Journal of Clinical Microbiology最新文献

Systemic shifts in antimicrobial resistance rates can be due to epidemiologic shifts in microbial susceptibility patterns or artifactual shifts introduced by technical biases in antimicrobial susceptibility testing (AST)-both ultimately leading to changes in antimicrobial prescribing. To reduce technical variability, quality control (QC) criteria for AST are published by manufacturers and standards organizations. However, traditional QC metrics, in isolation, are fallible. In this study, we describe a systematic shift in daptomycin AST results between 2022 and 2025 in isolates tested in two independent health systems. Comprehensive analysis of clinical isolate AST results and retrospective mining of QC data from this period revealed a subtle shift that led to a 5%-22% decrease in overall susceptibility rates for certain organisms, most notably Enterococcus faecium. As daptomycin is a key treatment option for these difficult-to-treat infections, this increase in resistance rates paralleled a decrease in prescribing daptomycin for infections with these organisms. Importantly, this trend was undetectable through routine QC processes and only became apparent through systematic review of patient data. Our findings highlight the opportunity to integrate routine patient data analysis into microbiology QC practices to enhance detection of subtle but clinically relevant changes in AST performance.

Importance: In this study, we report a critical incident of technical variability using daptomycin gradient diffusion methodology that was undetectable using routine quality control metrics. More broadly, this study underscores the opportunity to incorporate additional modalities, such as clinical patient results, into a comprehensive quality assurance plan to ensure high-quality antimicrobial susceptibility testing results. Given the dynamic spread of multidrug resistance in bacteria, accurate susceptibility testing results are critical to identify and respond to shifts in local epidemiology.

Bovine tuberculosis, a zoonotic disease caused primarily by Mycobacterium bovis, poses a significant threat to cattle health and farming livelihoods within the United Kingdom (UK). Disease control in cattle is complicated by the persistence of M. bovis in European badgers, the UK's principal wildlife reservoir. Accurate diagnostic tools for both species are essential for effective surveillance and disease control. Many existing badger serodiagnostic tests, which include MPB70, MPB83, and ESAT6-CFP10 antigens, have relatively modest sensitivities (~50%-60%), limiting their utility in surveillance. To address this issue, we used an unbiased and comprehensive antigen discovery approach to identify new diagnostic targets. This strategy identified Rv3616c as a novel antigen with promising diagnostic test potential for M. bovis infection in badgers. Overlapping peptides spanning the full Rv3616c amino acid sequence were screened to identify the most diagnostically informative epitopes. A pool of four Rv3616c peptides, used in an indirect enzyme-linked immunosorbent assay (ELISA), had a sensitivity of 85.71% (95% CI: 77.19-91.96), a specificity of 94.80% (95% CI: 90.35-97.59), and a diagnostic accuracy of 91.51% (95% CI: 87.54-94.54). The existing validated Badger M. bovis Ab Test, when used alone, had a sensitivity of 73.47% (95% CI: 63.59-81.88); however, parallel interpretation with the Rv3616c ELISA could increase overall sensitivity to 91.84% (95% CI: 84.55-96.41), with minimal loss of specificity. These findings support the use of Rv3616c-derived peptides in serodiagnostic tests to improve the detection of M. bovis infection in badgers and enhance tuberculosis surveillance in this wildlife reservoir.IMPORTANCEAccurate diagnosis of Mycobacterium bovis infection in wildlife reservoirs is essential for controlling bovine tuberculosis (bTB), a zoonotic disease that threatens human health, animal welfare, and farming livelihoods. In the United Kingdom, European badgers are the principal wildlife reservoir, complicating efforts to eradicate bTB in cattle. Existing serodiagnostic tests for badgers have moderate sensitivity, limiting effectiveness in surveillance. To address this, this study used an unbiased, comprehensive antigen discovery approach and identified several new diagnostic targets, including the Rv3616c protein. A test based on specific Rv3616c-derived peptides had a high diagnostic accuracy (91.51%) and, when used in parallel with a validated test, improved test sensitivity while maintaining specificity. These synthetic peptides are scalable, cost-effective, and adaptable to different diagnostic platforms. The findings reveal an antigen with diagnostic potential that could inform the development of new tests for bTB surveillance in wildlife, supporting One Health principles and global tuberculosis elimination strategies.

