Rosemary C. She

Carbapenem-resistant Acinetobacter baumannii (CRAB) is an extensively drug-resistant pathogen requiring new therapeutic strategies. Rifabutin (RBT), a U.S. Food and Drug Administration (FDA)-approved rifamycin, has demonstrated potent activity against CRAB; however, its activity is systematically underestimated by conventional antimicrobial susceptibility testing (AST) performed in conventional iron-rich Mueller-Hinton media, necessitating methods that accurately predict in vivo efficacy to guide its clinical use. Here, we describe the development of a disk diffusion method for RBT susceptibility testing against A. baumannii . Following Clinical and Laboratory Standards Institute (CLSI) M23 “Development of In Vitro Susceptibility Test Methods, Breakpoints, and Quality Control Parameters” guidance, multiple disk potencies were evaluated across standard and chelated Mueller-Hinton agar (MHA), including EDTA-supplemented media, and zone-of-inhibition (ZOI) diameters were correlated with MICs determined by agar dilution. A 20-µg RBT disk on chelated MHA, or equivalently on MHA supplemented with 100 mg/L EDTA, provided clear and reproducible discrimination of tentative wild-type and non-wild-type phenotypes, and preliminary quality control testing supported A. baumannii NCTC 13304 as a candidate quality control strain. While this investigational method has not been reviewed or approved by the CLSI, it was developed in concordance with the M23 guidance and addresses the key limitations of conventional AST, providing a robust framework for interlaboratory validation and future breakpoint determination. IMPORTANCE: Antimicrobial susceptibility testing is a critical tool for guiding clinical treatment decisions. Rifabutin exhibits potent activity against carbapenem-resistant Acinetobacter baumannii (CRAB) under the iron-restricted conditions encountered in vivo ; however, its activity is systematically underestimated by standard testing methods in iron-rich Mueller-Hinton media. Here, we describe a robust, simple, and reproducible disk diffusion method using a 20-µg rifabutin disk on chelated Mueller-Hinton agar, which enables accurate assessment of rifabutin activity against A. baumannii . Our method provides a practical foundation for standardized rifabutin susceptibility testing, future breakpoint development, and broader evaluation of rifabutin against CRAB to ultimately support its implementation in clinical microbiology laboratories.

Background: Coccidioides species are dimorphic fungi that exist in distinct morphological forms depending on environmental conditions, such as temperature. The parasitic phase, consisting of endosporulating spherules, is expected in vivo , while mycelial forms with arthroconidia seen in the environmental phase are expected at comparatively lower temperatures than mammalian body temperature. The incidence of Coccidioides fungemia is low but well documented in adults in cases of severe disseminated disease. The morphological description of Coccidioides spp. isolated from blood culture is limited in the published literature. Case Summary: We present the case of a 59-year-old man with a history of allogeneic hematopoietic stem cell transplantation who developed disseminated coccidioidomycosis with positive cultures from blood and bronchoalveolar lavage specimens. During laboratory work-up, blood cultures initially demonstrated septate hyphae on Gram stain of the broth isolate, which was subsequently identified as Coccidioides immitis/posadasii . The initial Gram stain result raised concerns of other mold infections, given the assumption that Coccidioides appears as the spherular rather than environmental mold form at body temperature. Conclusion: This case highlights the unexpected persistence of environmental phase morphology of Coccidioides spp. in blood culture despite incubation at 35°C, demonstrating that temperature alone is insufficient to trigger the parasitic phase transition and that additional host-specific factors are required for spherule formation.

