Journal of Clinical Microbiology最新文献

Serological assays for antibody detection have contributed significantly to the diagnosis and control of infectious diseases. African swine fever is the most devastating infectious disease of domestic pigs and wild boars, severely threatening the global pig industry in recent years. Here, we developed a rapid, simple, and sensitive immunoassay based on the split-luciferase system to detect IgG antibodies against African swine fever virus (ASFV). In this assay, the p30 protein of ASFV was genetically coupled to the LgBiT and SmBiT subunits of nanoluciferase, which were used as fusion probes for specific antibodies. Target engagement of the probes results in the reconstitution of a functional nanoluciferase, which further catalyzes bioluminescent reactions. Different orientations of the LgBiT and SmBiT-p30 fusion sensors were designed and investigated, and N-LgBiT/p30 and N-SmBiT/p30 were identified as a promising sensor pair for reforming active nanoluciferase in the presence of specific antibodies. After optimization, this split-luciferase complementation assay showed high sensitivity and specificity for the detection of ASFV antibodies. The analytical sensitivity of the assay was 16 times greater than that of the blocking enzyme-linked immunosorbent assay (ELISA) by the detection of serial dilutions of serum, and no cross-reaction was observed with other swine pathogens. As demonstrated in clinical samples, its performance is highly consistent with that of a commercial ELISA kit, with a concordance rate of 98.19%. This assay is simple and easy to perform, providing a more flexible and efficient approach for the measurement of ASFV antibodies in clinical applications.

Importance: The study is about a homogeneous split-luciferase assay for antibody detection. Split nanoluciferase biosensors for the detection of ASFV antibodies were designed. This sensor platform enables the sensitive and specific detection of antibodies. The split-luciferase assay is simple, rapid, and easy to use.

The MBT Pathfinder is an automated colony-picking robot designed for efficient sample preparation in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This article presents results from three key experiments evaluating the instrument's performance in conjunction with MALDI Biotyper instrument. The method comparison experiment assessed its clinical performance, demonstrating comparable results with gram-positive, gram-negative, and anaerobic bacteria (scores larger than 2.00) and superior performance over simple direct yeast transfer (score: 1.80) when compared to samples prepared manually. The repeatability experiment confirmed consistent performance over multiple days and labs (average log score: 2.12, std. deviation: 0.59). The challenge panel experiment showcased its consistent and accurate performance across various samples and settings, yielding average scores between 1.76 and 2.19. These findings underline the MBT Pathfinder as a reliable and efficient tool for MALDI-TOF mass spectrometry sample preparation in clinical and research applications.

Rapid detection is crucial for tuberculosis (TB) control. GeneXpert (Cepheid) is a widely used PCR system, known for its simplicity, random access, and point-of-care compatibility. SD BIOSENSOR recently introduced a similar system, STANDARD M10, including a Mycobacterium tuberculosis (MTB) and rifampicin (RIF) and isoniazid resistance (herein, MDR-TB) assay and an MTB/nontuberculous mycobacteria (NTM) assay. We evaluated these assays for the potential to replace the established Xpert MTB/RIF Ultra assay in a low-TB incidence setting. We analyzed 160 clinical respiratory samples (45 MTB-positive and 35 NTM-positive) and further 24 drug-resistant MTB, 30 mycobacterial species (2 MTB, 28 NTM), and 37 non-mycobacterial isolates. Compared with culture, clinical sensitivities and specificities for MTB detection were 88.9% (95% confidence interval [CI] = 76.1-95.6%) and 97.4% (CI = 92.3-99.4%) with Xpert Ultra, 88.9% (95% CI = 76.1-95.6%) and 98.3% (CI = 93.5-99.9%) with M10 MDR-TB, and 84.4% (CI = 70.9-94.4%) and 98.3% (CI = 93.5-99.9%) with M10 MTB/NTM, respectively. For NTM detection, M10 MTB/NTM showed sensitivity and specificity of 65.7% (CI = 49.1-79.2%) and 96.8% (CI = 91.8-99.0%). Compared with phenotypic drug susceptibility testing (DST), sensitivity and specificity for detecting RIF resistance were 100% (CI = 77.3-100%) and 95.6% (CI = 84.4-99.6%) with Xpert Ultra, and 100% (CI = 74.9-100%) and 95.5% (CI = 84.0-99.6%) with M10 MDR-TB. M10 MDR-TB showed 92.3% sensitivity (CI = 74.7-99.0%) and 100% specificity (CI = 87.3-100%) for detecting isoniazid resistance. All discrepancies in DST by PCR were concordant with whole-genome sequencing. While M10 MDR-TB demonstrated great potential as an alternative to Xpert Ultra, M10 MTB/NTM had limitations in NTM screening. Additionally, the M10 sputum pretreatment did not inactivate MTB efficiently, which should be considered in process risk assessment.

