Journal of Clinical Microbiology

Rapid and accurate identification of cultured molds is important to determine clinical significance and therapeutic decision-making. Conventional mold identification uses phenotypic macroscopic and microscopic characterization; however, this can take days or weeks for colony maturity and definitive microscopic structure formation, be limited to genus-level identification, and be misidentified due to morphologic mimics or similarities between closely related species. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI–TOF MS) revolutionized bacterial and yeast identification but remains uncommon for molds in part because of limited reference libraries. Here, a retrospective 5-year review at a large teaching hospital found that 88.6% of identified molds were in the Bruker Filamentous Fungi Library 3.0 and 91.5% in the VITEK Knowledge Base Library 3.2.0. A prospective evaluation was also performed on early growth from 205 consecutive, working clinical isolates. Each mold was processed using the VITEK chemical extraction method and modified NIH chemical plus bead-beating extraction method; both extractions were tested on both systems. When compared to conventional identification, more molds were identified using VITEK extractions over NIH extractions using the VITEK (65 and 59%) and Bruker (56 and 54%) systems, respectively, using the ≥1.5 log Bruker threshold. VITEK MS identified more molds, regardless of the extraction method. Isolates without consensus agreement ( n = 116) underwent sequence-based identification, which demonstrated that conventional identification had the highest genus-level (84%) but lowest species-level (3%) identification rates compared to VITEK (59 and 52%, respectively) and Bruker (52 and 36%) using VITEK extractions. Taken together, our findings suggest both MALDI-TOF systems can supplement conventional mold identification to optimize identification rates with species-level distinction. IMPORTANCE Mold identification using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) remains uncommon in clinical laboratories. Contributing concerns include limited genus/species spectra in the MALDI-TOF MS libraries, varying success rates in the literature regarding extraction methods and instrumentation, and the lack of practical performance evaluations using early mold colony growth, which would be used in a clinical mycology laboratory. This study used multiple approaches to improve our understanding of the clinical utility and performance of MALDI-TOF MS mold identification.

The World Health Organization (WHO) 2030 roadmap for schistosomiasis calls for development of highly sensitive and specific diagnostic tools to continue and sustain progress towards elimination. Serological assays are excellent for sensitive detection of primary schistosome infections and for schistosomiasis surveillance in near- and post-elimination settings. To develop accurate assay formats, it is necessary to identify defined antibody targets with low cross-reactivity and potential for standardized production. Here we aim to identify such target(s) with focus on defined schistosome glycan antigens. Target identification was performed by assessing antibody responses in well-characterized cross-sectional and cohort sample sets ( n = 366 individuals) on tailor-made antigen microarrays. IgM and IgG binding to candidate diagnostic targets was measured for serum/plasma samples from controlled human schistosome infection models, schistosome-infected travelers, soil-transmitted helminth-infected individuals, and non-infected individuals. We found that antibodies to a schistosome gut-associated glycan, the circulating anodic antigen (CAA), identify schistosome infection with high sensitivity (IgM ≥100%, IgG ≥97%) and specificity (IgM ≥93%, IgG ≥97%) in the test samples. Infection dose affected timing of anti-CAA antibody isotype switch. Furthermore, we demonstrate that other non-specific glycan epitopes in crude schistosome cercarial and egg antigen preparations can contribute to generation of false schistosomiasis positives, which is relevant for current serological assays based on these antigen mixtures. In conclusion, CAA is an excellent single glycan antigen target for development of highly sensitive and specific tools for schistosomiasis serology with use cases for travelers and surveillance in near- and post-elimination settings, as well as emerging transmission zones. IMPORTANCE The WHO 2030 roadmap deems diagnostics developments for schistosomiasis critically needed. Here we present identification of an antibody target with superior performance compared to traditionally used crude antigens in schistosomiasis serology. Access to unique controlled human infection model samples, traveler samples, and negative controls enabled this discovery, which forms the basis for development of new diagnostic tools urgently needed in travel medicine, surveillance in emerging transmission zones driven by climate change, and in pre- and post-elimination scenarios.