HIV serologic responses measured by clinical screening assays may be blunted when antiretroviral treatment (ART) suppresses HIV replication from the initial infection stages, with further decay following long-term treatment. Sensitivity may vary according to the timing of ART initiation after HIV acquisition, antigens (Ags) utilized to detect anti-HIV antibodies (Abs), and HIV subtype. We obtained samples from long-term ART-suppressed persons with HIV (PWH) who started ART at acute/early or during chronic infection, from blood donors with undetectable HIV RNA and nonreactive serology, and from blood donors with positive RNA and reactive serology (presumably untreated infections) spanning multiple HIV subtypes to compare patterns of serologic reactivity using two Ag/Ab screening assays (Alinity and VITROS). We found that earlier ART initiation was associated with progressively lower signal-to-cutoff ratios (S/CO) in both assays. Serologic reactivity in clinical samples from participants initiating ART at Fiebig 2 was somewhat lower for CRF01_AE (67%) than other HIV subtypes (91%) on the Alinity platform. In the late-ART initiation group, reactivity was high for both assays despite long-term treatment, with higher S/CO values in samples from blood donors with presumably untreated infection. We observed no strong evidence of S/CO waning >96 weeks after ART initiation in the early-ART initiation group. Our findings suggest potential limitations for HIV infection ascertainment in clinical samples from long-term treated PWH who started ART at early infection stages using currently approved serologic tests.IMPORTANCEThis manuscript increases our understanding of how the timing of initiation of HIV treatment affects our ability to detect the infection using commercial blood tests that measure HIV antigens or antibodies. Being able to detect HIV is important for clinical diagnostics and blood screening, and HIV is not as easily detected in persons who begin therapy shortly after infection using serological tests. In contrast to prior work, we show that current clinical HIV tests have stable reactivity over time in persons on long-term HIV treatment.

Recognizing and updating bacterial names is key to communication between the veterinary clinical microbiology laboratory, veterinarians, and clients. Moraxella oculi sp. nov., distinct from Moraxella bovis and Moraxella bovoculi, was isolated from a cow with infectious bovine keratoconjunctivitis. Several respiratory pathogens were recognized, including Neisseria leonii sp. nov. described from rabbits, Mannheimia indoligenes sp. nov. described from cattle in Europe, and Moraxella haemolytica sp. nov. described from a goat in China. Stenotrophomonas forensis sp. nov. is a novel designation within the Stenotrophomonas maltophilia complex associated with isolates derived from horses. New additions to the Campylobacter genus included Campylobacter californiensis sp. nov., recovered from bovine feces during a raw milk-associated outbreak of campylobacteriosis in humans. Taxonomic revisions were most notable in Gram-positive organisms. A species within genus Jeotgalicoccus has been renamed, and the subspecies designation Kocuria rosea subsp. polaris comb. nov. has been created. Streptococcus suis was revised to Streptococcus suis subsp. suis subsp. nov. in recognition of the initial description of Streptococcus suis subsp. hashimotonensis subsp. nov., which was isolated from people in Japan with bite wounds from boars. Chlamydophila caviae, which causes respiratory disease and abortion in guinea pigs and is zoonotic, has been revised to Chlamydia caviae comb. nov.

