Journal of Clinical Microbiology

This review summarizes novel taxon designations ascribed to prokaryotes derived from human primary clinical material during calendar year 2025, as well as proposed revisions to existing taxonomy. Major activity took place in the Streptococcus genus, as more than one dozen novel species were validly and effectively published, with two of these later classified as synonyms of Streptococcus thalassemiae. Moreover, whole genome sequencing and phylogenetic investigation of Streptococcus mitis group organisms resulted in a proposal to designate five species-level Streptococcus spp. taxa as Streptococcus mitis and an additional taxon as Streptococcus oralis subsp. dentisani. More than one dozen taxa were newly included in order Enterobacterales in 2025. Select novel taxa within genera Providencia and Enterobacter commonly possessed genotypes that conferred resistance to carbapenem and/or higher-generation cephem agents. Stenotrophomonas muris sp. nov. and Terrisporobacter muris sp. nov., initially characterized in gnotobiotic murine systems within the past 4 years, had clinical significance in human infection that was demonstrated in primary literature. The vast majority of the more than 70 novel obligate anaerobic taxa were derived from microbiome studies and had little ascribed clinical significance. Four novel obligate anaerobic Gram-negative taxa were shown to be of greater abundance in persons with Parkinson's disease than in those without. Updates to taxa previously published in the Journal of Clinical Microbiology compendia reveal that several could serve as reservoirs for multiple antimicrobial resistance determinants. One example is the non-glucose fermentative Gram-negative bacillus Pseudomonas juntendi.

iFAST is a label-free rapid antimicrobial susceptibility test (AST) that delivers qualitative results in under 4 h from a positive blood culture bottle. The technique measures changes in the electrical properties of thousands of individual bacteria following exposure to antibiotics. We report the diagnostic accuracy from the first clinical study of the iFASTone rapid AST system in three different centers according to ISO 20776-2:2021. Compared to the reference laboratory broth microdilution method, categorical agreement was 97.8% and 97.2% for the Enterobacterales and non-fermenting gram-negative species, respectively. iFAST specificity and sensitivity for the Enterobacterales were 98.2% and 96.8%, respectively, and 96.9% and 98.4% for the non-fermenting species, exceeding the acceptance criteria (≥95% sensitivity and specificity). Reproducibility was assessed with 12 isolates across 3 sites, in triplicate, on 3 separate days, with different operators at each site. Agreement was 95.7%. IMPORTANCE:Even short delays in the initiation of antimicrobial therapy in patients with bloodstream infections are associated with increased morbidity and mortality. Consequently, current guidelines recommend initiation of empiric antimicrobial therapy within 1 h of presentation. However, empiric broad-spectrum therapy may not always be appropriate and can contribute to the selection of resistant organisms. Antimicrobial susceptibility testing (AST) is therefore critical for antimicrobial stewardship and the timely selection of targeted therapy, but conventional methods typically require 1 to 2 days to generate results. This study evaluated the diagnostic accuracy of iFAST, a new low-cost, label-free rapid AST using 375 Enterobacterales and non-fermenter isolates tested directly from positive blood cultures, according to ISO-20776-2:2021. Compared with broth microdilution, iFAST demonstrated high sensitivity, specificity, and categorical agreement, with a time to result of less than 4 h. These findings support the implementation of iFAST for same-shift AST results.

HIV-1 and HIV-2 share transmission and replication characteristics, but require clinical differentiation. HIV-2 exhibits lower viral loads and slower disease progression, and most patients remain long-term non-progressors. Differentiation is crucial for selecting appropriate viral load monitoring tests and avoiding ineffective antiretroviral treatments, particularly non-nucleoside reverse transcriptase inhibitors, and certain protease inhibitors that do not work against HIV-2. Increased PrEP use may result in new infections testing negative for viral markers. This study aims to identify circulating tRNA expression profiles that could serve as non-viral biomarkers to detect and differentiate HIV-2 from HIV-1 infections. An initial pilot study analyzed 88 tRNAs in plasma samples from HIV-1- (n = 4) and HIV-2 (n = 4)-infected individuals, plus four healthy controls, using a tRNA-based PCR array for target identification. Eighteen selected tRNAs were further analyzed in a larger independent group of samples: HIV-1 (n = 20), HIV-2 (n = 20), and healthy controls (n = 20) for independent verification. A plasma tRNA diagnostic model was developed from these results and evaluated in 24 independent samples to detect and differentiate HIV-2 from HIV-1 infection. We identified a tRNA-based model (Model-II) containing six differentially expressed tRNAs (Leu-TAA, mt-Asp-GTC, mt-Ala-TGC, mt-Lys-TTT, mt-LeuT-AA, and Gly-CCC-1) that could detect HIV-2 infection and distinguish it from HIV-1 infection and healthy controls with 100% accuracy, sensitivity, and specificity. We also found that other blood-borne viruses such as HTLV, HBV, and HCV had no cross-reactivity with HIV-2, demonstrating that Model-II is specific for HIV-2 infection. This study establishes proof of concept for tRNA expression profiles as HIV diagnostic biomarkers. The tRNA-based Model-II represents a promising diagnostic tool for identifying HIV-2 infection and differentiating it from HIV-1.IMPORTANCEHIV-2 infection requires accurate differentiation from HIV-1 to guide appropriate antiretroviral therapy, as HIV-2 exhibits intrinsic resistance to non-nucleoside reverse transcriptase inhibitors and certain protease inhibitors. Current diagnostic challenges include the absence of FDA-approved HIV-2 RNA quantification assays and potential false-negative results in LEVI (low-level viremia with evolving viral infection) syndrome patients on pre-exposure prophylaxis (PrEP), in whom viral markers may be undetectable. We developed a novel diagnostic approach using circulating transfer RNA (tRNA) expression profiles as host-derived, non-viral biomarkers. Our six-tRNA panel (Model-II) achieved 100% accuracy, sensitivity, and specificity in detecting HIV-2 infection while distinguishing it from HIV-1 and other blood-borne viral infections (HTLV, HBV, and HCV). This represents the first tRNA-based diagnostic model for HIV, offering a promising complementary tool for early detection and accurate viral differentiation, particularly in clinical scenarios where traditional viral markers are absent or unreliable.

