Understanding the burden of pertussis and pertussis-related mortality is important in enabling a better understanding of population vulnerability and supporting vaccination program planning. However, changes in laboratory practice can have important implications for public health surveillance. This study considers the impact of increasing use of multiplex panel testing for respiratory pathogens on the ascertainment of pertussis-related non-infant deaths, drawing on multiple surveillance data sources from England. Information was sourced from the Office of National Statistics cause of death records, public health case management records, laboratory results collated nationally, and death notifications received via General Practices. Figures covered the period 2023/24, coinciding with a large, national outbreak in England, and were compared with data from 2012-the last major outbreak in England. Of 34 deaths identified as potentially pertussis-related among individuals aged >12 months in 2023/24, 11 ultimately met the pre-defined criteria to be classified as pertussis-attributable; none of the five possible cases identified in 2012 did so. A total of 29 of the 34 (85%) tested positive for pertussis via multiplex panel testing, of which 17 out of the 29 (59%) were ultimately excluded as pertussis-related deaths. Findings highlight significant complexity in the attribution of pertussis-related mortality in an era of widespread multiplex panel testing.IMPORTANCECorrectly identifying deaths caused by pertussis (whooping cough) is important for timely public health action but is not straightforward. We aimed to find out how many deaths potentially caused by pertussis that are notified to national authorities in England are really caused by the infection, in a context where many hospitalized patients are now being tested using multiplex panel testing. Overall, 68% of those cases identified as potential pertussis deaths through regular pertussis surveillance processes in England proved not to have been caused by the infection. This finding highlights challenges to surveillance of pertussis-related deaths in the era of widespread multiplex panel testing use. It may point to the need for clearer guidance concerning the use of this testing approach.

This dual-center study evaluated the impact of artificial intelligence (AI) on urine culture turnaround times in Canadian diagnostic laboratories using microbiology laboratory automation. Data were collected before and after the implementation of PhenoMATRIX (PM), an AI-based software that provides continuous culture sorting and result interpretation support. In both a low-volume tertiary care hospital and a high-volume community laboratory, PM enabled earlier availability of interpretable results; however, reductions in time to result reporting (TTRR) were contingent on workflows for result release. At the tertiary care site, implementation of PM alone was associated with increased TTRR, reflecting delays between result availability and reporting. Integration of PM+ enabled automated release of negative results as they became available, resulting in a TTRR reduction of approximately 1.3 h. At the community laboratory, where PM+ was not implemented, a TTRR improvement of approximately 5.3 h was achieved by advancing manual screening workflows (08:00 vs 16:00), facilitating earlier review and release of PM-generated results. These findings indicate that AI-driven culture assessment reduces TTRR when coupled with processes that enable timely result release, either through automated reporting or optimized laboratory review workflows.IMPORTANCEAdvances in artificial intelligence (AI), coupled with the power of laboratory automation, is the next step in the evolution of the clinical microbiology laboratory. We demonstrated that bacterial culture assessment (urine cultures) by AI decreases the time required to release results to clinicians and reduces the hands-on time required by technologists to analyze and finalize laboratory analysis. This study was performed in two different laboratories that differed not only in geography but also in scope and scale. The same ultimate benefits were seen in both institutions demonstrating that this technology is widely applicable for most laboratories.

Frontline hospitals are required to care for patients with suspected viral hemorrhagic fever (VHF), yet guidance on laboratory preparedness remains fragmented and incomplete. We conducted a multidisciplinary risk assessment of our institutional capacity to perform routine diagnostic testing for VHF persons under investigation (PUI), focusing on the feasibility of using automated core laboratory instruments. Our assessment revealed substantial gaps between CDC guidance (which permits core lab testing) and the practical ability to implement it safely. Public health mandates for VHF preparedness have not been accompanied by granular guidance on biosafety, laboratory infrastructure, or regulatory clarity necessary for implementation. Community hospitals, which would benefit most from safely using their existing automated core laboratory instruments, lack the infrastructure, staffing expertise, and clear guidance to do so, while well-resourced tertiary centers are often best positioned to develop dedicated point-of-care testing (POCT)-based workflows. Federal and state authorities must provide explicit, validated examples of acceptable mitigation strategies for testing using core lab instrumentation and reconcile conflicting recommendations across guidance documents. Without such authoritative clarity, frontline hospitals cannot confidently meet their mandated VHF preparedness obligations.