Piperacillin-tazobactam (TZP) is a commonly used broad-spectrum agent. OXA-1 β-lactamases drive global Enterobacterales TZP resistance and raise MICs to the clinical breakpoints (8/4–16/4 µg/mL), making susceptibility testing challenging. Two TZP disks are used globally. The first with 100 µg piperacillin and 10 µg tazobactam, established in 1992, is used by laboratories following U.S. FDA and Clinical and Laboratory Standards Institute (CLSI) standards. The second, with 30 µg piperacillin and 6 µg tazobactam, was developed by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). When CLSI updated Enterobacterales TZP MIC breakpoints in 2022, it became apparent that the 100/10 µg disk may not accurately predict TZP resistance or susceptibility. In this study, we performed a disk mass titration study using 100 contemporary Enterobacterales isolates, including 40 harboring bla OXA-1 . Relative to reference broth microdilution with CLSI breakpoints, categorical agreement (CA) for the 100/10 µg disk was 68% with 0.6% major errors (MEs) and 32% minor errors. The CA for the 30/6 µg disk against EUCAST breakpoints was 88% with 15% very major errors and 10% ME. A third disk developed in this study, 20/5 µg, yielded 81% CA with CLSI MIC breakpoints and disk breakpoints generated using the error-rate-bounded method. CA was 62.4%, 63.2%, and 70.9% for isolates with bla OXA-1 using the 100/10, 30/6, and 20/5 µg disks, respectively, whereas it was 71.6%, 86.9%, and 83.1% for isolates without bla OXA-1 . Decreasing TZP disk potency improved the overall performance of disk diffusion and improved separation between susceptible and non-susceptible isolates, particularly those harboring OXA-1, but no disk yielded optimal results. IMPORTANCE In this article, we address major gaps in contemporary data for piperacillin-tazobactam (TZP) susceptibility testing and evaluate the performance of disk diffusion. TZP is the most common empiric broad-spectrum agent against Gram-negative pathogens and is used as a carbapenem-sparing regimen. OXA-1 β-lactamases drive global Enterobacterales TZP resistance and raise MICs to the clinical breakpoints, making susceptibility testing challenging. In 2022, CLSI revised the Enterobacterales TZP MIC breakpoints. Due to the lack of contemporary correlates, disk diffusion breakpoints were revised using outdated historical data from 1991 and 2003 and yielded unacceptable error rates. Additionally, there is a lack of global consensus on disk potency. The EUCAST TZP disks contain 30 μg piperacillin and 6 μg tazobactam. The CLSI disks, established after TZP approval in 1979 and prior to the widespread prevalence of ESBLs, contain 100 μg piperacillin and 10 μg tazobactam. Here, we evaluate disk diffusion using the 100/10 and 30/6 μg TZP disks with 100 contemporary Enterobacterales isolates, including 40 harboring bla OXA-1 . We conducted a disk development study to determine if an alternative potency might address accuracy issues with TZP susceptibility testing. We demonstrate that decreasing TZP potency improves the performance of disk diffusion and improves separation between susceptible and non-susceptible isolates, particularly those harboring OXA-1, but no disk yielded optimal results. The alternative 20/5 μg disk yielded the lowest errors using CLSI MIC breakpoints and the best separation between susceptible isolates and isolates harboring bla OXA-1 . Our study addresses an unmet need, shows that further optimization of the TZP disk potency is possible, and provides clinical laboratories with a better understanding of the performance of TZP disks using contemporary, challenging isolates. A larger, multicenter study is needed for further optimization but has been limited by a lack of funding for an off-patent antimicrobial. Our struggles in accessing funding underscore the frequent challenge with AST for older but heavily used antimicrobials.

Enterobacterales (mostly Klebsiella pneumoniae , Escherichia coli ) with OXA-48-like carbapenemases (e.g., OXA-48, -181, -232, -244) are undermining the global efficiency of carbapenem therapy. In the Middle East, North Africa, and some European countries, OXA-48-like carbapenemases are the most common types of carbapenemases among Enterobacterales . Currently, OXA-48 is endemic in the Middle East, North Africa, Spain, France, and Belgium; OXA-181 is endemic in Sub-Saharan Africa and the Indian Subcontinent, while OXA-232 has been increasing in the Indian Subcontinent. European countries (e.g., Germany, Denmark, Switzerland, France) are experiencing community outbreaks with E. coli ST38 that produce OXA-244, and these strains have been introduced into Norwegian, Polish, and Czech hospitals. The global ascendancy of OXA-48-like genes is due to the combination of carbapenemases with horizontal spread through promiscuous plasmids (e.g., IncL, IncX3, ColE2) and vertical spread with certain high-risk multidrug-resistant clones (e.g., K. pneumoniae ST14, ST15, ST147, ST307; E. coli ST38, ST410). This is a powerful “gene survival strategy” that has assisted with the survival of OXA-48-like genes in different environments including the community setting. The laboratory diagnosis is complex; therefore, bacteria with “difficult to detect” variants (e.g., OXA-244, OXA-484) are likely underreported and are spreading silently “beneath the radar” in hospital and community settings. K. pneumoniae and E. coli with OXA-48-like carbapenemases are forces to be reckoned with.

Urinary tract infections (UTIs) impose a substantial burden on patient quality of life and urine testing accounts for the majority of workload in many clinical microbiology laboratories. Traditional UTI diagnosis relies on symptoms, urinalysis, and culture which are interpreted based on historical guidelines. This approach, while foundational, presents limitations, particularly in complex cases. Low-level bacteriuria and the presence of fastidious organisms are often overlooked or entirely missed in standard urine culture, stressing the need for novel diagnostic methods and technologies. This mini-review summarizes the existing state of UTI diagnostics in 2024 and covers current and upcoming technologies including rapid molecular-based pathogen identification, next-generation sequencing, and advanced antimicrobial susceptibility testing. However, these methods represent unique challenges, and as they are implemented, they will require the field to adapt to new concepts to avoid misdiagnosis and overtreatment.

Human papillomavirus (HPV) genotype predicts cervical cancer risk, and genotyping could help guide the management of HPV positives as part of cervical screening. An isothermal amplification HPV extended genotyping test (ScreenFire HPV RS assay) can assay up to 96 samples/controls in 1 hour plus preparation time. A novel format with pre-aliquoted reagents and an anti-contamination component (Zebra BioDome) could simplify the HPV testing process and reduce the chances of post-amplification contamination. We validated the Zebra BioDome formulation prior to its clinical use. Residual provider-collected cervical samples ( n = 450) from a population-based study in rural Nigeria were retested with ScreenFire, once using the standard assay version (liquid reagents combined onsite) and twice with Zebra BioDome. HPV results with adequate DNA ( N = 427) were analyzed channel-by-channel and using the cervical cancer risk-based hierarchy of HPV type channels (HPV16, else 18/45, else 31/33/35/52/58, else 39/51/56/59/68, else high-risk HPV negative) to evaluate Zebra BioDome repeatability and accuracy against the standard version. Zebra BioDome reduced the number of pipetting steps to run the ScreenFire HPV assay. Following amplification, the BioDome material formed a sealant layer above the reaction components. Zebra BioDome had excellent repeatability and agreement with the standard version, both at the channel-specific analysis (positive percent agreement between 88.4% [HPV39/51/56/59/68] and 100% [HPV16]; negative percent agreement between 97.8% [HPV31/33/35/52/58] and 100% [HPV39/51/56/59/68]) and hierarchical analysis (overall agreement 97.2%). The assay version utilizing Zebra BioDome performed similarly to the previously validated standard version of the ScreenFire HPV assay and is now undergoing field evaluation. This solution has the potential to reduce assay preparation time and risk of contamination, providing a simpler, low-cost, near-point-of-care HPV testing and extended genotyping solution for cervical screening in lower-resource settings. The potential application of Zebra BioDome technology to other PCR assays should be considered. IMPORTANCE This work validates a novel pre-packed formulation for the ScreenFire human papillomavirus (HPV) assay, which has the potential to simplify the HPV testing process and to reduce the chances of post-amplification contamination, providing a simpler, low-cost, near-point-of-care HPV testing, and extended genotyping solution for cervical screening in resource-limited settings as part of the ultimate public health goal to accelerate cervical cancer prevention. This technology can also have broad applications for other DNA amplification assays beyond HPV.