Rapid pathogen identification optimizes antibiotic therapy in bacteremia and can be achieved through molecular-based diagnostic tests. This study assesses the clinical impact after the implementation of an updated multiplex PCR panel (BIOFIRE Blood Culture Identification 2, BCID2). This single-center, retrospective, quasi-experimental study compared outcomes in adult patients with gram-negative bloodstream infections (GN-BSIs) pre and post BCID2 implementation. Data were collected from 1 March 2022 to 1 March 2024. Patients were included if they were ≥18 years of age, had a gram-negative organism identified by the panels, and received ≥48 h of antibiotics. The primary outcome was time to effective antimicrobial therapy; secondary outcomes included time to appropriate therapy, 30-day mortality, and hospital length of stay. A total of 180 patients were included (90 per BCID and BCID2 groups). Baseline characteristics were similar: median age 62 years, mostly non-critically ill, Charlson Comorbidity Index 4, and Pitt Bacteremia score 1 in both groups. Median time to effective therapy was reduced in the BCID2 vs BCID group (1 vs 4 h, P = 0.004) as well as time to appropriate therapy (25 vs 49 h, P = 0.627). In the ESBL subgroup, BCID2 significantly reduced time to effective therapy (0.5 vs 3 h, P = 0.006) and time to appropriate therapy (16 vs 44 h, P = 0.016). There was no significant difference in mortality (6.7% vs 8.9%, P = 0.756) or length of stay (10 vs 14 days, P = 0.134) between BCID and BCID2 groups. The implementation of BCID2 significantly reduced time to effective therapy in GN-BSIs, particularly among resistant organisms. IMPORTANCE:This study evaluated the impact of transitioning from BCID to BCID2 panel in gram-negative bloodstream infections to address gaps in resistance detection and assess whether expanded molecular diagnostics improve time to antibiotic therapy and clinical outcomes.

Detection of GES-type carbapenemases remains challenging because of their low prevalence and frequently weak hydrolytic activity against carbapenems. Carbapenem inactivation method (CIM)-based assays are widely used as phenotypic screening tools for carbapenemase detection; however, their performance in large collections of GES producers has not been systematically evaluated. We assessed the performance of CIM, modified CIM (mCIM), and CIM-Tris in a diverse collection of GES-producing clinical isolates, including 110 Enterobacterales and 108 Pseudomonas aeruginosa, recovered from Spanish hospitals (2010-2024), and 10 Acinetobacter baumannii isolates, mostly obtained from a hospital in Egypt. Whole-genome sequencing was carried out for species confirmation and resistome analysis. Meropenem MICs were determined by broth microdilution. Overall, 92.1% of isolates were GES-carbapenemase producers (CP), whereas 7.9% expressed GES-type extended-spectrum β-lactamases (ESBLs). In Enterobacterales (predominantly carrying blaGES-6), mCIM improved sensitivity compared with CIM (63.6% vs 40.0%), although many isolates remained undetected due to low meropenem MICs (MIC50, 0.5 µg/mL). In CP-P. aeruginosa (mainly blaGES-5), CIM, mCIM, and CIM-Tris showed sensitivities of 89.1%, 94.6%, and 100%, respectively; however, CIM-Tris yielded false-positive results in 50% of non-CP isolates (mostly blaGES-1 producers). Meropenem MICs in P. aeruginosa were higher (MIC50, >32 µg/mL). In A. baumannii, CIM-Tris improved sensitivity compared with CIM (100% vs 25.0%). These findings indicate that CIM-based methods can detect GES-type carbapenemases, but performance varies according to bacterial species and GES variant, and reduced specificity may occur in isolates producing GES-type ESBLs. Complementary molecular testing may therefore be necessary to ensure accurate detection of GES-type carbapenemases in routine clinical laboratories. IMPORTANCE:GES-type carbapenemases represent an important but underrecognized diagnostic challenge due to their low global prevalence, heterogeneous hydrolytic activity, and the limited performance data available for routine phenotypic detection methods. Although CIM-based assays are widely implemented in clinical microbiology laboratories for carbapenemase screening, their performance against GES-producing organisms has not been comprehensively evaluated across different bacterial genera and GES variants. In this study, we evaluated the performance of CIM, modified CIM (mCIM), and CIM-Tris in a large multicenter collection of well-characterized GES-producing clinical isolates, including Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii. Our findings demonstrate substantial variability in assay performance according to bacterial species and GES variant. Notably, mCIM improved sensitivity among Enterobacterales with low meropenem MICs, whereas CIM-Tris achieved excellent sensitivity in P. aeruginosa and A. baumannii but at the expense of reduced specificity in isolates producing GES-type ESBLs. To the best of our knowledge, this is the first study directly comparing multiple CIM-based approaches in such a large and taxonomically diverse collection of GES-producing isolates.