Macrolide-resistant Mycoplasma pneumoniae (MRMP) is increasingly detected worldwide, with increasing prevalence even in the United States. Detection of resistance within M. pneumoniae has meaningful clinical and stewardship implications. Resistance is most commonly mediated by point mutations within 23S rRNA and can be reliably identified by molecular assays, not just on isolates butalso on primary respiratory samples like swabs. Multiple studies associate macrolide resistance with prolonged fever, delayed clinical response, increased healthcare utilization, and greater antibiotic exposure due to delay or treatment failure. The detection of resistance can meaningfully influence the clinical course and overall patient outcomes. Though clinical disease is not any more severe in infections with resistant isolates, failure to report may perpetuate unnecessary macrolide use, leading to delay in transition to effective second-line agents (tetracyclines or fluoroquinolones when appropriate), and contribute to selective pressure. Beyond individual patient care, routine reporting provides valuable local epidemiologic data to inform empiric therapy, support outbreak recognition, and guide antimicrobial stewardship initiatives. Even so, a significant clinical gap remains as most commercially available diagnostic assays do not routinely detect or report resistance markers in Mycoplasma pneumoniae. Moreover, even among assays designed to detect resistance, performance can vary considerably with reduced sensitivity for organism detection and incomplete identification of key 23S rRNA mutations reported in comparative evaluations, as reported by Hénin N, Silvant A, Gardette M, Balcon C, Guiraud J, Bébéar C, Pereyre S, J Clin Microbiol 64: e0149125, 2026, https://doi.org/10.1128/jcm.01491-25. Focused efforts are, therefore, needed to close existing diagnostic and reporting gaps to ensure resistance detection is more consistently integrated into routine clinical practice.

Human herpesvirus 6 (HHV-6) comprises two genetically and biologically distinct species, HHV-6A and HHV-6B, yet species-level differentiation is rarely performed in clinical practice. We conducted a retrospective study of 119 immunocompromised adults with detectable HHV-6 DNA between 2020 and 2025 to characterize species distribution, compare epidemiological, clinical, and virological features, and evaluate the diagnostic relevance of genotyping. Viral species were detected by real-time polymerase chain reaction (PCR) across multiple sample types, and suspected chromosomal integration was confirmed through detection of endogenous HHV-6 (eHHV-6) DNA in hair follicles by quantitative real-time PCR. HHV-6B accounted for ~96% of infections and was significantly associated with organ involvement, predominantly gastrointestinal disease, irrespective of plasma viral load. In contrast, HHV-6A was less frequently detected in tissues and was consistently associated with viremia and chromosomal integration, independent of viral load. Genotypic concordance across plasma, biopsy, cerebrospinal fluid, bronchoalveolar lavage, and hair follicle samples from the same patient was complete. Incorporating species-level genotyping and targeted testing for chromosomal integration (eHHV-6) into diagnostic workflows may improve interpretation of viral detection and help prevent unnecessary antiviral therapy.IMPORTANCESpecies-specific identification of human herpesvirus 6 (HHV-6A/B) has critical diagnostic implications in immunocompromised patients, although it is rarely performed in diagnostic laboratories. Our study showed that HHV-6A detection is associated with chromosomal integration, whereas HHV-6B is the main species linked to clinically relevant disease and invasive tissue infection. This study emphasizes that implementing species-specific PCR and testing for eHHV-6 in clinical microbiology laboratories may help interpret viral DNA detection, prevent misclassification of integrated virus as active infection, and avoid unnecessary antiviral therapy.