Cholera rapid diagnostic tests (RDTs) are vulnerable to virulent bacteriophage predation. We hypothesized that an enhanced cholera RDT that detects the common virulent bacteriophage ICP1 might serve as a proxy for pathogen detection. We previously developed a monoclonal antibody (mAb) to the ICP1 major capsid protein. Our objective was to design and assemble a first-of-its-kind RDT that detects both a bacterial pathogen ( Vibrio cholerae ) and associated virulent bacteriophage (ICP1). Candidate mAbs were expanded to increase design options and evaluated by immunological assays (ELISA; western blot). A subset of mAbs were selected for gold conjugation and printing on the RDT. The detection limit of the prototype RDTs was determined in diarrheal stools with the addition of ICP1. Three mAb candidates were developed and evaluated for the capsid decoration protein (ORF123) and tail fiber protein (ORF93), and the prior mAb for the major capsid protein (ORF122). A single mAb sandwich RDT prototype for ORF122 was able to detect ICP1; RDTs with mAbs to ORF123 and ORF93 failed to detect ICP1 in single- or dual-sandwich configurations. Biologically relevant concentrations for ICP1 were detected only after boiling the stool with ICP1; analysis by electron microscopy (EM) suggested increased epitope availability after boiling. In this study, we demonstrate a proof of concept for a functional RDT that can detect both the primary pathogen and a common virulent bacteriophage as a proxy for pathogen detection. Further optimization is required before scaled production and implementation. IMPORTANCE This paper represents an important step forward to address the vulnerability of cholera RDTs to the effects of phage predation on the target Vibrio cholerae . The assembly and evaluation of an RDT that detects both the primary pathogen and a phage as a proxy for the primary pathogen is an innovative solution. When optimized and evaluated in clinical studies, this tool may become critical in the cholera response tool kit as well as represent a diagnostic proof-of-concept for other infectious agents.

The performance of the Liofilchem Compact Antimicrobial Susceptibility Panel (ComASP) Cefiderocol was evaluated in a multicenter study. Enterobacterales, Acinetobacter baumannii , and Pseudomonas aeruginosa clinical isolates and challenge isolates were tested by three and one sites, respectively. Minimum inhibitory concentration (MIC) testing was performed by the Clinical and Laboratory Standards Institute (CLSI) broth microdilution and ComASP, which included two reading endpoints (CLSI read; MIC is the first well in which reduction of growth is <1 mm or light haze/faint turbidity] and ComASP [ComASP read; MIC is the first well at which 100% inhibition of growth occurs]). Each site performed reproducibility and quality control (QC) by ComASP and broth microdilution (BMD). Reproducibility was excellent (97.4% within ±1 dilution of modal MIC). All QC results were within CLSI QC ranges by BMD and ComASP, except for two E. coli ATCC 25922 results from one site. Essential agreement for combined clinical and challenge Enterobacterales was 84.3% (CLSI read) and 95.7% (ComASP read), P. aeruginosa was 83.3% (CLSI read) and 93.7% (ComASP read), and A. baumannii was 78.3% (CLSI read) and 96.7% (ComASP read). Categorical agreement for Enterobacterales was 92.4% for both CLSI read and ComASP read, for P. aeruginosa was 89.7% (CLSI read) and 92.1% (ComASP read), and for A. baumannii was 72.8% (CLSI read) and 91.3% (ComASP read). There were no very major errors using the ComASP read. One very major error for P. aeruginosa occurred using the CLSI read method. Three very major errors for A. baumannii occurred using the CLSI read method. ComASP Cefiderocol was shown to be a reliable method for testing cefiderocol MIC against relevant clinical isolates when ComASP read is used. IMPORTANCE There are very limited commercial methods available to clinical laboratories for cefiderocol minimum inhibitory concentration (MIC) testing. The Compact Antimicrobial Susceptibility Panel (ComASP) Cefiderocol method includes iron-depleted cation-adjusted Mueller–Hinton broth, which eliminates variability in cefiderocol MIC results based on iron levels. The lyophilized multi-well format of ComASP also provides for room temperature storage. In comparison to what an individual lab may do for method verification, this multi-site, multi-isolate study provides a robust evaluation and greater assurance to clinical microbiologists of the method's accurate and reproducible performance.