The rise of multidrug-resistant microorganisms highlights the need for rapid antimicrobial susceptibility testing (AST) to guide early and effective therapy. This study evaluated a novel MALDI-TOF MS-based assay (MBT FAST, Bruker, Germany), which employs the direct-on-target microdroplet growth assay (DOT-MGA) for phenotypic AST. A total of 243 Enterobacteriaceae isolates representing diverse species and resistance patterns were tested against a broad panel of antibiotics. Each isolate was incubated in 6-µL microdroplets of cation-adjusted Mueller-Hinton broth, with and without antibiotics at two-fold serial dilutions, at 35°C for 6 h inside a humidity-controlled chamber (MBT FAST Shuttle, Bruker). Broth was removed using a prototype device (MBT FAST Stamp, Bruker), and MALDI-TOF MS measurements were performed with the MALDI Biotyper Sirius system (Bruker). Results were compared with broth microdilution as the standard for determining minimum inhibitory concentrations (MIC). Valid growth control was achieved for 100% of isolates. Essential agreement (EA), defined as MICs within ±1 two-fold dilution step of the standard method, was as follows: amoxicillin/clavulanic acid 96.3%, amikacin 97.5%, aztreonam 95.5%, ceftazidime/avibactam 95.5%, ceftazidime 94.6%, cefepime 92.2%, ciprofloxacin 91.8%, colistin 92.2%, ceftriaxone 95.5%, cefotaxime 94.2%, gentamicin 95.5%, imipenem 91.8%, levofloxacin 97.9%, meropenem 87.2%, meropenem/vaborbactam 94.2%, piperacillin/tazobactam 90.9%, trimethoprim/sulfamethoxazole 90.9%, temocillin 93.3%, and tobramycin 97.5%. The overall EA across all antimicrobials tested reached 96.0%. The research-use-only MBT FAST assay provides reliable AST within 6 h, substantially reducing turnaround time. Building on technology widely implemented in diagnostic laboratories, this approach holds promise for integration into microbiology practice. IMPORTANCE:Standard antimicrobial susceptibility testing requires prolonged incubation, delaying targeted treatment and de-escalation. This study evaluated MBT FAST, a MALDI-TOF MS-based assay that measures bacterial growth directly on the MALDI target plate in the presence of antibiotics. In a diverse collection of Enterobacteriaceae tested against a panel of 19 antibiotics, the assay provided reliable minimum inhibitory concentration results after 6 h and showed high agreement with standard broth microdilution. Because MALDI-TOF MS instruments are widely used in clinical microbiology laboratories, this approach could make rapid phenotypic susceptibility testing easier to integrate into routine workflows.