Major advances in hepatitis C virus (HCV) treatment have made timely and accurate diagnosis a critical determinant for United States elimination goals. Recently authorized point-of-care (POC) HCV RNA testing enables faster diagnosis than the traditional two-step algorithm but comes with higher laboratory costs. We analyzed HCV testing data from 2017 to 2024 across three medical centers in Seattle, Washington, to evaluate strategies for integrating new rapid direct detection tests. HCV antibody testing volumes increased 72% over the study period, with outpatient settings accounting for 76.0% of tests and a 2.7% positivity rate. Emergency department testing increased by 682% to 5,654 tests in 2024, with a 10.3% positivity rate, such that one-third of all HCV diagnoses in our medical system now originate from the public county hospital emergency department. Adoption of sample-to-answer HCV RNA testing in 2024 reduced median collection-to-result turnaround times for antibody-positive specimens from 84 h (IQR 58-120) to 45 h (IQR 28-57). Using a viral load cut-off of 10,000 IU/mL, HCV antigen testing was estimated to detect 98% of infections. Converting all HCV testing to POC RNA would increase laboratory costs by 260% (+$6,439 per HCV infection detected), while restricting POC RNA to the public county hospital emergency department would increase costs by 22.3% (+$552 per HCV infection detected). Reflexing antibody-positive samples to antigen testing slightly reduced costs. These findings highlight the significant laboratory costs associated with POC HCV RNA testing and the need for specific reimbursement and funding mechanisms for new HCV testing algorithms.IMPORTANCEHepatitis C virus (HCV) elimination in the United States requires rapid and reliable diagnosis, yet current testing pathways are too slow for treatment initiation in a single clinical visit. Analysis of more than 325,000 HCV test results from 2017 to 2024 from our academic medical system in Seattle, Washington, highlights the growing role of emergency departments, particularly those serving safety-net populations, in making new HCV diagnoses. While point-of-care HCV RNA testing can enable connection to treatment, it substantially increases laboratory costs when implemented broadly. Targeted use of point-of-care HCV RNA in high-yield settings such as safety-net emergency departments is essential to maximize public health impact while preserving laboratory resources. These findings highlight the need for policy and reimbursement frameworks that support cost-effective deployment of new HCV diagnostic technologies.

Stool is a recommended sample for the diagnosis of Mycobacterium tuberculosis (MTB) using Xpert MTB/RIF (Ultra) (GXU) in children, and the Simple One-Step (SOS) stool method is one of the recommended processing methods. We investigated modifications to the protocol of the SOS stool method to make it fit for testing stool with Truenat MTB Plus and MTB-RIF Dx (Truenat) and Xpert MTB/XDR (GXX). Experiments were conducted using stool spiked with different MTB concentrations and using modified versions of the SOS stool processing method. The established protocol was then validated on routine clinical stool samples from presumptive TB patients, comparing the performance of Truenat and GXX against GXU. The concordance for MTB detection between GXU and Truenat using 50, 100, and 150 mg of stool was 100%. Truenat did not provide valid results at 300 mg. In routine stools, concordance for MTB detection was 89.3% and 90.6% when comparing Truenat-100 mg to GXU-100 and 600 mg, respectively. For rifampicin detection, 44.8% of results were indeterminate on Truenat, but none on GXU (except Trace calls). GXX stool testing gave valid results, although for low bacterial loads, GXX less often detected MTB. We conclude that stool can be tested on Truenat to detect MTB using the SOS stool Xpert method adapted to the test composition of Truenat. In addition, the SOS protocol, as originally developed for GXU, can be used for GXX. This provides opportunities to expand access to rapid molecular testing to detect TB and resistance to first- and second-line anti-TB drugs for children and adults who cannot produce sputum.IMPORTANCEFollowing World Health Organization recommendations, countries commenced stool testing on GeneXpert as the primary test to improve tuberculosis (TB) case finding among children who cannot produce sputum. To further expand access to rapid molecular testing, we adapted the Simple One-Step (SOS) stool method for GeneXpert to the test kit composition of the Truenat platform through a series of laboratory experiments. Truenat is being introduced on a large scale by countries for near point-of-care testing as it can operate better in more remote settings. As part of our experiments, we also concluded that stool can be tested with the Xpert MTB/XDR cartridge using the original SOS protocol. Our findings further expand access to rapid molecular testing to detect TB and resistance to diverse anti-TB drugs for children and adults who cannot produce sputum. Increased access to TB testing in these vulnerable populations will support TB case finding efforts and timely and appropriate treatment.