Tongue swab (TS) sampling combined with quantitative PCR (qPCR) to detect Mycobacterium tuberculosis (MTB) DNA is a promising alternative to sputum testing for tuberculosis (TB) diagnosis. In prior studies, the sensitivity of tongue swabbing has usually been lower than sputum. In this study, we evaluated two strategies to improve sensitivity. In one, centrifugation was used to concentrate tongue dorsum bacteria from 2-mL suspensions eluted from high-capacity foam swab samples. The pellets were resuspended as 500-µL suspensions, and then mechanically lysed prior to dual-target qPCR to detect MTB insertion elements IS 6110 and IS 1081 . Fractionation experiments demonstrated that most of the MTB DNA signal in clinical swab samples (99.22% ± 1.46%) was present in the sedimentable fraction. When applied to archived foam swabs collected from 124 South Africans with presumptive TB, this strategy exhibited 83% sensitivity (71/86) and 100% specificity (38/38) relative to sputum microbiological reference standard (MRS; sputum culture and/or Xpert Ultra). The second strategy used sequence-specific magnetic capture (SSMaC) to concentrate DNA released from MTB cells. This protocol was evaluated on archived Copan FLOQSwabs flocked swab samples collected from 128 South African participants with presumptive TB. Material eluted into 500 µL buffer was mechanically lysed. The suspensions were digested by proteinase K, hybridized to biotinylated dual-target oligonucleotide probes, and then concentrated ~20-fold using magnetic separation. Upon dual-target qPCR testing of concentrates, this strategy exhibited 90% sensitivity (83/92) and 97% specificity (35/36) relative to sputum MRS. These results point the way toward automatable, high-sensitivity methods for detecting MTB DNA in TS. IMPORTANCE Improved testing for tuberculosis (TB) is needed. Using a more accessible sample type than sputum may enable the detection of more cases, but it is critical that alternative samples be tested appropriately. Here, we describe two new, highly accurate methods for testing tongue swabs for TB DNA.

Determination of antimicrobial resistance (AMR) in pneumococcal isolates is important for surveillance purposes and in a clinical context. Antimicrobial susceptibility testing (AST) of pneumococci is complicated by the need for exact minimal inhibitory concentrations (MICs) of beta-lactam antibiotics. Two next-generation sequencing (NGS) analysis tools have implemented the prediction of AMR in their analysis workflow, including the prediction of MICs: Pathogenwatch ( https://pathogen.watch/ ) and AREScloud (OpGen). The performance of these tools in comparison to phenotypic AST following EUCAST guidelines is unknown. A total of 538 Streptococcus pneumoniae isolates were used to compare both tools with phenotypic AST for penicillin, amoxicillin, cefotaxime/ceftriaxone, erythromycin, trimethoprim-sulfamethoxazole, and tetracycline. Disk diffusion was performed for all isolates, and broth microdilution was performed for isolates with reduced beta-lactam susceptibility. Demultiplexed FASTQ files from Illumina sequencing, covering the whole genome of pneumococci, were used as input for the NGS tools. Categorical agreement (CA), major error (ME), and very major error (VME) rates were calculated. For beta-lactam antibiotics, CA was high (>94%) associated with none or only one ME and VME (<1%). For erythromycin and tetracycline, CA was >93% for predictions by AREScloud, while for Pathogenwatch, this ranged around 88%. For trimethoprim-sulfamethoxazole, CA was for both tools <86%. High VME rates were observed for erythromycin and tetracycline, higher for Pathogenwatch (53.6% and 47.0%, respectively) compared to AREScloud (14.3% and 19.1%, respectively). Both tools performed excellently despite the complexity of predicting beta-lactam resistance in pneumococci. Further optimization and validation are needed for non-beta-lactams since high (very) major error rates were observed.

The aim of this study was to identify parameters influencing DNA extraction and PCR amplification efficiencies in an attempt to standardize Mucorales qPCR. The Fungal PCR Initiative Mucorales Laboratory Working Group distributed two panels of simulated samples to 26 laboratories: Panel A (six sera spiked with Mucorales DNA and one negative control serum) and Panel B (six Mucorales DNA extracts). Panel A underwent DNA extraction in each laboratory according to the local procedure and were sent to a central laboratory for testing using three different qPCR techniques: one in-house qPCR assay and two commercial assays (MucorGenius and Fungiplex). Panel B DNA extracts were PCR amplified in each laboratory using local procedures: nine in-house qPCR assays and two commercial kits (MucorGenius and MycoGENIE). All data were compiled and anonymously analyzed at the central laboratory. For Panel A, a total of six different automated platforms and five manual extraction methods were used. Positive rates were 64%, 70%, and 89%, for the MucorGenius, Fungiplex, and the in-house qPCR assay, respectively. Using a large volume of serum for DNA extraction provided the highest analytical sensitivity (82.5% for 1 mL compared with 62.7% for smaller volumes, P < 0.01). For Panel B, five in-house qPCR assays and two commercial kits had >78% positivity. Using larger PCR input volumes (≥7 µL) was associated with the highest sensitivity at 95.5% compared to 58.3% when lower input volumes were used ( P < 0.01). Using larger sample volumes for nucleic acid extraction and DNA template volumes for PCR amplification significantly improves the performance of Mucorales qPCR when testing serum. IMPORTANCE Mucormycosis is a life-threatening mold infection affecting immunosuppressed patients but also other patients with diabetes or trauma. Better survival is linked to shorter delays in diagnosis and treatment initiation. Detection of Mucorales-free DNA in serum or plasma using quantitative PCR allows a prompt diagnosis and earlier treatment. Several techniques and protocols of quantitative Mucorales PCR are used in Europe, and improving performance remains a common objective of laboratories participating in the fungal PCR Initiative Working Group. This study, which combined results from 26 laboratories in Europe, showed that the main parameters underpinning sensitivity are the preanalytical variables (volume of serum used for DNA extraction and DNA template volume), irrespective of the extraction platforms and qPCR assay/platform.