Purpureocillium lilacinum, a common environmental fungus, can cause hyalohyphomycosis with varied clinical manifestations. Mortality rates can reach 20% with invasive disease. P. lilacinum is intrinsically resistant to amphotericin B, leaving few treatment options. In the absence of clinical breakpoints, epidemiological cutoff values (ECVs) can aid clinicians in monitoring antifungal resistance trends and in guiding initial therapy. We performed antifungal susceptibility testing (AFST) on P. lilacinum isolates to establish minimum inhibitory concentration (MIC) and minimum effective concentration (MEC) distributions and ECVs. Data were collected from 13 laboratories located in seven countries. AFST was performed by broth microdilution (CLSI M38 standard) for itraconazole, voriconazole, posaconazole, isavuconazole, terbinafine, flucytosine, amphotericin B, natamycin, and manogepix. ECVs were established using the iterative statistical method with ECOFFinder (v2.1) following CLSI M57 guidelines. Results from 479 P. lilacinum isolates were analyzed. ECVs were 1 μg/mL for voriconazole, 2 μg/mL for posaconazole, 4 μg/mL for isavuconazole, and 2 μg/mL for terbinafine. Itraconazole displayed a trimodal MIC distribution, although almost all MICs were ≥0.5 µg/mL (mode, 16 μg/mL). Amphotericin B, natamycin, and flucytosine MICs were truncated at the high end of the range and consistent with intrinsic resistance. The manogepix modal MEC was 0.008 μg/mL. These proposed P. lilacinum ECVs and MIC/MEC distributions provide baseline data to monitor resistance trends. Our results confirm that P. lilacinum is intrinsically resistant (IR) to amphotericin B and likely IR to natamycin and flucytosine. Caution is needed in interpreting itraconazole results due to high interlaboratory variability.IMPORTANCEPurpureocillium lilacinum is a hyaline mold commonly found in the environment and used as an agricultural biopesticide. Infections caused by P. lilacinum can range from fungal eye infections to life-threatening pulmonary and disseminated disease, and mortality rates can be high (up to 20%). Single-center studies examining antifungal susceptibility testing (AFST) data found reduced susceptibility to certain antifungals, but more robust global AFST studies are needed to help interpret MICs and MECs. We performed AFST according to the Clinical Laboratory and Standards Institute reference method in thirteen independent laboratories on 479 P. lilacinum isolates to establish MIC/MEC distributions and epidemiological cutoff values (ECVs) for nine antifungals, including those recommended by global treatment guidelines. The calculated ECVs were 1 μg/mL for voriconazole, 2 μg/mL for posaconazole, 4 μg/mL for isavuconazole, and 2 μg/mL for terbinafine. Our results also confirm that P. lilacinum is intrinsically resistant to amphotericin B and likely intrinsically resistant to natamycin and flucytosine. Caution is needed in interpreting itraconazole AFST results due to high interlaboratory variability. These antifungal in vitro susceptibility data suggest limited options to treat P. lilacinum infections.

Invasive fungal diseases are difficult to diagnose because of the limited sensitivity of culture. Panfungal PCR amplicon sequencing assays (targeting ribosomal RNA, such as 18S, 28S, ITS) are recommended for fungal identification in histopathology samples showing fungal elements. However, data describing its overall performance and consistency are lacking. This systematic literature review and meta-analysis assessed the performance of panfungal PCR on formalin-fixed paraffin-embedded (FFPE) and non-fixed (fresh or frozen) tissue samples. A systematic literature search was performed to include studies reporting the use of panfungal PCR for fungal identification in FFPE or non-fixed tissue samples. PCR sensitivity and specificity were assessed using the reference standard of histopathology showing fungal elements. Quality assessment was performed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool. Pooled estimates were obtained using random-effects meta-analysis. Twenty-eight studies were included. In FFPE samples (18 studies, 852 samples), sensitivity and specificity were 75.4% (95% confidence interval [CI], 59.2-86.6) and 93.5% (70.2-98.9), respectively. Sensitivity in non-fixed samples (13 studies, 207 samples) was 86.5% (74.7-93.3), while specificity could not be assessed (insufficient data). Comparative analyses showed a significantly higher sensitivity of panfungal PCR over culture (88.2%; 76-94.7 vs 52.2%; 39-65, P = 0.001). Sub-analyses could not demonstrate the superiority of one PCR target over another due to limited data. Panfungal PCR exhibited adequate sensitivity and good specificity in FFPE samples. Sensitivity was even higher in non-fixed samples and largely superior to culture. Nevertheless, large interstudy variability was observed, warranting interlaboratory studies to define the optimal PCR target and standardized protocols. IMPORTANCE:Invasive fungal diseases are difficult to diagnose because of the low sensitivity of culture. Panfungal PCRs are widely used for fungal identification in tissue specimens but suffer from heterogeneous procedures and performance. This meta-analysis shows an acceptable sensitivity (75.4% and 86.5% in fixed and non-fixed samples, respectively) and good specificity (93.5%) of panfungal PCR, supporting its use, not only on histopathology-positive fixed samples but also in non-fixed samples concomitantly with other diagnostic tools (cultures and fungal-specific PCRs if available). These results provide a strong basis for further standardization of panfungal PCR techniques via interlaboratory assays to assess reproducibility and optimize analytical protocols. CLINICAL TRIALS:This study is registered with PROSPERO as CRD42023461148.