Smear microscopy using carbolfuschin- or auramine-based staining is widely employed for rapid mycobacterial detection from patient specimens. While auramine stains are preferred to detect M. tuberculosis complex (MTBC), there are little data on the relative performance of these stains for the detection of nontuberculous mycobacteria (NTM). This retrospective study evaluated the diagnostic accuracy of Kinyoun and auramine-rhodamine (AR) stains on clinical specimens from a tertiary cancer center (2017–2025), using culture-based identification as the gold standard. Of 16,294 specimens, 651 (4.0%) recovered mycobacteria, predominantly Mycobacterium avium complex (MAC) (56.2%) and MTBC (14.4%). Both stains demonstrated high specificity (>99.9%) but limited sensitivity (17.4% overall, 11.2%–11.8% among index cases) for mycobacteria, with higher sensitivity for MTBC (40.4%–43.6%) than NTM (12.9%–13.5%) ( P < 0.0001). 1.7% of mycobacteria-positive specimens had discordant qualitative AR vs Kinyoun smear results: AR detected three MTBC and three MAC cases missed by Kinyoun stain, while Kinyoun stain detected five rapid-growing mycobacteria (RGM) cases not detected by AR, though differences were not statistically significant ( P > 0.05). Semi-quantitatively (1-4+), of 107 AR+/Kinyoun+ smears, AR was one grade higher in 9 specimens (3 MTBC, 5 MAC, 1 RGM), while Kinyoun was one grade higher in 3 specimens (2 MAC, 1 RGM). A significant increase in frequency of NTM isolation in culture over the study period was observed (P = 0.02), but MTBC frequency remained stable. Given time and resource considerations, even where NTM predominates in prevalence, AR is appropriate as the primary mycobacterial stain, but carbolfuschin-based staining should be considered when RGM is suspected. IMPORTANCE: Accurate and rapid detection of mycobacteria is essential for timely patient management, especially in vulnerable immunocompromised patients. This study analyzes more than 16,000 clinical specimens over 8 years and provides a direct comparison of auramine rhodamine and Kinyoun stains for mycobacterial detection on smear microscopy. Both stains show high specificity and comparable overall performance, but Kinyoun exhibits a modest trend toward improved the detection of rapid growing mycobacteria. These findings offer guidance for clinical laboratories performing smear microscopy for both tubercular and non-tuberculous mycobacteria.

Diagnostic stewardship (DxS) is a key activity for providing effective and efficient testing strategies for patient care. Complex test menus and result interpretation, high costs, limited reimbursements, and other factors necessitate DxS practices in clinical microbiology. We conducted a national survey of clinical microbiologists on their perceptions and practice of DxS in infectious diseases (ID) diagnostics. The 119 respondents (83 clinical directors, 33 operational leaders, and three trainees) indicated that DxS was practiced at their institution “some” (50.4%) or “most” (23.5%) of the time. Most respondents (>95%) had infection prevention and control and antimicrobial stewardship committees at their institutions. Academic medical center laboratories (AMCs) more often reported having microbiology consultation service, DxS/laboratory utilization committee, or official laboratory formulary than community hospital laboratories ( P ≤ 0.001). Respondents identified ID providers (79.0%) and clinical laboratory directors (74.8%) as the most common participants in their DxS activities. Leveraging informational technology (IT) was considered the most effective strategy (78.2%), and antimicrobial stewardship and/or ID collaboration (52.1%) was the most valuable resource for supporting DxS. The biggest challenge was time and personnel resources (52.1%). Respondents highlighted molecular testing, including syndromic panels (22.7%) and sequencing-based assays (21.0%), as a top priority for DxS efforts, followed by culture-based testing (20.2%). AMC respondents more likely considered sequencing assays as highest priority than other respondents ( P < 0.001). These data provide a valuable baseline for understanding the state of DxS in clinical microbiology laboratories. Key challenges include personnel resources and opportunities include engaging with ID providers and utilization of IT tools for DxS initiatives. IMPORTANCE: At a time when microbiology test menus are expanding with costly molecular diagnostics while workforce shortages and reimbursement constraints persist, we conducted a national survey-based study of clinical microbiology professionals to capture their perceptions and practice of diagnostic stewardship. Results from 119 clinical microbiologists identified several common challenges as well as key differences between respondents from different institutional types as well as between respondents fulfilling different professional roles. The survey additionally identified diagnostic stewardship interventions regarded as most effective, factors most important in prioritization of stewardship efforts, and breadth of roles participating in diagnostic stewardship for microbiology tests. These unique observations into the state of diagnostic stewardship in clinical microbiology laboratories help establish a baseline for future research and guideline development in infectious disease testing.