Despite first-void urine (FVU) being increasingly recognized as a credible specimen for human papillomavirus (HPV) detection, there is a lack of well-validated testing methods providing full quantitative genotyping required for vaccine impact monitoring from FVU samples. The Allplex HPV28 assay, capable of individually detecting 28 HPV genotypes, presents a promising method. We aimed to evaluate its genotype-specific performance on FVU samples, following optimization of FVU preanalytics. We selected 701 FVU samples collected using a Colli-Pee device (20 mL, with UCM), enriched for HPV-positivity ( n = 630) based on previous testing with GP5+/6+-PCR-based reverse line blot (GP5+/6+ RLB) and E7-MPG after Amicon filtration (AF). We first evaluated the comparability and agreement of Allplex HPV28 genotype-specific positivity according to different preanalytics. Subsequently, we conducted the genotype-specific comparison of Allplex HPV28 with GP5+/6+ RLB AF and E7-MPG AF. No significant differences in HPV positivity by Allplex HPV28 testing were observed when comparing pre-centrifuged versus non-centrifuged DNA extraction, nor when comparing manual versus automated DNA extraction. Good genotype-specific agreement was observed between Allplex HPV28 and GP5+/6+ RLB AF, with Allplex HPV28 being slightly more sensitive for all 28 HPV genotypes (average Allplex HPV28:GP5+/6+ RLB AF ratio 1.729). Compared to E7-MPG AF, Allplex HPV28 exhibited lower sensitivity for all 21 overlapping HPV genotypes (average Allplex HPV28:E7-MPG AF ratio 0.588). The findings of this study, combined with practical considerations for real-world implementation, support the use of Allplex HPV28 testing after automated or manual DNA extraction without the requirement for pre-centrifugation, for HPV studies based on FVU samples, most notably those for vaccine impact monitoring on HPV prevalence. IMPORTANCE This study provides the first analytical validation of the Allplex HPV28 genotyping assay for use in first-void urine samples, offering a reliable, non-invasive, and practical alternative to cervical samples for human papillomavirus (HPV) detection. It demonstrates a validated approach that supports the assay’s potential application in real-world settings, including low- and middle-income countries, where non-invasive and widely acceptable sampling methods are crucial for maximizing population coverage and representativity. Given the urgent need for accurate and practical tools to monitor HPV vaccination impact, these findings offer a timely and impactful contribution to the field.

Initial workup [e.g., identification (ID) and/or antimicrobial susceptibility testing (AST)] of bacterial growth on solid media traditionally occurs 16–24 h after sub-culturing of positive blood cultures (BC). Early ID and AST can be facilitated by reviewing digital images captured using a Microbiology Laboratory Automation (MLA) system. The goal of this study was to evaluate the utility of images captured at 4 h on the WASPLab MLA system for rapid bacterial ID and AST. Retrospective review of all positive BCs results between 1 January 2021 and 31 July 2022 was performed. WASPLab App data were extracted to determine the decision (e.g., perform ID and/or AST or re-incubate plates) made from the 4 h image. Culture results were extracted from the laboratory information system (LIS). A total of 6,845 BCs flagged positive during the study period. The 4 h images for 1,476 cultures (21.6%) were reviewed: 1,200 cultures were re-incubated due to insufficient growth and 276 cultures (4.0%) were sent for ID and/or AST. ID by mass spectrometry was in 100% agreement with that of the molecular BC identification panels. Overall categorical agreement between AST results from the 4 h and overnight growth was 98%. The 4 h images for the remaining 5,369 cultures (78.4%) were not available for review during the day shift. Implementing early reading times for BCs on MLA allows for rapid and accurate ID and AST results. However, optimization of the reading schedule to align with the laboratory’s operation schedule is key to realizing the full potential of early reading times. IMPORTANCE In recent years, an increasing number of clinical microbiology laboratories have adopted laboratory automation for processing and incubation of specimens submitted for bacterial culture. At our institution, we implemented the Copan WASPLab in 2018 for all cultures, including positive blood cultures. Given that positive blood cultures start with a higher biomass of organisms, the first image capture was set up to occur after 4 h of incubation. In this study, we investigated the utility of this early 4 h image by capturing and calculating the percentage of useful actions taken based on growth identified on the image and the yield of both new identification by MALDI-TOF MS and valid and accurate antimicrobial susceptibility testing (AST) results. We found that while the 4-hour time point provided accurate, early identification and AST results, the overall yield was minimal. From a practical standpoint, this review prompted us to discontinue capture and review of this time point. While our staffing model is likely responsible for this low yield, we hope that our experience would help other laboratories decide how to implement WASPLab workflow for positive blood cultures. Thus, we believe that this information will be of interest to the readers of JCM.

Aspergillus fumigatus is a common cause of pulmonary and invasive mold infections among immunocompromised hosts. Mortality in immunocompromised hosts with invasive Aspergillus infections (IAI) has been reported to be as high as 80%. Therefore, appropriate therapy is essential in treating IAI. Both isavuconazole and voriconazole are first-line agents in treatment guidelines for IAI, but isavuconazole has favorable properties, often leading it to be preferred over voriconazole, given the lengthy duration of treatment. It is difficult to perform mold antifungal susceptibility testing, which often requires a reference lab and several weeks to determine results. Therefore, use of surrogate markers can be helpful to infer susceptibility when testing is not possible or delayed. We performed isavuconazole and voriconazole broth microdilution susceptibility testing by the Clinical and Laboratory Standards Institute (CLSI) method on a collection of 976 non-duplicate A. fumigatus isolates from a global surveillance program between 2017 and 2022. We found that voriconazole and isavuconazole have a very high essential agreement within two doubling dilutions at 99.9% and a categorical agreement of 92.7% with no very major errors, one major error (0.11%), and <10% minor errors. Many of the minor errors were in the setting of voriconazole testing at a MIC of 0.5 mg/L (susceptible) but isavuconazole at 2 mg/L (intermediate). Genetic analysis of cyp51 genes confirmed that isavuconazole and voriconazole susceptibility testing identified isolates with cyp51A and cyp51B mutations. Voriconazole can be used to predict the isavuconazole susceptibility testing result when A. fumigatus is tested by CLSI broth microdilution methodology.