Candida auris is a multidrug-resistant fungal pathogen associated with healthcare outbreaks and high mortality. Its accurate and timely identification is critical for infection prevention, yet conventional culture-based methods are limited by slow growth, morphological ambiguity, and potential misidentification. This multicenter study evaluated the performance of the Simplexa C. auris Direct assay using 2,020 axilla/groin swab specimens collected from six clinical sites compared to culture followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Specimens were tested prospectively (n = 1,930) and retrospectively (n = 90), with demographic data spanning inpatient, intensive care unit, emergency department, and long-term acute care hospital settings. Discordant results were resolved by bi-directional sequencing, targeting the internal transcribed spacer and D1/D2 regions of the 28S ribosomal DNA gene. Compared to culture followed by MALDI-TOF MS identification, the Simplexa assay demonstrated an overall sensitivity of 94.8% and specificity of 98.7%, with a Cohen's kappa of 0.80, indicating strong agreement. Discordance rates were low (1.4%), and diagnostic accuracy was high (98.6%). The limit of detection was 127 CFU/mL for Clade I and 260 CFU/mL for Clade IV. Mean cycle threshold values were significantly lower in culture-positive specimens (25.1, 95% CI, 23.6-26.7) compared to culture-negative ones (33.6, 95% CI, 32.1-35.0; P < 0.0001). Comparative analysis with four lab-developed molecular tests (LDTs) showed low rates of discordance: 0.9% for LDT 1 and 2, 1.5% for LDT 3, and 0% for LDT 4, indicating a high level of agreement between the molecular methods. The Simplexa C. auris Direct assay provides a rapid and reliable alternative to traditional methods, providing results in under 2 h, supporting early detection and containment of C. auris in healthcare settings.

Blood culture contamination can lead to false-positive blood culture results, potentially contributing to increased costs and unneeded antibiotic utilization. Initial specimen diversion devices (ISDD) have been proposed to reduce blood culture contamination rates. A single-center, observational study conducted at Mayo Clinic Rochester compared blood culture contamination rates using Steripath or Kurin ISDDs versus standard blood culture collection protocols. Over 255 phlebotomists were trained in the use of the ISDDs, with 31,215 blood cultures, 410 of which were contaminated, collected from adults in the emergency department and inpatient wards. Baseline contamination rates were 1.29%-2.53% with standard collection methods. The Steripath ISDD with direct-to-media inoculation of blood culture bottles (Steripath-DTM) reduced blood culture contamination compared to standard practice (0.34% vs. 1.43%, respectively, P < 0.001). Follow-up studies with the alternative Steripath configuration with an integrated syringe (Steripath-syringe), and then the Kurin ISDD with syringe collection (Kurin-syringe), did not meet the primary endpoint of <0.5% contamination (Steripath-syringe: 1.28%; Kurin-syringe: 1.76%). All three ISDD configurations failed to meet our goal of >75% utilization, with an average measured utilization of 26% for the Steripath-DTM, 26.4% for the Steripath ISDD (combined DTM and syringe) in a follow-up study, and 41.5% in the Kurin study. Additionally, a survey of phlebotomists revealed low staff satisfaction with both Steripath ISDD configurations. Staff reported multiple ergonomic barriers limiting the Steripath ISDD's utility, which precluded implementation of the Steripath-DTM in routine clinical practice.