Patients undergoing hematopoietic cell transplantation (HCT) who receive fluoroquinolone prophylaxis are at high risk for bacteremia due to fluoroquinolone-resistant viridans group streptococci (FQ-R VGS), particularly Streptococcus mitis. Detecting colonization with FQ-R VGS may identify neutropenic patients at risk for streptococcal bacteremia during FQ prophylaxis, but the optimal specimen screening site and microbiologic screening method are unknown. We therefore simultaneously collected buccal, oropharyngeal, and perianal swabs from patients upon admission for HCT, and performed selective culture with both direct plating and broth enrichment. FQ-R VGS were detected in 62 (90%) of 69 participants. Direct plating resulted in a positive percent agreement (PPA) of 79% (95% confidence interval [CI]: 67%-88%) for detecting FQ-R VGS from buccal swabs, and a PPA of 86% (95% CI: 74%-93%) from oropharyngeal swabs compared to detection by any specimen or method. Broth enrichment yielded a PPA of 97% (95% CI: 89%-100%) for both buccal and oropharyngeal swabs. Perianal swabs had low PPA. For detecting FQ-R S. mitis group, the direct plating PPA was 74% (95% CI: 60%-85%) for buccal and 72% (95% CI: 58%-84%) for oropharyngeal swabs, while the broth enrichment PPA was 82% (95% CI: 69%-91%) and 58% (95% CI: 43%-72%), respectively. Combining direct plating and broth enrichment of buccal swabs had a PPA of 96% (95% CI: 86%-100%) for detecting FQ-R S. mitis group. Buccal swabs are a reliable specimen to detect FQ-R VGS colonization when using broth enrichment, and combining direct plating and broth enrichment may optimize detection of FQ-R S. mitis group.IMPORTANCEViridans group streptococci (VGS) are important causes of bloodstream infections in patients with cancer undergoing hematopoietic cell transplantation. These infections frequently occur despite using prophylactic fluoroquinolone antibiotics, suggesting that some patients are colonized with fluoroquinolone-resistant VGS (FQ-R VGS). A test that reliably detects carriage of FQ-R VGS might help identify patients at highest risk of streptococcal infection, but the optimal methods to detect FQ-R VGS colonization were previously unknown. In this study, we compared different body sites and culture methods for detecting colonization with FQ-R VGS. We found that buccal (cheek) swabs reliably detected these resistant bacteria, including members of the Streptococcus mitis group, and adding a broth enrichment step further improved detection from buccal samples. These findings support buccal sampling with broth enrichment as a practical approach for detecting colonization with FQ-R VGS and establish a foundation for future studies to define the clinical significance of FQ-R VGS colonization.

Dermatophytosis is a highly prevalent superficial fungal infection, yet antifungal susceptibility testing results remain difficult to interpret because clinical breakpoints are unavailable, and epidemiological cutoff values (ECVs) are still limited for most dermatophyte-drug combinations. We conducted a multicenter study of 305 Trichophyton mentagrophytes and Trichophyton interdigitale isolates collected between 2019 and 2024 at six tertiary hospitals across geographically diverse regions of China. Antifungal susceptibility testing was performed using the CLSI M38 broth microdilution method under standardized quality control, and provisional ECVs were derived following CLSI M57 guidance. For T. mentagrophytes, fluconazole showed a non-unimodal MIC distribution and was not assigned a provisional ECV. The remaining seven agents yielded ECVs of 1 µg/mL for ciclopirox olamine and griseofulvin, 0.5 µg/mL for itraconazole, 0.06 µg/mL for voriconazole, 0.25 µg/mL for posaconazole and amorolfine, and 0.016 µg/mL for terbinafine. For T. interdigitale, fluconazole showed a complex non-unimodal distribution, whereas itraconazole and posaconazole showed unstable low-MIC shoulders precluding ECV estimation. The remaining five agents yielded ECVs of 1 µg/mL for ciclopirox olamine, 0.25 µg/mL for griseofulvin, 0.12 µg/mL for voriconazole and amorolfine, and 0.016 µg/mL for terbinafine. Isolates above the provisional ECVs were uncommon for most combinations (≤2.5%), except for griseofulvin against T. interdigitale (20.5%). Overall, the two species showed distinct MIC distribution profiles, underscoring the value of species-level analysis and continued surveillance. These multicenter CLSI-based data and provisional ECVs provide a contemporary baseline for species-specific interpretation of dermatophyte susceptibility data in China. IMPORTANCE:Reliable interpretation of dermatophyte antifungal susceptibility testing remains limited because clinical breakpoints and epidemiological cutoff values (ECVs) are unavailable for most dermatophyte-drug combinations. This is particularly important for the Trichophyton mentagrophytes/Trichophyton interdigitale species complex, which is increasingly implicated in refractory dermatophytosis and may display species-specific susceptibility patterns. In this multicenter study, we established provisional epidemiological cutoff values for most tested species-agent combinations and showed that these two closely related species differ in MIC distribution profiles and in the detection of non-wild-type isolates. These data provide practical laboratory thresholds for recognizing isolates with reduced susceptibility, highlight the limitations of complex-level interpretation, establish a contemporary baseline for dermatophyte susceptibility surveillance in China, and may help support laboratory-informed antifungal decision-making.