Formulating safe and effective diagnostic stewardship guidance for blood cultures in immunocompromised patients is challenging due to limited data and high risk of infectious complications. A 2024 global shortage in blood culture bottles (BD Diagnostics) necessitated the implementation of blood culture stewardship, including at our tertiary care cancer center. This was a retrospective pre-post intervention study of the effects of diagnostic stewardship at a stand-alone cancer center during a 5-month period of the 2024 blood culture bottle shortage compared to the same 5-month unaffected period in 2023. Interventions included discontinuation of an ordering set for persistent fever, modification of an ordering set for new fever to recommend two sets of blood cultures for initial workup, and issuing revised blood culture ordering guidance to providers, phlebotomists, and nurses with repeated educational efforts. Stewardship interventions led to decreased blood culture utilization by 36.3% (from 14,021 to 8,932), while the number of patients tested remained similar. Greatest reductions in utilization occurred in inpatient encounters and with follow-up blood cultures. There was a trend toward increasing blood culture positivity following intervention, excluding potential contaminants (7.1% vs 7.6%, P = 0.08). The frequency of sepsis-coded encounters and 30-day mortality rate were similar between the two time periods. No adverse outcomes related to blood culture stewardship were reported. A significant reduction in blood culture utilization is achievable in a high-complexity cancer patient population without negative clinical impact through limiting the number of initial blood cultures and eliminating pre-emptive ordering of blood culture orders for persistent fever. IMPORTANCE: Cancer and transplant patient populations are frequently excluded from experimental studies on decreasing blood culture utilization, but a recent global supply shortage in blood culture bottles provided a valuable opportunity to evaluate the effects of stewardship interventions on a large scale. This study, centered on a high-complexity cancer patient population, found that a significant 36.3% reduction in blood culture utilization was achievable, safe, and effective through multidisciplinary diagnostic stewardship efforts.

The increasing emergence of antifungal resistance poses potential clinical challenges, particularly among immunocompromised patients with cancer at risk of invasive mold infections, but data on antifungal susceptibility trends specific to this population are few. We evaluated distributions of minimal inhibitory concentrations (MIC), including minimal effective concentrations (MEC) for echinocandins, of 11 antifungal agents for 523 mold isolates (395 Aspergillus spp.) from cancer patients. Based on published Clinical and Laboratory Standards Institute guidelines, isavuconazole had notably high rates of non-wild-type MICs for A. fumigatus (19.6%), A. flavus/oryzae (34.8%), A. niger complex (26.1%), and A. terreus complex (8.33%). Persistent low baseline resistance of A. fumigatus to voriconazole was observed across multiple years (2.4–11.5% per year, average 8.41%) without significant trends in MIC change over time. Itraconazole and posaconazole demonstrated the lowest MIC distributions (MIC₅₀ ≤ 0.06–0.5 µg/mL) of the azoles against Aspergillus spp. Amongst the A. niger complex, 29.4% (27/92) demonstrated non-wild-type MICs to itraconazole. While the A. nidulans group was less frequent (n = 24), bimodal peaks in MIC/MEC were noted for caspofungin (≤0.06 and 1 µg/mL). Non-Aspergillus molds of significance (Zygomycetes, Fusarium spp., Scedosporium spp., and Lomentospora prolificans) demonstrated variable but increased MICs to antifungal agents as previously described. Our results highlight increased rates of non-wild type MICs for Aspergillus spp. to isavuconazole and voriconazole, which are commonly used antifungal agents in cancer patients. Such AST trends should be closely monitored in populations with frequent antifungal use and encourage increased antifungal stewardship efforts.

The diagnosis of infective endophthalmitis is supported by microbiologic work-up. Rapid work-up is critical to confirm clinical suspicion and enable appropriate antimicrobial therapy. We report the novel use of an automated liquid culture processing system (Fast System, Qvella Corporation) in a vitreous fluid culture. A 59-year-old patient with post-operative endophthalmitis presented with acute right eye pain and blurred vision. Vitreous fluid collected for microbiology culture was of limited quantity and only inoculated to thioglycolate broth. Broth culture recovered beta-hemolytic, group G Streptococcus dysgalactiae susceptible to penicillin and vancomycin. Experimental application of the Fast System to isolate and concentrate the organism from the broth culture yielded the same identification and susceptibility test results but one day sooner than the standard workflow. Despite prompt treatment with appropriate antibiotics including vancomycin and ceftriaxone, disease progressed rapidly and required enucleation to achieve a stable therapeutic outcome. Use of automated processors such as the FAST System for monomicrobial broth cultures has thus far focused on positive blood culture broths, but could potentially include other liquid-based cultures such as for sterile body fluids of critical nature.