Laboratory-based diagnosis of active hepatitis C virus (HCV) infection currently requires a positive antibody test and a confirmatory RNA test. Many institutions in the United States continue to require two separate sample collections, partially due to concerns for carryover contamination. Reflexive RNA testing of antibody-positive samples reduces loss to follow-up, improves linkage to care, and is a recommended operational strategy by the Centers for Disease Control and Prevention. This study describes false positivity rates of reflexive RNA testing in a reference laboratory before and after introduction of a risk mitigation strategy that requires medical director review for reflexed HCV viral loads < 1,000 IU/mL (defined as low-positive). Initial HCV antibody testing was performed at one of five chemistry laboratories; reflex HCV RNA testing was performed using the cobas HCV assay on cobas 8800 (Roche) at a central reference laboratory. A total of 4,079 consecutive positive RNA results were included pre-intervention, and 4,433 were included post-intervention. All low-positive results (N = 157) were retrospectively adjudicated as true-positive (N = 66), false-positive (N = 38), or unknown (N = 53), based on clinical history and redraw results. The pre-intervention false-positive rate was 0.44%, which was significantly reduced to 0.14% post-intervention, P = 0.008. Median viral load for those clinically adjudicated as true-positive was significantly higher at 97 IU/mL (IQR: 24, 330 IU/mL) compared to 17 IU/mL (IQR: 14, 58 IU/mL) classified as false-positive or 16 IU/mL (IQR: 14, 62 IU/mL) classified as unknown. This study supports wider implementation of reflexive RNA testing for antibody-positive samples and provides a blueprint for a risk mitigation strategy.IMPORTANCEMost patients infected with hepatitis C virus are unable to spontaneously clear the virus and develop chronic infection. Chronic hepatitis C virus (HCV) infection can lead to life-threatening complications including liver cirrhosis, liver failure, and liver cancer. Therefore, accurate diagnosis of hepatitis C infection and linkage to treatment is paramount. To increase linkage to care, current Centers for Disease Control and Prevention (CDC) guidelines recommend single-visit sample collection to test for antibodies and, if antibody positive, viral RNA. However, almost half of the laboratories do not use reflexive testing strategies, in part due to concerns for carryover contamination. Our study describes a low risk of false-positive RNA results and suggests a risk mitigation intervention to further minimize false positives. Our work reaffirms CDC guidelines and provides real-world data to encourage other facilities to follow a reflex testing algorithm to increase linkage to care.

Fonsecaea monophora is a dematiaceous fungus with worldwide distribution that can cause chromoblastomycosis, an implantation mycosis and a neglected tropical disease. F. monophora also causes phaeohyphomycosis, including central nervous system infections like brain abscesses, with high mortality rates. In the absence of clinical breakpoints, epidemiological cutoff values (ECVs) can aid clinicians in monitoring antifungal resistance trends and guide initial antifungal selection. To establish F. monophora MIC distribution and ECVs, we performed antifungal susceptibility testing (AFST) on F. monophora isolates. AFST data on F. monophora isolates were collected at 14 laboratories during September 2023-May 2026. Species identification was previously confirmed by DNA sequence analysis. AFST was performed by CLSI M38 standard broth microdilution method for itraconazole, voriconazole, posaconazole, isavuconazole, ketoconazole, terbinafine, flucytosine, and amphotericin B. The ECVs were established using the iterative statistical method with ECOFFinder (v2.1) following CLSI M57 guidelines. We analyzed MIC results from 137 F. monophora isolates. The calculated ECVs were itraconazole, 2 μg/mL; voriconazole, 0.25 μg/mL; posaconazole, 1 μg/mL; isavuconazole, 0.25 μg/mL; and terbinafine, 0.5 μg/mL. Amphotericin B and flucytosine had bimodal distributions, and no ECVs were set. Ketoconazole did not have data from ≥100 isolates. These F. monophora ECVs can detect non-wild-type isolates to reevaluate antifungal treatment and investigate treatment failure associated with potential clinical resistance. Established through a global multicenter collaboration, these values provide a baseline to better understand the in vitro antifungal susceptibility profile of this species and monitor resistance.IMPORTANCEFonsecaea monophora is a dematiaceous fungus with worldwide distribution that can cause chromoblastomycosis, an implantation mycosis and neglected tropical disease of the skin and subcutaneous tissue. F. monophora also causes phaeohyphomycosis, including central nervous system infections like brain abscesses with high mortality rates. The paucity of antifungal susceptibility testing data on F. monophora complicates interpretation of minimum inhibitory concentration (MIC) values. We performed antifungal susceptibility testing on 137 F. monophora isolates to establish MIC distributions and epidemiological cutoff values (ECVs) for eight antifungals, including those commonly used to treat F. monophora infections. The calculated ECVs for commonly used antifungals were 2 μg/mL for itraconazole, 0.25 μg/mL for voriconazole, 1 μg/mL for posaconazole, and 0.5 μg/mL for terbinafine. ECVs can assist in selecting potential treatments for F. monophora and tracking antifungal resistance trends.