Timely initiation of appropriate antimicrobial therapy is crucial for patients with Gram-negative (GN) blood stream infections. In this study, the performance of VITEK REVEAL (bioMérieux, USA), an FDA cleared in vitro diagnostic automated system for antimicrobial susceptibility testing (AST) directly from positive blood culture (BC), was compared to that of Accelerate Pheno (Accelerate Diagnostics, USA), as standard of care method. 128 GN positive BCs were analyzed according to manufacturer recommendations, comparing time to result (TTR) and AST results. The categorical agreement (CA) rate between VITEK REVEAL and Accelerate Pheno was 94.3% and the essential agreement (EA) rate was 96.0%. Very major discrepancies (VMD), major discrepancies (MD), and minor discrepancies (miD) rates between the systems were 7.5%, 0.5%, and 4.1%, respectively. Lastly, we observed a mean sequential TTR (reporting of AST results per antibiotic in real-time) on VITEK REVEAL of 6.1 h (3.0-8.2) and mean final TTR of 7.9 h (6.5-8.2), compared to Accelerate Pheno, with a significantly shorter mean final TTR of 7.1 h (6.8-7.7). Sequential TTR on VITEK REVEAL was significantly shorter for resistant isolates (those with ≥1 'Resistant' antimicrobial-organism combination interpretation, using FDA STIC 2024 or CLSI M100 criteria) compared to susceptible/intermediate ones, with a mean difference of 1.4 h (P  <  0.001). Overall, compared to Accelerate Pheno, VITEK REVEAL displayed high %CA and %EA for AST of GN bacteria directly from positive BC. Also, unlike Accelerate Pheno, VITEK REVEAL reports MIC results in real time, allowing an earlier release of actionable information useful for antimicrobial stewardship.

Importance: Previous studies have compared the performance of the VITEK REVEAL, system for fast antimicrobial susceptibility testing (AST) to conventional, non-rapid microbiology methods. To evaluate how the VITEK REVEAL correlates to similarly available fast, direct-from-blood culture AST technologies, this study aimed to compare it to the Accelerate Pheno system, as standard of care method. High categorical agreement (94.3%) and essential agreement (96.0%) between the two systems were observed, underscoring their reliability. The VITEK REVEAL has the advantage of real-time AST reporting, unlike the Accelerate Pheno. Ultimately, this study supports the need for and continued optimization of fast diagnostic technologies for AST, contributing to the goal of advancing antimicrobial stewardship-focused treatment strategies and improving the prognosis of patients with bloodstream infections.

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.IMPORTANCECancer 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.

Mycoplasma pneumoniae is a major cause of community-acquired pneumonia and increasingly exhibits macrolide resistance due to the A2063G and A2064G mutations in the 23S rRNA gene. We developed a loop-mediated isothermal amplification (LAMP) assay with hybridization-based TaqMan-style probes (HyTaq) to simultaneously detect M. pneumoniae wild-type strains and macrolide resistance mutations (A2063G and A2064G) within a single reaction (hereafter referred to as the MP-R HyTaq-LAMP assay). Analytical performance was evaluated using plasmid standards, and clinical validation was conducted with 224 nasopharyngeal swab samples. The MP-R HyTaq-LAMP assay detected wild-type strains and the A2063G and A2064G mutations with a limit of detection of 1 × 10³ copies/μL. In clinical samples, the assay demonstrated 95.79% sensitivity for A2063G and 100% sensitivity for wild-type strains, along with 100% specificity against 103 non-infection samples. Additionally, no cross-reactivity was observed with 16 other common respiratory pathogens. The MP-R HyTaq-LAMP assay provides rapid, multiplex detection of M. pneumoniae and associated macrolide resistance mutations without thermal cycling, demonstrating its strong potential as a point-of-care diagnostic tool.

Importance: Macrolide-resistant Mycoplasma pneumoniae is a significant cause of treatment failure in community-acquired respiratory infections, particularly in children and adolescents. Early and accurate detection of resistance-associated mutations, such as A2063G and A2064G, is essential for guiding effective antibiotic therapy. In this study, we developed an MP-R HyTaq-based loop-mediated isothermal amplification assay capable of simultaneously detecting and discriminating wild-type strains and macrolide resistance mutations (A2063G and A2064G) in a single-tube, isothermal reaction. Its high specificity, rapid turnaround time, and minimal equipment requirements make this assay suitable for point-of-care testing in diverse clinical settings.