Journal of Clinical Microbiology最新文献

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.

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.

We hypothesized that bighorn sheep ewes with chronic nasal Mycoplasma ovipneumoniae carriage are the source of infection that results in fatal lamb pneumonia. We tested this hypothesis in captive bighorn ewes at two study facilities over a 5-year period, by identifying carrier ewes and then comparing lamb fates in groups that did (exposed pens) or did not (non-exposed pens) include one or more carrier ewes. Most (23 of 30) lambs born in exposed pens, but none of 11 lambs born in non-exposed pens, contracted fatal pneumonia. In addition, surviving lambs in exposed pens showed obvious signs of respiratory disease while lambs in non-exposed pens did not. In crossover experiments, individual non-carrier ewes had lambs that experienced fatal pneumonia in years when housed in exposed pens, but not in years when housed in non-exposed pens. The results of these studies clearly associate lamb pneumonia to exposure to M. ovipneumoniae carrier ewes, consistent with a necessary role for this agent in epizootic pneumonia of bighorn sheep. These data specifically highlight the role of chronic M. ovipneumoniae carriage by some bighorn ewes in the epidemiology of this population-limiting wildlife disease.IMPORTANCEBighorn sheep populations, historically important in mountain and canyon ecosystems of western North America, declined precipitously following European settlement of North America and remain depressed today. One factor contributing to these declines and lack of recovery is epizootic pneumonia caused by the bacterium Mycoplasma ovipneumoniae. This pathogen arrived with settlers' domestic sheep and goats and spilled over to infect bighorn sheep, a process that continues to this day. Bighorn losses from this disease include high rates of mortality (median, approaching 50%) of all ages of bighorn sheep on initial exposure, followed in subsequent years to decades by mortality largely limited to young lambs. The source of infection causing persistent lamb losses is the focus of the research described here. Conducting these studies on groups of captive bighorn sheep enabled demonstration of clear linkage between largely asymptomatic nasal carriage of M. ovipneumoniae by ewes and outbreaks of fatal pneumonia in lambs.

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.

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.IMPORTANCEThis 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.

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.IMPORTANCEThis 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.

An accurate diagnosis of invasive aspergillosis (IA) in patients with underlying hematological malignancies relies heavily on galactomannan detection. In this study, we compared the VirCLIA chemiluminescence immunoassay (CLIA) with the frequently used Platelia enzyme-linked immunosorbent assay (ELISA) on serum from hematology patients with suspected IA. Patients were categorized according to EORTC/MSGERC 2020 definitions into proven/probable IA and possible/no IA. The first cohort included 161 patients at four centers, and the VirCLIA manufacturer's cutoff of 0.200 was evaluated. Next, the optimal cutoff was determined using the Youden's index. In a second independent cohort of 189 patients from four centers, this optimal cutoff was evaluated again. In the first cohort, sensitivities and specificities for probable/proven IA were 21.1% and 100.0% for ELISA (1.0 cutoff) and 36.6% and 95.6% (0.5 cutoff), compared to 11.3% and 97.8% for CLIA (0.200 cutoff). In the second cohort, the sensitivities of ELISA and CLIA were comparable (ELISA ≥ 1.0: 33.3%, CLIA ≥ 0.200: 38.1%). The area under the ROC curve was lower for CLIA than for ELISA in the first cohort (65.0% vs 78.7%, P = 0.005) but comparable in the second cohort (79.5% vs 81.3%, P = 0.649). Youden's index identified 0.100 as the optimal CLIA cutoff with sensitivities of 35.2% and 61.9% in cohorts 1 and 2, respectively, at slightly reduced specificities of 85.6% and 90.5%. While the sensitivity of both assays was low to moderate at best, in patients with a high pre-test probability, we suggest 0.100 as the cutoff for the VirCLIA assay.IMPORTANCEThis study demonstrates a comparable performance of the novel chemiluminescence immunoassay (CLIA) and the conventionally used enzyme-linked immunosorbent assay for galactomannan serum testing in hematological patients at high risk for invasive aspergillosis. In patients with a high pre-test probability, a lower CLIA cutoff of 0.100 is preferred.