To establish a laboratory detection assay for a human neutralizing antibody (NAb) against rabies virus and to evaluate its efficiency in human serum samples. A pseudovirus pXN2‑RABV‑G harboring the RABV G gene was applied. The precision, robustness, linearity, stability, inclusivity, and limit of detection of the assay were evaluated. The consistency and correlation of the assay with ELISA and rapid fluorescent focus inhibition test (RFFIT) were analyzed. A pseudovirus-based neutralizing antibody (pVNA) detection system based on the pXN2‑RABV‑G was successfully developed and showed broad robustness (culture media: χ2 = 4.00, P > 0.05, cell passages: χ2 = 0.25, P > 0.05), precision (independent detection: P > 0.05), lower/upper limit of linearity (0.09 to 93.73 IU/mL, R2 > 0.95), and stability (P > 0.05). The limit of detection (LOD) was 2.63 IU/mL (95% CI: 1.81-3.84), expressed as the lowest human sera antibody level reliably distinguishable from background signals, and the coefficient of variation was less than 20%. Compared with the commercial ELISA and the gold standard RFFIT assay, the kappa values were 0.74 and 0.82, respectively, and the results were highly consistent. Linear correlation analysis revealed a strong correlation between these variables (R2 > 0.90). A total of 103 recipients from 2019 to 2023 were included in the serological testing and the following analysis. NAb titers were significantly correlated with vaccine dose (P < 0.001) and the most recent vaccination time (P < 0.001). The geometric mean titer (GMT) of the 5-dose vaccination group was greater than that of the 1-dose, 2-dose, 3-dose, and 4-dose groups (3.15 vs 3.19 vs 6.18 vs 12.21 vs 13.40 IU/mL). The established pVNA assay for human rabies NAb detection has a detection capability similar to that of the gold standard RFFIT, but it has the advantages of reduced biosafety requirements, laboratory activity, and time, which highlight its safety, high efficiency, and feasibility for screening and monitoring rabies virus antibodies after vaccination. IMPORTANCE:Rabies is a lethal disease with no effective clinical treatment. Vaccination is the only way to prevent it routinely, which makes post-vaccination antibody testing essential. The current standard test requires a highly equipped laboratory, biosafety measures, and is time-consuming. This study develops a safe, efficient test for detecting rabies neutralizing antibodies using pseudovirus technology. It is comparable to the gold standard test in accuracy but easier to implement in routine labs, which enables broader use in screening and monitoring antibodies post-vaccination. This advantage addresses a key gap in rabies prevention, reducing the uncertainty for individuals with immunodeficiency who are nonresponsive to vaccination and at risk of rabies exposure.

Numerous studies describe increased rates of sexually transmitted infections (STIs) in women with symptomatic vaginitis from various causes. We present a comparative analysis of the diagnostic utility of STI nucleic acid amplification testing (NAAT) for Trichomonas vaginalis (TV), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Mycoplasma genitalium (MG) in 1,039 women with symptomatic vaginitis, including 823 women diagnosed with bacterial vaginosis (BV) and/or vulvovaginal candidiasis (VVC) by in-office health care provider (HCP) clinical assessment, conventional laboratory methods (Nugent Score/Amsel Criteria/culture), or BV- and VVC-NAAT methods. Among 508 women with a conventional laboratory BV diagnosis, significantly more STIs were detected in women with a positive BV-NAAT result (147/154, 95.5%) compared to women with HCP diagnosis of BV (134/154, 87.0%; P = 0.0408). For women with a culture-positive VVC diagnosis, a higher STI-NAAT positivity rate correlated with a positive VVC-NAAT result (55/59, 93.2%) compared to HCP VVC diagnosis (18/59, 30.5%), although this difference was not significant. A positive vs negative BV-NAAT result had a significantly higher relative risk for any STI infection (RR: 1.85; 95% confidence interval [CI] 1.43-2.39, P < 0.0001), while relative risks for STI-NAAT positive infections were not elevated significantly in VVC. Finally, STI NAAT positivity rates were higher in women with positive BV/VVC NAAT vaginitis diagnoses compared to women diagnosed with BV/VVC by conventional clinician empirical diagnosis. These results indicate combined molecular testing for STIs, and BV/VVC has improved diagnostic yield compared to conventional laboratory methods or traditional HCP assessment for these disorders.IMPORTANCEAn all-molecular (nucleic acid amplification test [NAAT]-based) approach to vaginitis and sexually transmitted infection (STI) testing of vaginal swabs from women with symptomatic vaginitis provides optimal STI case finding comparable to using STI NAATs after traditional lab-based methods for vaginitis diagnosis and better than using STI NAATs following a clinician's assessment for vaginitis diagnosis. This study demonstrates that a combined molecular testing strategy to identify the causes of vaginitis and STIs can improve risk stratification, support guideline-aligned STI testing, help clinicians provide the most appropriate and comprehensive care, and streamline clinical and laboratory workflows using a single specimen and single test method (NAAT).

