according to CLSI EP09-A3. Precision studies used a panel of four native and one contrived serum/lithium-heparin plasma samples. One aliquot of each sample pool was tested in duplicate in two runs per day &amp;gt;2 h apart on each analyzer for &amp;gt;20 days with two reagent lots on two systems. For the method comparison, individual native human lithium-heparin plasma samples were analyzed using the Atellica IM TnIH assay on both the Atellica IM and Atellica CI 1900 Analyzers. Detection capability studies including limit of blank (LoB), limit of detection (LoD), and 20-day functional sensitivity (LoQ) were performed according to CLSI EP17-A2 guidelines. Expected values of normal healthy individuals and hook effect were also verified on the Atellica CI 1900 Analyzer. Results Repeatability and within-lab %CVs ranged from 0.7% to 4.4% and 1.3% to 5.1%, respectively, at TnIH concentrations ranging from 13.73 to 23 197.12 ng/L. Method comparison between the Atellica IM TnIH assay on the Atellica CI 1900 Analyzer vs the Atellica IM Analyzer showed a regression slope of 1.00 and an intercept of 0.04 ng/L (n = 170). In detection capability studies, four lots of the Atellica IM TnIH assay on two Atellica CI 1900 analyzers showed a LoB of 0.35 ng/L and an LoD of 0.76 ng/L. LoQ, defined as the lowest amount of analyte in a sample at which within-lab %CV = 20%, was 1.10 ng/L; %CV was approximately 10% at a TNIH concentration of 2.57 ng/L. In addition, &amp;gt;50% of healthy individuals tested had TnIH values &amp;gt;LoD. The 99th percentile for the Atellica IM TnIH assay (45.20 ng/L) was verified on Atellica CI 1900 Analyzer and was consistent with the Atellica IM Analyzer. No interference from biotin was observed up to a concentration of 3500 ng/mL, and no hook effect was observed up to a concentration of 500 000 ng/L. Conclusion Evaluation of the Atellica IM TnIH assay using the Atellica CI 1900 Analyzer demonstrated reproducible assay precision, sensitive detection capabilities, and equivalent performance compared to the same assay on the Atellica IM Analyzer. This evaluation shows that the Atellica IM TnIH assay on Atellica CI 1900 Analyzer meets the definition of a true high-sensitivity troponin I assay as defined by the International Federation of Clinical Chemistry (IFCC) Task Force on Clinical Applications of Cardiac Biomarkers. *The products/features mentioned here are not commercially available in all countries. Their future availability cannot be guaranteed.

Erratum for Humphries et al., "CLSI Methods Development and Standardization Working Group Best Practices for Evaluation of Antimicrobial Susceptibility Tests".

Erratum for Humphries et al., "CLSI Methods Development and Standardization Working Group Best Practices for Evaluation of Antimicrobial Susceptibility Tests".

Genotyping Analysis of Cryptococcus deuterogattii and Correlation with Virulence Factors and Antifungal Susceptibility by the Clinical and Laboratory Standards Institute and the European Committee on Antifungal Susceptibility Testing Methods.

Data about the relationship between their molecular types, virulence factors, clinical presentation, antifungal susceptibility profile, and outcome are still limited for Cryptococcus deuterogattii. This study aimed to evaluate the molecular and phenotypic characteristics of 24 C. deuterogattii isolates from the southeast region of Brazil. The molecular characterization was performed by multilocus sequence typing (MLST). The antifungal susceptibility profile was obtained according to CLSI-M27-A3 and EUCAST-EDef 7.1 methods. The virulence factors were evaluated using classic techniques. The isolates were divided into four populations. The molecular analysis suggests recombinant events in most of the groups evaluated. Resistance and susceptibility dose-dependent to fluconazole were evidenced in four isolates (16%) by EUCAST and in four isolates (16%) by CLSI methods. The agreement at ±two dilutions for both methods was 100% for itraconazole, ketoconazole, and voriconazole, 96% for amphotericin B, and 92% for fluconazole. Significant differences in virulence factor expression and antifungal susceptibility to itraconazole and amphotericin B were found. The mixed infection could be suggested by the presence of variable sequence types, differences in virulence factor production, and decreased antifungal susceptibility in two isolates from the same patient. The data presented herein corroborate previous reports about the molecular diversity of C. deuterogattii around the world.

Four uncommon clinical fungi, Lodderomyces elongisporus, Kodamaea ohmeri, Cyberlindnera fabianii and Wickerhamomyces anomalus, isolated in superficial samples from Côte d'Ivoire.

The rare yeast species Lodderomyces elongisporus, Kodamaea ohmeri, Cyberlindnera fabianii, and Wickerhamomyces anomalus are increasingly implicated in severe mycoses in immunocompromised patients. This study aimed to assess the prevalence of uncommon yeast species in Côte d'Ivoire. The yeast isolates from superficial samples, mainly vaginal swabs, were collected at the Pasteur Institute of Abidjan in a study on the molecular epidemiology of clinical yeast species. Identification relied on MALDI-TOF MS and ITS sequence analysis. Antifungal susceptibility testing was performed using the CLSI method. Of the 315 strains analysed from 227 outpatients, 14 belonged to 4 uncommon species: Lodderomyces elongisporus, Kodamaea ohmeri, Cyberlindnera fabianii, and Wickerhamomyces anomalus. None exhibited elevated fluconazole, amphotericin B, caspofungin, ketoconazole, or flucytosin MIC. The presence of these rare yeasts represents a risk in immunocompromised people. Their adequate and timely identification is a priority. Overall, enhancing the mycoses diagnostic capacities in Côte d'Ivoire, and more generally in African clinical laboratories with limited resources is a critical aim.