Rapid and accurate diagnosis of sepsis is of paramount importance to reduce associated morbidity and mortality. The Qvella FAST System is a new instrument that concentrates and purifies bacteria from positive-flagged blood culture bottles (PFBCBs) to produce a “liquid” colony comparable to a subcultured colony in less than 40 min for rapid ID and calibrated antibiotic susceptibility testing (AST). In this study, we evaluated performances of the FAST System workflow and our rapid routine manual workflow (bacterial pellet obtained after lysis, cleaning, washing, and centrifugation for ID; AST by disc diffusion by direct inoculation after dilution) by comparison to the reference method based on 24-h bacterial subcultures. Two panels of PFBCBs were studied: panel A (including 107 prospective BCs from septic patients, October–November 2022) and panel B (including 102 BCs spiked with difficult-to-identify bacteria [mostly streptococci] and multidrug-resistant isolates), resulting in a total of 209 evaluable samples. The FAST System provided a correct ID to the species level in 178/209 (85.2%) of cases. For AST, the categorical agreement (CA) of the FAST System was 99.4%, with rates of very major (VME), major (ME), and minor (mE) errors of 0.59%, 0.20%, and 0.26%, respectively. Our rapid routine workflow based on manual methods show similar results for ID (86.2%) and AST (CA, 99.6%; VME, 0.50%; ME, 0.16%; mE, 0.13%). In conclusion, the Qvella FAST system, a promising tool that can reduce diagnostic time by approximately 1 day, shows excellent performances for rapid ID and AST.

Urgent improvements in the diagnosis and management of Mycobacterium tuberculosis infection are required to reach End TB goals. Conventional interferon-gamma release assays (IGRAs), such as QuantiFERON-TB Gold Plus (QFT-Plus), require substantial laboratory infrastructure and large blood volumes, limiting use in high-burden settings. The QIAreach QuantiFERON-TB (QIAreach QFT) was developed to overcome these challenges but has not previously been evaluated in field conditions in a low-income, high-burden country, or at scale in children. We performed a diagnostic evaluation of QIAreach QFT against QFT-Plus, in a cross-sectional IGRA survey in Blantyre, Malawi. We recruited a population-representative sample of children aged 1–4 years and adolescents and adults aged 10–40 years, from households and primary care. We calculated sensitivity, specificity, and Cohen’s kappa for QIAreach QFT against QFT-Plus, and constructed Bayesian hurdle-categorical models to compare quantitative test results. A total of 1,049 participants were recruited (64%: 1–4 years; 13%: 10–19 years; and 23%: 20–40 years). More participants had a positive QIAreach QFT result (32%) compared to QFT-Plus (15%). Over half of positive QIAreach QFT results had time-to-positivity of exactly 20 min, the assay cutoff. There was minimal agreement between QFT-Plus and QIAreach QFT results ( κ = 0.26), which was lowest in children aged 1–4 years ( κ = 0.13). Sensitivity and specificity of QIAreach QFT relative to QFT-Plus were 62% and 74%, respectively, with poor correlation between quantitative results. The suboptimal performance of QIAreach QFT, particularly in young children, suggests that it cannot currently be recommended for wider use and that the urgent need for an accessible test of Mtb infection remains unmet. IMPORTANCE Almost a quarter of the world’s population has evidence of Mycobacterium tuberculosis (Mtb) infection. Monitoring and addressing this substantial burden of so-called “latent” tuberculosis (TB) infection will be critical to reach End TB targets. However, current interferon-gamma release assays (IGRAs) for Mtb infection are costly, and require a large volume of venous blood and significant laboratory processing, which are major barriers to their wider use in low-income countries. The novel QIAreach QuantiFERON-TB (QIAreach) assay has been designed as a more accessible alternative. We sought to evaluate it against a reference standard of QuantiFERON-TB Gold Plus, in a large cross-sectional survey in Blantyre, Malawi. To our knowledge, this is the first diagnostic evaluation of QIAreach QFT to be performed in a population-based survey in a low-income high-incidence setting, and to specifically focus on young children (a priority group for interventions targeting Mtb infection). In contrast to previous studies in other settings, we observed poor performance of QIAreach QFT, particularly in young children where there was little correlation between the novel test and the reference standard. This leads us to conclude that this test cannot be widely recommended for use in its current form; indeed manufacture is currently suspended. We believe our findings are of urgent importance to policymakers, clinicians, and researchers and underscore the importance of careful evaluation of new diagnostics in the contexts where they are intended to be used.

This study aimed to evaluate a new protocol of the bile solubility test performed directly on the blood from positive blood culture bottles to identify Streptococcus pneumoniae rapidly. Seventy-five positive blood cultures (PBC) showing Gram-positive cocci in pairs or chains on Gram stain, including 32 S. pneumoniae isolates and three reference American Type Culture Collection (ATCC) isolates were included to evaluate the performance of a modified bile solubility test (MBST). One milliliter of blood from the PBC bottle was mixed with 0.5 mL of 10% desoxycholate or a saline solution. Both suspensions were analyzed after 10 min of incubation through a Gram stain to detect solubilization. This technique was compared with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification, performed on PBC following extraction or on colonies after short or standard incubation, and the optochin susceptibility test on colonies. The capsular serotypes were determined for all S. pneumoniae , and the Belgian National Reference Center confirmed the identification. All 32 clinical isolates and the ATCC isolate of S. pneumoniae were solubilized on the desoxycholate-treated slides, while the other species tested remained visually unchanged on both, the test and control slides. The MBST test demonstrated a 100% sensitivity and specificity with a mean turnaround time (TAT) of just 39 min, making it 14 h and 56 min faster than the optochin susceptibility test. This rapid variant of the bile solubility test appears to be a reliable method to identify S. pneumoniae directly from positive blood culture bottles, with a TAT of 39 min. It is a cost-effective, easy-to-perform, and time-efficient technique. Negative results should be interpreted cautiously, as they may result from mixed infections with S. pneumoniae and other Gram-positive cocci.