The diagnosis of infective endophthalmitis is supported by microbiological work-up. Rapid work-up is critical to confirm clinical suspicion and appropriate antimicrobial therapy. We report the novel use of an automated liquid culture processing system (FAST system, Qvella, ON, Canada) in a vitreous fluid culture. A 59-year-old patient with post-operative endophthalmitis presented with acute right eye pain and blurred vision. Vitreous fluid collected for microbiology culture was of limited quantity and only inoculated to thioglycolate broth. The broth recovered beta-haemolytic, group G Streptococcus dysgalactiae susceptible to penicillin and vancomycin. Experimental application of the FAST system to purify the organism from broth culture yielded the same identification and susceptibility test results but 1 day sooner. Despite prompt treatment with appropriate antibiotics, including vancomycin and ceftazidime, disease progressed rapidly and required enucleation to achieve a stable therapeutic outcome. Use of automated processing of monomicrobial broth cultures has thus far focused on positive blood culture broths, but could potentially include other liquid-based cultures such as for sterile body fluids of critical nature.

INTRODUCTION: Absolute polymorphonuclear leukocyte (PMN) count (PMN-C) ≥250 cells/mm³ in ascites is the diagnostic hallmark of spontaneous bacterial peritonitis (SBP) and is associated with high morbidity and mortality. However, the clinical significance of ascitic PMN percentage (PMN-%) and PMN-C in the absence of SBP as additional biomarkers for mortality and future incidence of SBP has not been determined. METHODS: This retrospective cohort included adults with cirrhosis undergoing first-recorded paracentesis with initial PMN-C < 250 cells/mm³ at 2 tertiary medical centers between 2015 and 2020. Patients with prior SBP were excluded. Outcomes were death and SBP development. Cox regression estimated hazard ratios (HRs) for risk of death and SBP development and Akaike information criterion to compare model fit. RESULTS: Three hundred eighty-four adults (73% male, median age 58 years, 67% with alcohol-associated cirrhosis, median PMN-C 14 cells/mm³ [interquartile range 5–34], and median PMN-% 10% [interquartile range 4–20]) were included in this study. Univariate risk of death increased 10% per 25-unit increase in PMN-C (95% confidence interval 1.01–1.21, P = 0.03) and 19% per 10-unit increase in PMN-% (95% confidence interval 1.06–1.33, P = 0.003) with PMN-% demonstrating better model fit in assessing mortality risk (Akaike information criterion: 1,044 vs 1,048, respectively). In models adjusted for age, chronic hepatitis C virus infection, and Model for End-Stage Liver Disease-Sodium, PMN-% was associated with risk of death (PMN-% 10%–29%, HR 1.17, P = 0.50; PMN-% ≥ 30% group, HR 1.94, P = 0.03; vs PMN-% < 10%) and SBP development (PMN-% 10%–29%, HR 1.68, P = 0.07; PMN-% ≥ 30%, HR 3.48, P < 0.001; vs PMN-% < 10%). DISCUSSION: Our results suggest PMN-% at first paracentesis represents a better biomarker compared with PMN-C for assessing risk of death and future SBP development in patients with PMN-C < 250 cells/mm³.

The diagnosis of infective endophthalmitis is supported by microbiologic work-up. Rapid work-up is critical to confirm clinical suspicion and enable appropriate antimicrobial therapy. We report the novel use of an automated liquid culture processing system (Fast System, Qvella Corporation) in a vitreous fluid culture. A 59-year-old patient with post-operative endophthalmitis presented with acute right eye pain and blurred vision. Vitreous fluid collected for microbiology culture was of limited quantity and only inoculated to thioglycolate broth. Broth culture recovered beta-hemolytic, group G Streptococcus dysgalactiae susceptible to penicillin and vancomycin. Experimental application of the Fast System to isolate and concentrate the organism from the broth culture yielded the same identification and susceptibility test results but one day sooner than the standard workflow. Despite prompt treatment with appropriate antibiotics including vancomycin and ceftriaxone, disease progressed rapidly and required enucleation to achieve a stable therapeutic outcome. Use of automated processors such as the FAST System for monomicrobial broth cultures has thus far focused on positive blood culture broths, but could potentially include other liquid-based cultures such as for sterile body fluids of critical nature.