Importance: The molecular diagnostic STANDARD M10 system is highly analogous to the widely established GeneXpert system, which significantly increases the relevance of this evaluation study in the field of rapid detection of M. tuberculosis. To our knowledge, this is the first clinical evaluation describing the performance of the STANDARD M10 MDR-TB and MTB/NTM assays, including an extensive analytical specificity panel (inclusivity and exclusivity) for the detection of M. tuberculosis, drug resistance, and nontuberculous mycobacteria.

We investigated the performance of the targeted next-generation sequencing (tNGS)-based Oxford Nanopore Diagnostics AmPORE TB assay, recently approved by the World Health Organization (WHO) as tuberculosis (TB) diagnostic test for the detection of drug resistance on respiratory specimens. A total of 104 DNA samples from Xpert MTB/RIF-positive TB sputum specimens were tested using the AmPORE TB kit, with the GenoScreen Deeplex Myc-TB as a comparative tNGS assay. For AmPORE TB, DNA samples were divided into five sequencing runs on the MinION device. Data analysis was performed using proprietary software. The WHO catalog of mutations was used for drug resistance interpretation. The assay achieved a high validity rate of 98% (102/104 DNA samples), homogeneous mean reads coverage across TB-positive specimens, and 100% positive and negative agreements for detecting mutations associated with resistance to rifampicin, pyrazinamide, fluoroquinolones, ethambutol, and capreomycin compared with Deeplex Myc-TB. The main discrepancies for the remaining drugs were attributable to the different assay panel designs. The AmPORE TB turnaround time was approximately 5-6 hours from extracted DNA to tNGS reporting for batches of 22 DNA samples. The AmPORE TB assay drastically reduced the time to tNGS reporting from days to hours and showed good performance for drug-resistant TB profiling compared with Deeplex Myc-TB.

Importance: Targeted next-generation sequencing (tNGS) of Mycobacterium tuberculosis provides comprehensive resistance predictions matched to new multidrug-resistant/rifampicin-resistant tuberculosis regimens and received World Health Organization approval for clinical use in respiratory samples in 2024. The advanced version of the Oxford Nanopore Diagnostics AmPORE TB tNGS kit was evaluated in this study for the first time and demonstrated good performance, flexibility, and faster turnaround time compared with the existing solutions.

There is a clear medical need for an accurate diagnostic test for typhoid that can be performed at point of care. Two antigens (lipopolysaccharide [LPS] and hemolysin E [HlyE]) have recently been identified that can distinguish typhoid from other bacterial infections. Here, we present the results of a diagnostic accuracy study of the Dual Path Platform (DPP) Typhoid assay (Chembio) that detects IgA to both LPS and HlyE using blood culture as the reference standard. This was a retrospective, observational, laboratory study conducted at the Aga Khan University research laboratory, Pakistan, to evaluate the sensitivity and specificity of the DPP Typhoid assay, using archived frozen serum samples collected during a previous typhoid diagnostic accuracy study (NCT04801602). The sensitivity, specificity, and accuracy (area under the receptor operating characteristics curve [AUC]) were then assessed using the manufacturer's and Youden's optimal thresholds. In total, 385 samples were included in the analysis. Using the manufacturer's thresholds, the sensitivity, specificity, and AUC were 97.8% (95% confidence interval [CI] 94.6-99.2), 65.3% (95% CI 58.5-71.6), and 81.5% (95% CI 75.5-85.3), respectively. At Youden's optimal threshold, the overall sensitivity of the DPP Typhoid assay was 89.7% and the specificity was 82.2%. In latent class modeling compared with other nine rapid diagnostic tests evaluated from the same cohort sample, the DPP Typhoid assay demonstrated the highest balanced accuracy (89.2%). The DPP Typhoid assay demonstrated a high diagnostic accuracy for typhoid fever. However, further adjustment to new thresholds is recommended to enhance its performance capabilities.

Importance: Currently available diagnostic tests for typhoid have several limitations, including low sensitivity and specificity. Dual Path Platform Typhoid assay is a multiplex rapid test that detects IgA antibodies to lipopolysaccharide and hemolysin E antigen. It is considered to have high sensitivity and specificity, and its results were found to be highly correlated with ELISA results. However, very few studies have been conducted to evaluate this test and limited information about the accuracy of this test is present. Hence, this study evaluated the new typhoid test.