Genotypic methods for detecting antibiotic resistance in Helicobacter pylori infection offer a rapid, convenient, and accurate approach for tailored therapy. However, existing studies predominantly examine single sample types and lack comparative analyses across different samples. This study comprehensively detects and compares genotypic resistance to clarithromycin and levofloxacin in gastric mucosa, gastric fluid, and fecal samples from the same patients. The study enrolled 183 participants, comprising 124 H . pylori -positive and 59 H . pylori -negative patients. All participants provided fecal samples and underwent gastroscopy for the collection of gastric mucosa and gastric fluid. Real-time PCR was employed to detect genotypic resistance to clarithromycin and levofloxacin in conjunction with bacterial culture and antibiotic susceptibility testing. Genotypic resistance detection rates for clarithromycin were 100% in gastric mucosa, 99.2% in gastric fluid, and 79.8% in fecal samples. For levofloxacin, detection rates were 97.6%, 96.8%, and 72.6%, respectively. The results showed that PCR detection for clarithromycin exhibited high sensitivity (0.94–0.95) and specificity (0.88–0.89) across all sample types. However, PCR detection for levofloxacin demonstrated slightly lower sensitivity (0.79–0.89) and specificity (0.79–0.83). The comparison of genotypic resistance results by PCR among the three sample types showed that gastric mucosa and gastric juice exhibited higher consistency, while the consistency between feces and both gastric mucosa and gastric juice was lower. This study confirmed good consistency between genotypic and phenotypic resistance in clarithromycin and levofloxacin. While both gastric mucosa and gastric fluid samples demonstrated high detection performance, the efficiency of detecting fecal samples was constrained by challenges in DNA extraction. IMPORTANCE This study, with a large sample size, comprehensively tested both Helicobacter pylori-negative and -positive patients, including rapid urease test, histopathological evaluation and staining, bacterial culture, susceptibility testing, and resistance gene mutation analysis. By simultaneously examining gastric mucosa, gastric juice, and fecal samples from the same individuals, we minimized confounding factors arising from different sample sources, ensuring the reliability of our results. This approach effectively delineated the differences and characteristics in detection performance among different sample types, offering crucial reference data for selecting appropriate detection samples and identifying areas for improvement. The findings revealed robust concordance between genotypic and phenotypic resistance, with both gastric mucosa and gastric juice samples demonstrating excellent detection performance. However, the efficiency of detecting resistance in fecal samples was hampered by challenges in DNA extraction.

Mosquito-borne viruses represent a large global health burden. With geographic expansion of competent vectors for chikungunya virus (CHIKV), dengue virus (DENV), and Zika virus (ZIKV) in Europe, it is anticipated that the number of autochthonous cases of these tropical viruses in Europe will increase. Therefore, regular assessment of diagnostic capabilities in Europe is important. Our aim was to evaluate the mosquito-borne virus molecular detection capability of expert European laboratories by conducting an external quality assessment in October 2023. Molecular panels included 12 plasma samples: one alphavirus (CHIKV), four orthoflaviviruses (ZIKV, yellow fever virus [YFV], DENV, and Japanese encephalitis virus [JEV]), and two negative control samples. Mosquito-borne virus detection was assessed among 36 laboratories in 24 European countries. Adequate capabilities were lacking for YFV and JEV. Many laboratories relied on a mix of laboratory-developed tests (some of which were pan-orthoflavivirus or pan-alphavirus in combination with sequencing) and commercial assays. 47.2% of laboratories characterized all external quality assessment (EQA) samples correctly. Correct result rates were 100% for CHIKV and ZIKV and >99% for DENV, but laboratories lacked capacity, specificity, and sensitivity for JEV and YFV. Three of the viruses in this panel emerged and transiently circulated in Europe: CHIKV, ZIKV, and DENV. Molecular detection was excellent for those viruses, but <50% is accurate for the remainder of the panel. With the possibility or continuation of imported cases and a growing global concern about climate change and vector expansion, progress toward rapid, accurate mosquito-borne virus diagnostics in Europe is recommended, as well as regular EQAs to monitor it. IMPORTANCE The external quality assessment (EQA) focused on Aedes -borne viruses: chikungunya virus (CHIKV), dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV). Japanese encephalitis virus, an orthoflavivirus that is spread by mosquito species belonging to the genus Culex , was included in the quality assessment as well. CHIKV, DENV, and ZIKV have proven potential for transient and limited circulation in Europe upon introduction of viremic travelers returning to Aedes albopictus -endemic regions. Results of this EQA were excellent for those viruses, but <50% is accurate for the remainder of the panel (YFV and Japanese encephalitis virus). Considering imported cases and the threat of climate change and competent vector expansion, progress toward rapid, accurate mosquito-borne virus diagnostics in Europe is recommended.