Distinguishing tuberculous meningitis (TBM) from other causes of meningitis remains challenging in high-burden settings because clinical features overlap and existing assays often delay etiologic confirmation. We evaluated MeltArray CNS, a highly multiplexed PCR assay targeting 85 meningitis-associated pathogens, for etiologic diagnosis in patients with suspected meningitis. Analytical performance was assessed using pre-characterized targets. MeltArray was compared head-to-head with metagenomic next-generation sequencing (mNGS) in 79 cerebrospinal fluid (CSF) specimens, with discrepant results adjudicated by Sanger sequencing, and was then prospectively evaluated in 255 consecutive patients with suspected meningitis. Biomarker-based models using routine CSF and serum parameters were also explored. The assay achieved limits of detection of 5 copies/reaction for Mycobacterium tuberculosis (MTB) and 50 copies/reaction for other targets, with no false-positive results in analytical specificity testing. Within its targeted range, MeltArray yielded more confirmed detections than mNGS. In the prospective cohort, MeltArray showed 100.00% sensitivity for definite TBM (35/35; 95% confidence interval [CI], 90.11-100.00%) and 100.00% specificity for MTB detection among non-TBM meningitis cases (39/39; 95% CI, 89.32-100.00%). Among 49 MTB-positive TBM cases, 21 (42.86%) showed co-detections of additional pathogens. Test areas under the curve (AUCs) were 0.877 (95% CI, 0.760-0.994) for distinguishing infectious meningitis from noninfectious mimics and 0.776 (95% CI, 0.617-0.934) for distinguishing MTB-positive from MTB-negative cases. MeltArray enables rapid etiologic confirmation and may facilitate earlier TBM diagnosis in high-burden settings. Co-detections highlight microbiologic complexity with potential treatment implications, although viral findings should be interpreted cautiously. Biomarker models may aid triage and risk stratification but do not replace pathogen confirmation. IMPORTANCE:Tuberculous meningitis (TBM) is a life-threatening infection that requires a rapid and accurate diagnosis to guide effective treatment. Conventional diagnostic methods are often slow or insufficiently sensitive, leading to delays in therapy and potential exposure to unnecessary medications. In this study, we evaluated a rapid multiplex molecular assay for patients with suspected tuberculous meningitis. Rapid detection of Mycobacterium tuberculosis together with alternative infectious causes of meningitis was achieved within approximately 2.5 h, supporting earlier etiologic clarification during initial clinical evaluation. Detection of additional pathogens in some patients further supported the value of broad-spectrum molecular testing in the differential diagnosis of central nervous system infections in high-burden settings. Routine laboratory biomarkers may assist clinical triage but do not replace rapid pathogen confirmation.

Accurate drug susceptibility testing (DST) is crucial for designing effective regimens for multidrug-resistant (MDR) and pre-extensively drug-resistant tuberculosis (pre-XDR TB). Sensititre MYCOTB enables simultaneous determination of minimum inhibitory concentrations (MICs) for multiple drugs, but its diagnostic performance varies across studies. This meta-analysis evaluated the diagnostic performance of Sensititre MYCOTB for key MDR and pre-XDR TB drugs. The protocol was registered in PROSPERO (CRD420251230599). PubMed, Cochrane, Google Scholar, Scopus, ONOS, Web of Science, ScienceDirect, and registries were systematically searched for studies published between 2010 and 2025. Studies comparing the Sensititre MYCOTB with reference DST for Mycobacterium tuberculosis complex (MTBC) were included. Bias assessment and pooled diagnostic accuracy estimates were generated. Fourteen studies, including 1,728 isolates, were analyzed. Rifampicin and isoniazid demonstrated high sensitivity (0.976 [95% CI: 0.94-0.99] and 0.977 [95% CI: 0.95-0.99]) and specificity (0.958 [95% CI: 0.84-0.98] and 0.957 [95% CI: 0.83-0.99], respectively) with low heterogeneity. Amikacin, kanamycin, and ofloxacin demonstrate good diagnostic accuracy, with high specificity (>0.98 [95% CI]). Moderate diagnostic accuracy was observed for ethambutol, streptomycin, ethionamide, and rifabutin. Cycloserine, moxifloxacin, and para-aminosalicylic acid showed inconsistent performance despite excellent specificity (>0.97 [95% CI]). Sensitivity analysis partially improved pooled sensitivity for moxifloxacin 0.801 (95% CI: 0.585-0.924) and para-aminosalicylic acid 0.76 (95% CI: 0.518-0.894), whereas cycloserine remained at 0.436 (95% CI: 0.190-0.725), although heterogeneity persisted. Sensititre MYCOTB DST demonstrates high diagnostic accuracy for MDR-TB and pre-XDR-TB drugs, while caution is required with cycloserine, moxifloxacin, and para-aminosalicylic acid. These findings support the integration of MIC-based testing into clinical decision-making.