Tuberculosis (TB) remains a major global health challenge, requiring rapid, reliable molecular tests to detect the Mycobacterium tuberculosis complex (MTBC) and resistance to the drugs that cause multidrug resistance, i.e., rifampicin and isoniazid. Although many moderate-complexity automated nucleic acid amplification tests use multiple targets to improve sensitivity, previous versions of FluoroType MTBDR (FTv2old; Bruker-Hain) relied on a single MTBC DNA target, often necessitating the use of an initial multilocus MTBC assay in the workflow to achieve comparable sensitivity. This prospective head-to-head study compared FTv2old with an updated version (FTv2new) that includes the multi-copy IS6110 insertion sequence to improve MTBC detection. Using 1,483 clinical specimens from pulmonary and extrapulmonary sources, with culture and whole-genome sequencing (WGS) as reference standards, FTv2new showed a significant 17-percentage-point increase in overall sensitivity, reaching 87% compared with 70% for FTv2old (P = 0.008). The improvement was particularly notable in microscopy-negative, culture-positive samples, where the sensitivity increased from 50% to 78% (P = 0.008). Specificity remained high and comparable between versions at 99%. Because the assay's resistance-conferring targets remained unchanged, the capability for resistance profiling did not improve significantly. These findings indicate that inclusion of the IS6110 target closes the performance gap of the FluoroType platform, eliminating the need for reflex testing and streamlining laboratory workflows for high-throughput tuberculosis screening.IMPORTANCETuberculosis remains a major global health threat, and rapid, accurate diagnosis is essential for disease control. This study evaluates an updated molecular assay for detecting the Mycobacterium tuberculosis complex (MTBC). Earlier versions of this assay relied on a single genetic target and were less sensitive than competing methods. The inclusion of a multi-copy target significantly improves MTBC detection, particularly in specimens with low bacterial loads that are frequently microscopy-negative. This improvement is clinically relevant, enabling faster, more reliable diagnosis while eliminating the need for additional, time-consuming reflex testing, thereby streamlining laboratory workflows and supporting timely initiation of appropriate treatment, thereby supporting global efforts to eliminate TB.

Implant-associated infections, including periprosthetic joint infections (PJI) and fracture-related infections (FRI), are severe complications following orthopedic surgery. Conventional tissue cultures often yield false-negative results, especially after antibiotic initiation or in chronic biofilm-associated infections. Sonication of explanted implants enhances bacterial recovery, but heterogeneous protocols limit comparability. This scoping review maps sonication protocols currently reported in the literature. A comprehensive literature search across three databases (PubMed, Embase, and Scopus) was performed. Clinical studies that reported on sonication protocols used in the microbiological workup of PJIs and FRIs published from 2007 to October 2025 were eligible for inclusion. Eligible studies were screened for the following information: container size and material and fluids, sonication device and settings, pre- and post-sonication handling, culture conditions, reporting format, and/or diagnostic thresholds. Data were extracted and synthesized descriptively. After duplicate removal, 312 records were screened, and 146 studies were included. Reporting was incomplete for several key parameters. Container material was not specified in 43.2% of studies, container type in 30.8%, and fluid type in 31.0%. Among reported protocols, hard plastic containers (54.8%) and Ringer's lactate or saline (40.4%) were most frequently used, with fluid volumes ranging from 50 to 800 mL. Sonication frequency was reported in 50.7% of studies, most commonly 40 kHz (28.8%). Sonication time was not reported in 34.2%, with reported durations ranging from 1 to 10 min and most frequent duration as 5 min (30.1%). Vortexing and centrifugation were applied in 55.5% and 45.9% of studies, respectively. CFU thresholds varied widely, ranging from any growth to ≥50 CFU/mL or per plate. Time-to-culture and storage conditions were rarely reported. Sonication protocols vary greatly across almost all parameters, with detailed descriptions often lacking, and certain variables rarely reported. Standardizing these protocols is essential to ensure reproducibility and to establish clinically reliable diagnostic cut-offs.