Assaying for the effectiveness and efficiency of some local plant extracts in the control of dairy farm-associated bacteria pathogens using in vitro well diffusion technique

Though antimicrobial agents have been used for the effective control of infectious agents, there is a growing concern about the emergence and re-emergence of multidrug-resistant bacteria pathogens against commonly prescribed antibiotics. Consequently, there is gross reduction in the effectiveness of the control mechanism thereby increasing health care costs and its associated consequences. Plant parts and extracts have been used in crude forms for the local control of several ailments including those of animals. Two-phase research was set-up. One, to isolate and identify multi-drug bacteria pathogens from some small-scale farms in Kwali, FCT, Abuja and their drug resistance status. The second part was to screen and evaluate the antimicrobial effects of common phytochemicals from some local plants, viz; guava (Psidium guajava), ginger (Zingiber officinale), neem (Azadirachta indica), and moringa (Moringa oleifera) against the previously isolated MDR bacteria (Salmonella spp. Clostridium sp. Staphylococcus aureus, Streptococcus sp. and E. coli.). The aqueous, ethanolic and methanolic extracts of the plants were used for susceptibility test, minimum inhibitory concentration and minimum bactericidal concentrations using in vitro agar disc diffusion protocol based on the CLSI methods. The results showed significant Inhibitory zones range of 23-32mm. This is in agreement with several research previously done using plant extracts. The type of solvent used also influenced the quality of the extract and the output. Based on the results, we concluded that the plant extracts have some antimicrobial activities comparable to the currently prescribed modern drugs tested. It is justifiable that the therapeutic agents derived from these plants would be effective in the control of emerging diseases and also contribute to the growth of scientific knowledge about herbal medicines as important alternatives or complementary treatment of animal diseases. It is hereby recommended that further research (The third phase) carry out studies on the clinical efficacy trial, safety, toxicity and affordability analyses as well as optimization and upscaling parameters so as to proceed to the final step for the mass production of the tested products.

Analytical validation of white blood cell differential and platelet assessment on the Sysmex DI-60 digital morphology analyzer.

Digital morphology analyzers are increasingly replacing light microscopy in laboratory hematology practice. This study aimed to perform the analytical validation of the white blood cell (WBC) differential and of reliability of platelet assessment on Sysmex DI-60 (Kobe, Japan). Validation included determination of within-run and between-run precision for WBC differential according to the CLSI EP15-A3 protocol, accuracy and method comparison with light microscopy and with the automated WBC differential from the Sysmex XN-10 hematology analyzer, reliability of platelet clump detection and platelet count estimation. Standard deviations of both pre- and post-classification mostly satisfied manufacturer's criteria for imprecision. Accuracy assessment revealed that only eosinophil count (1.4%) in one peripheral blood smear (PBS) remained outside the declared range (2-10%) after reclassification. Method comparison between DI-60 and light microscopy yielded Spearman's correlation coefficients from 0.37 (basophils) to 0.94 (neutrophils and lymphocytes), minor proportional difference for bands, constant difference for monocytes, both constant and proportional difference for lymphocytes and statistically significant biases for bands, lymphocytes, monocytes and basophils. Diagnostic sensitivity (Se) and specificity (Sp) of DI-60 in detecting immature/pathological cells were 88.7% (95%CI:81.1-94.0) and 83.0% (95%CI:78.7-86.7), respectively, with the area under the curve (AUC) of 0.86 (95%CI:0.82-0.89). Agreement in detection of platelet clumps was 94.8% (kappa coefficient = 0.67, 95%CI:0.53-0.80). Se and Sp of DI-60 to detect platelet clumps were 65.7% (95%CI: 47.8-80.9) and 96.9% (95%CI: 93.9-98.6), respectively, while AUC was 0.81 (95%CI: 0.76-0.86). DI-60 provides reliable WBC differential and platelet assessment. In doubtful cases, the use of light microscopy is still mandatory.

Methods for Antifungal Susceptibility Testing of the Cryptococcus neoformans/C. gattii Complex: Strengths and Limitations

When method-dependent categorical endpoints are available, namely either BPs or ECVs, MICs could aid in selecting the best treatment agent(s). BPs can categorize an isolate as either susceptible or resistant while the ECVs/ECOFFs can distinguish the wild type (WT, no known resistance mechanisms) from the Non-WT (NWT, harboring resistant mechanisms). Our literature review focused on the Cryptococcus species complex (SC) and the available methods and categorization endpoints. We also covered the incidence of these infections as well as the numerous Cryptococcus neoformans SC and C. gattii SC genotypes. The most important agents to treat cryptococcal infections are fluconazole (widely used), amphotericin B, and flucytosine. We provide data from the collaborative study that defined CLSI fluconazole ECVs for the most common cryptococcal species or genotypes and modes. EUCAST ECVs/ECOFFs are not yet available for fluconazole. We have summarized the incidence of cryptococccal infections (2000-2015) where fluconazole MICs were obtained by reference and commercial antifungal susceptibility tests. This occurrence is documented all over the world and those fluconazole MICs are mostly categorized by available CLSI ECVs/BPs as "resistant" instead of non-susceptible strains, including those by the commercial methods. As expected, the agreement between the CLSI and commercial methods is variable because SYO and Etest data could yield low/variable agreement (<90%) versus the CLSI method. Therefore, since BPs/ECVs are species and method dependent, why not gather sufficient MICs by commercial methods and define the required ECVs for these species?