Broth microdilution (BMD) susceptibility testing with Acinetobacter spp. can be challenging with skipped wells and trailing observed for some agents. This also applies to cefiderocol, where isolates can display different growth patterns, such as trailing, regrowth, or hazy growth. To better understand the growth patterns observed with cefiderocol BMD testing, the content of wells was evaluated microscopically. Analysis of the trailing, hazy, and regrowth wells above the MIC endpoints showed disturbed growth with long filamentous and spherical cells characteristic of cell envelope instability observed with other β-lactams. When a cell metabolic activity reagent was used to evaluate the viability of the cells in wells above the MIC, inconclusive results were obtained, but when plated, substantial growth was noted for most wells, demonstrating that those cells were able to escape cefiderocol's bactericidal activity. Replicate testing (n = 5) showed that aberrant growth patterns were reproducible, with some MIC variability observed with isolates showing regrowth or trailing, and did not correlate with growth patterns that can be noted in cefiderocol disk diffusion testing (inner colonies, ghost zones, or fuzzy edges). When the prevalence of the different BMD growth patterns was evaluated with 455 isolates from Europe and the United States tested as part of a surveillance program for cefiderocol, about 60% of the isolates exhibited unclear endpoints, and these were noted in most countries, Acinetobacter species, and different genetic backgrounds. Unclear endpoints are not uncommon when testing Acinetobacter spp. against cefiderocol, and tolerability of cells in the presence of cefiderocol seems to be the cause of this. IMPORTANCE:Cefiderocol susceptibility testing for Acinetobacter spp. is challenging due to the aberrant growth patterns that occur in the broth microdilution method, including trailing, regrowth, and haziness. We initiated this study to better understand these phenomena and assess if these growth patterns were reproducible. We concluded that cefiderocol is not bactericidal against certain isolates and that, despite the cell membrane biosynthesis process being impaired, the cells do not undergo lysis as expected with β-lactam agents. We also evaluated the prevalence of these aberrant growth patterns and evaluated the genetic traits of the isolates producing them, showing that these patterns are common and not related to geographic origin or specific genetic traits.

Hepatitis B virus (HBV) infection remains a major global health challenge, necessitating rapid and accurate diagnostic methods for effective disease management. Although quantitative real-time PCR (qPCR) is widely used, its detection time is relatively long. We developed an ultra-fast HBV-DNA quantification system based on rapid VPCR technology, which, combined with a 10-min nucleic acid extraction process, reduces the entire workflow to approximately 35 min. Crucially, despite this significant increase in speed, the system maintains analytical sensitivity, specificity, and reproducibility comparable to those of conventional qPCR. The system's performance was then evaluated using 143 clinical samples and compared to that of standard qPCR, demonstrating that the developed real-time VPCR system significantly shortened the amplification time while maintaining excellent performance, with 100% sensitivity and 97.09% specificity. Additionally, a cost-effective qualitative endpoint visual detection method based on probe cleavage was established, offering an alternative readout strategy suitable for HBV screening in resource-limited settings without requiring expensive instruments. Collectively, this study presents a rapid quantification system and a qualitative visual detection method that significantly enhance diagnostic efficiency, providing strong technical support for clinical HBV management and screening.IMPORTANCEThe rapid hepatitis B virus (HBV) quantification system established in this study completes the entire workflow within 35 min and delivers excellent performance, meeting the demand for rapid decision-making in time-sensitive scenarios such as emergency care and acute liver failure. Its "test-and-treat" workflow is particularly suitable for patients in remote areas, allowing diagnosis and treatment in a single visit. Meanwhile, the accompanying visual qualitative method provides a low-cost screening strategy for resource-limited settings. This system greatly improves detection efficiency and clinical accessibility, showing important application value for the rapid diagnosis of hepatitis B and other pathogens.

Herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV) are associated with a range of clinical manifestations depending on the anatomical site of infection. HSV and VZV lesions often exhibit similar phenotypical characteristics, making clinical differentiation challenging. Detection and differentiation of HSV-1, HSV-2, and VZV are essential for diagnosis and treatment management. An automated multiplex real-time PCR assay, Alinity m HSV 1 & 2/VZV assay, was developed to detect and differentiate HSV-1, HSV-2, and VZV. The assay was validated for use with cutaneous and mucocutaneous lesion swab specimens collected in commercial viral transport media from symptomatic patients. The assay demonstrated a limit of detection of 5.90 TCID50/mL for HSV-1, 2.07 TCID50/mL for HSV-2, and 0.055 TCID50/mL for VZV. Testing with clinical swab specimens demonstrated greater than 95% clinical sensitivity and specificity for all three analytes. The Alinity m HSV 1 & 2/VZV assay provides a reliable and accurate method for detection and differentiation of HSV-1, HSV-2, and VZV in lesion swab specimens for improved diagnostic accuracy and patient management.IMPORTANCEHerpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV) can cause skin and mucocutaneous lesions that often appear clinically similar, making accurate diagnosis difficult without laboratory testing. Rapid and reliable identification of the causative virus is important because patient counseling, treatment decisions, and infection management differ among these infections. In this multicenter study, a fully automated molecular assay was evaluated that simultaneously detects and differentiates HSV-1, HSV-2, and VZV from a single-lesion swab specimen. Testing of more than 1,200 clinical specimens demonstrated high sensitivity and specificity for all three viruses. The assay also offers a streamlined workflow with minimal hands-on time and rapid turnaround. These findings support the use of this multiplex assay as a practical diagnostic tool that can improve laboratory efficiency and provide clinicians with timely, accurate results to guide patient care.