The laboratory diagnosis of Clostridioides difficile infection (CDI) is controversial. Nucleic acid amplification tests (NAAT) and toxin enzyme immunoassays (EIA) are most widely used, often in combination. However, the interpretation of a positive NAAT and negative toxin immunoassay (NAAT+/EIA−) is uncertain. PubMed and EMBASE were searched for studies reporting clinical outcomes in NAAT+/EIA− versus NAAT+/EIA+ patients. Forty-six studies comprising 33,959 patients were included in this meta-analysis. All-cause mortality (RR 0.96, 95% CI 0.80–1.15), attributable mortality (RR 0.61, 95% CI 0.20–1.91), fulminant CDI (RR 0.83, 95% CI 0.57–1.20), radiographic evidence of CDI (RR 0.87, 95% CI 0.65–1.16), total CDI complications (RR 0.95, 95% CI 0.59–1.53), colectomies (RR 0.78, 95% CI 0.34–1.79), and ICU admission (RR 1.04, 95% CI 0.84–1.30) did not significantly differ between NAAT+/EIA− and NAAT+/EIA+ patients. However, rates of recurrent (RR 0.62, 95% CI 0.50–0.77) or severe (RR 0.74, 95% CI 0.63–0.88) CDI were significantly lower in NAAT+/EIA− patients than in NAAT+/EIA+ patients. The pooled prevalence of NAAT+/EIA− patients who were treated with antibiotics for CDI was 73.4% (pooled proportion 0.72, 95% CI 0.52–0.88). NAAT+/EIA− patients have lower rates of recurrence and are at reduced risk for severe CDI compared with NAAT+/EIA+ patients but have a risk of CDI-related complications and mortality comparable to that of NAAT+/EIA+ patients. Toxin results cannot rule in or rule out CDI, and the decision whether to treat symptomatic NAAT+/EIA− patients for CDI should be based on clinical presentation and not on the toxin result. IMPORTANCE Clostridioides difficile infection (CDI) is a common cause of healthcare-associated infections and the leading cause of antibiotic-associated diarrhea. However, the laboratory diagnosis of CDI, primarily done by nucleic acid amplification test (NAAT) and enzyme immunoassay (EIA), is controversial, especially in patients who test positive by NAAT but negative by EIA. In this systematic review, we compared the clinical outcomes of NAAT+/EIA− versus NAAT+/EIA+ patients and found that the two groups have similar risk of mortality and CDI-related complications. However, NAAT+/EIA− patients had significantly lower rates of recurrence and severe CDI than NAAT+/EIA+ patients, and most NAAT+/EIA− patients received CDI therapy. Toxin testing can help to predict the likelihood of CDI recurrence or severe infection, but the toxin result should not be a determining factor in the administration of CDI therapy. The decision on whether to treat NAAT+/EIA− patients should be based on clinical assessment.

Accurate identification of non-tuberculous mycobacterial (NTM) species is crucial for the diagnosis and appropriate management of NTM infections. This study aimed to evaluate the performance of two assays, FluoroType Mycobacteria VER 1.0 and Maldi BioTyper (MBT) Mycobacteria. The two assays were evaluated using 119 NTM, including 85 slow-growing mycobacteria and 34 rapid-growing mycobacteria, representing a total of 33 species isolated in three French clinical laboratories. We used the GenoType assays as reference method for species identification, followed by 16S rRNA gene sequencing if the GenoType kits returned Mycobacterium sp. Compared to the reference method, the FluoroType Mycobacteria assay provided correct species identification in 89.9% of cases (107/119). Among the most frequently encountered species in clinical settings, low concordance was obtained for Mycobacterium intracellulare (82.4%, 14/17), Mycobacterium gordonae (66.7%, 6/9), and Mycobacterium xenopi (75%, 6/8). Misidentification was obtained in two cases ( Mycobacterium smegmatis instead of Mycobacterium mageritense , and Mycobacterium mucogenicum instead of Mycobacterium phocaicum ). Using the MBT Mycobacteria assay, 78.1% (93/119) of NTM isolates were correctly identified at the species level. One Mycobacterium europaeum isolate was misidentified as M. intracellulare / Mycobacterium chimaera . In five cases, the assay provided more accurate NTM identification compared to GenoType assays, in which closely related species are identified as a group. The FluoroType Mycobacteria VER 1.0 and the MBT Mycobacteria assays are useful tools for NTM identification from positive cultures, reducing handling time compared to GenoType assays. Their routine use in laboratories must take into consideration their performance and limitations in clinical settings.

Bovine reproductive failure, which includes infertility, abortion, and stillbirth in cattle, leads to significant economic losses for beef and milk producers. Diagnosing the infectious causes of bovine reproductive failure is challenging as there are multiple pathogens associated with it. The traditional stepwise approach to diagnostic testing is time-consuming and can cause significant delays. In this study, we have developed a syndromic next-generation sequencing panel (BovReproSeq) for the simultaneous detection of 17 pathogens (bacteria, virus, and protozoa) associated with bovine reproductive failure. This targeted approach involves amplifying multiple pathogen-specific targets using ultra-multiplex PCR, followed by sequencing with the Oxford Nanopore platform and subsequent analysis of the data using a custom bioinformatic pipeline to determine the presence or absence of pathogens. We tested 116 clinical samples and found that BovReproSeq results matched with current diagnostic methods for 93% of the samples, and most of the disagreements occurring in samples with very low pathogen loads (Ct >35). At the optimal read-count threshold of 10 reads (minimum number of reads to classify the sample as positive), the clinical sensitivity of the assay was approximately 82%, while clinical specificity was 100%. The overall accuracy of the assay was 98.8%. Matthews correlation coefficient (correlation coefficient of binary classification) was approximately 0.90 and F1 score (harmonic mean of precision and recall) was 0.90, indicating excellent overall performance. Our study presents a significant advancement in detecting the infectious agents associated with bovine reproductive failure and the BovReproSeq panel’s ability to detect 17 pathogens makes it a promising tool for veterinary diagnostics. IMPORTANCE Bovine reproductive failure causes substantial economic losses to beef and milk producers, and infectious disease contributes significantly to this syndrome. Etiologic diagnosis is complicated since multiple pathogens can be involved and infections with some pathogens are asymptomatic or cause similar clinical signs. A stepwise approach to diagnostic testing is time-consuming and increases the risk of missing the correct diagnosis. BovReproSeq is a next-generation sequencing-based diagnostic panel that allows detection of 17 reproductive failure pathogens simultaneously.