Current laboratory protocols for periprosthetic joint infections (PJIs) involve a standard 10- to 14-day incubation period. However, recent evidence indicates considerable variability in the time to diagnosis (TTD) between acute and chronic PJIs. TTD is also influenced by the employed culture media and sample types. Enriched liquid media, such as broths and blood culture bottles, along with sonication fluid culture, are commonly used, though their incremental benefit for PJI diagnosis remains debated. We retrospectively analyzed 187 confirmed hip and knee PJIs, each with at least three intraoperative samples. Comparison of TTD among early acute (n = 68), late acute (n = 52), and late chronic (n = 67) PJIs revealed a significant difference, particularly between late acute and late chronic infections (P < 0.004). Early acute and late acute PJIs were diagnosed within 5 days in 97.1% and 98.1% of cases, respectively, contrasting with 14 days required for 97.1% of late chronic PJIs. Enriched liquid media significantly improved species detection, especially in polymicrobial and anaerobic infections. Pediatric and anaerobic blood culture bottles demonstrated superior efficacy over thioglycolate broths for diagnostic confirmation. Sonication fluid culture was essential for confirming diagnoses in 17.6% of cases. Our findings highlight that clinical presentation, rather than time since primary arthroplasty, should guide incubation duration: both early acute and late acute PJIs can be diagnosed within 5 days. Medical microbiology laboratories should consider shorter incubation times for acute PJIs to optimize diagnostic efficiency. The use of blood culture bottles and sonication fluid culture proves invaluable for accurate PJI diagnosis.

Importance: While molecular techniques are becoming increasingly employed, culture remains the gold standard for diagnosing periprosthetic joint infections. However, guidance for laboratory protocols is limited and highly variable. This article aims to increase diagnostic efficiency by providing concrete recommendations for medical microbiology laboratories.

Hepatitis E virus (HEV) is a globally prevalent zoonotic pathogen that is primarily spread through the fecal-oral route, such as by consuming undercooked or contaminated pork. HEV infection leads to an estimated 3.3 million symptomatic cases of viral hepatitis and 70,000 deaths in human populations each year. Therefore, a rapid and accurate method for detecting HEV in serum or stool samples is essential. In this study, we aimed to develop and evaluate two methods for the rapid and convenient detection of HEV RNA: reverse transcription recombinase-aided amplification with lateral flow dipstick (RT-RAA-LFD) and quantitative real-time reverse transcription recombinase-aided amplification (qRT-RAA). We optimized the reaction conditions and assessed their sensitivity and specificity. The RT-RAA-LFD assay completed its reaction at 39°C within 15 minutes, achieving a 95% limit of detection (LOD) of 247 copies/μL. The qRT-RAA assay, completed at 42°C within 20 minutes, had a 95% LOD of 25 copies/μL. Both assays demonstrated no cross-reactivity with other porcine pathogens and exhibited strong specificity. In testing 245 porcine bile and fecal samples, the RT-RAA-LFD assay showed a kappa value of 0.943 (P < 0.001) with a 97.14% (238/245) coincidence rate compared with quantitative reverse transcription PCR. Similarly, the qRT-RAA assay achieved a kappa value of 0.976 (P < 0.001) with a 98.78% (242/245) coincidence rate. In conclusion, these two RT-RAA assays show promising potential as effective diagnostic tools for broad and efficient screening of swine HEV in veterinary clinics.

Importance: Hepatitis E virus (HEV) is a globally widespread zoonotic pathogen that poses a significant public health risk. Swine serve as the primary natural host for zoonotic HEV. This study introduces a rapid and precise method for detecting swine HEV RNA, showcasing its potential as an effective diagnostic tool for comprehensive and efficient screening of swine HEV in veterinary clinics.