Artificial intelligence-based culture reading tools can potentially accelerate reading, improving reporting consistency in image-based interpretation, which is relevant for high-volume urine cultures. This study evaluated the BD Kiestra Urine Culture Application (UCA) in a centralized microbiology laboratory. Consecutive midstream and indwelling catheter urine specimens inoculated onto BD BBL CHROMagar Orientation were analyzed by the UCA. Growth score output at different reporting thresholds was evaluated, along with expert rule-based categories constructed using the app's growth score, purity score, and presumptive identification parameters, with blinded technologist image reads as the comparator. Of 1,304 cultures, 1,251 were evaluable for growth score comparison, and 1,239 for expert-rule assessment. The exact agreement across four growth categories (<10[5], 10[5], 10[6], ≥10[7] CFU/L) was 86.2%. Using a <10[6] CFU/L triage threshold, the overall agreement was 95.9%, with positive and negative percent agreement of 89.9% and 98.9%, respectively. In this dataset, 30.5% of cultures met this threshold for potential auto-release or batch-review. For presumptive identification in ≥10[7] CFU/L pure or predominant cultures, UCA performed best for E. coli (PPA 98.9%). For Enterococcus spp., PPA was 89.5%, but PPV was low (25.4%), reflecting frequent false-positive presumptive calls. UCA performed well at clinically relevant thresholds and could meaningfully reduce the burden of manual reading. Most discrepancies reflected differences between total colony count outputs and morphotype-based laboratory quantitation conventions, along with missed subtle growth. Expert-rule category discrepancies were primarily related to differences in purity output and presumptive identification, particularly fine light-blue growth being called presumptive Enterococcus spp., highlighting a priority area for improvement.IMPORTANCEUrine cultures are among the most frequently ordered culture tests in clinical microbiology. Timely, consistent plate interpretation impacts both patient care and laboratory capacity. Digital imaging of culture plates facilitates the application of artificial intelligence to urine culture specimens, but laboratories need clear evidence of where these tools perform reliably and where their outputs require caution. This study shows how an automated urine culture reading application can support safe triage of cultures with little or no growth, helping technologists focus on specimens that likely require workup and clinical action. At the same time, it highlights an important limitation: organism labeling can be vulnerable to look-alike growth patterns. In our dataset, fine or hazy, light-blue growth that was not typical of Enterococcus spp. was classified by the application as presumptive Enterococcus spp., a mismatch that could affect downstream decisions if not recognized. These findings reinforce that local validation and human oversight remain essential, even for common uropathogens, such as Escherichia coli.

Coproparasitological stool analysis based on microscopic examination is the reference diagnostic technique routinely performed in clinical microbiology laboratories. Automated image analysis systems could offer a suitable solution, reducing technologist workload and improving screening efficiency. The Human Fecal Ova & Parasite (O&P) Detection Solution (Techcyte) is an artificial intelligence (AI) software employing imaging-based algorithms to provide screening presumptive diagnostic results. This study aimed to validate the Wet Mount Iodine Solution software for detecting and diagnosing protozoan and helminthic infections in stool specimens. A total of 178 clinical stool specimens were retrospectively analyzed between July and October 2024 for comparative evaluation of the AI-based system. Positive specimens with confirmed mono (n = 116) and mixed (n = 31) protozoa and helminth infections, as well as negative specimens (n = 31), were included. Conventional microscopy results were compared with AI presumptive detection and identification outcomes. In addition, operator-reviewed AI results were evaluated against the reference diagnosis. The system achieved 96.62% positive percent agreement (PPA) and 93.33% negative percent agreement (NPA) for overall detection considering operator-reviewed interpretation. The kappa coefficient was 0.865, indicating strong agreement with conventional microscopy. The AI system detected additional parasites in 74/178 (41.57%) specimens, providing a diagnostic improvement compared to traditional microscopy. AI-assisted microscopy enhances intestinal parasite diagnosis, providing reliable results when combined with operator review. Despite limitations such as specimen low volume analysis and artifact detection, the system shows strong potential for improving diagnostic workflows in clinical microbiology laboratories.