Superficial fungal infections caused by dermatophytes are a prevalent global health concern. Rapid and accurate diagnosis of these pathogens through molecular tools would offer a substantial advantage for early detection and effective treatment. The conventional fungal culture presents inherent limitations, including extended result delivery delay and variable sensitivity. This study aimed to evaluate the performance of the multiplex real-time PCR Novaplex dermatophyte assay (Seegene) in comparison to traditional mycological methods including direct examination and culture. A total of 312 nail, skin, and scalp samples collected from patients with suspected superficial fungal infections for mycological diagnosis were retrospectively subjected to the Novaplex dermatophyte assay. Overall, 170 (54.6%) and 186 (59.6%) samples tested positive for dermatophyte culture and dermatophyte PCR, respectively. The concordance between PCR and culture for dermatophyte detection was 87.2%. There were 158 culture-positive/PCR-positive samples, 12 culture-positive/PCR-negative samples, and 28 culture-negative/PCR-positive samples. The sensitivity of PCR against culture varied according to the dermatophyte target, ranging from 90.5% (Trichophyton mentagrophytes/interdigitale/benhamiae), 91.2% (Trichophyton rubrum), to 100% (Microsporum spp. and Trichophyton tonsurans). When considering the final diagnosis using composite criteria, the sensitivity and specificity for the diagnosis of dermatophytosis were 92.9% and 96.6% for PCR, 86.7% and 100% for culture, and 95.4% and 92.2% for direct examination and culture combined, respectively. The Seegene Novaplex dermatophyte assay is an easy-to-use automated one-step extraction-PCR system that offers satisfactory performance for routine diagnosis of dermatophytoses in clinical laboratories, particularly in non-specialized centers. However, it cannot fully replace conventional mycology due to its inability to detect mold infections and to identify dermatophytes at the species level.

Medical microbiologists, defined as doctoral-level laboratory directors with subspecialty training in medical microbiology, lead the clinical laboratory operations through activities such as clinical consultations, oversight of diagnostic testing menu, institutional leadership, education, and scholastic activities. However, unlike their clinical colleagues, medical microbiologists are largely unable to bill for clinical consultations performed within the hospital and, therefore, unable to generate relative value units or a similar quantifiable metric. As hospital budgets tighten and justification of staffing becomes a necessity, this may present a challenge to the medical microbiologist attempting to prove their value to the organization. To aid in providing tangible data, the Personnel Standards and Workforce subcommittee of the American Society for Microbiology conducted a multi-center study across seven medical centers to document clinical consultations and their impact. Consults were generated equally from internal (laboratory-based) and external (hospital-based) parties, with the majority directly impacting patient management. Near universal acceptance of the medical microbiologist's recommendation highlights the worth derived from their expertise. External consults required more time commitment from the medical microbiologist than internal consults, although both presented ample opportunity for secondary value, including impact through stewardship, education, clinical guidance, and cost reduction. This study is a description of the content and impact of consultations that underscore the importance of the medical microbiologist as a key member of the healthcare team.

Importance: Medical microbiologists are invaluable to the clinical microbiology laboratory and the healthcare system as a whole. However, as medical microbiologists do not regularly generate relative value units, capturing and quantifying the value provided is challenging. As hospital budgets tighten, justification of staffing becomes a necessity. To aid in providing tangible data, the Personnel Standards and Workforce subcommittee of the American Society for Microbiology conducted a multi-center study across seven medical centers to document clinical consultations and their impact. To our knowledge, this is the first study to provide detailed evaluation of the consultative value provided by medical microbiologists.

The QIAstat-Dx BCID Panels (RUO) ("QIAstat," QIAGEN, Hilden, Germany) for identification of 13 Gram-negative bacteria and 18 antimicrobial resistance (AMR) gene groups was evaluated. The study was conducted in two phases; in phase 1, analytical performance was evaluated against 154 challenge isolates against whole genome sequencing data. In this phase, sensitivity and specificity of organism identification calls were 153/154 (99.3%) and 1,748/1,749 (99.8%), respectively. For AMR genes, sensitivity was 434/435 (99.8%) and specificity was 2,334/2,337 (99.9%). One false-negative bla_IMP, one false-positive bla_CTX-M, and two false-positive aac-6'-lb detections were noted in this challenge set of organisms. In phase 2, 101 clinical blood culture isolates of Gram-negative rods were evaluated by the multiplexed PCR versus reference broth microdilution, for the ability of identification combined with AMR genes to predict final susceptibility results. Negative predictive values were 92.8% for ampicillin resistance (100% for Escherichia coli), 93.4% for ceftriaxone, 97.4% for ceftazidime, and 98.7% for cefepime. In constrast, negative predictive values for current standard of care (identification plus detection of bla_CTX-M) ranged from 56.5% to 88.8%. This study demonstrated additive value of additional beta-lactamase genes for bacteria isolated from blood cultures.

Importance: Prediction of Gram-negative bacteria resistance through detection of resistance genes is complex. This study evaluated a novel, direct-from-blood or bacterial isolate multiplexed PCR for the detection of 17 resistance genes, and evaluated the prediction of antimicrobial susceptibility.

Sequencing of plasma microbial cell-free DNA (mcfDNA) has gained increased acceptance as a valuable adjunct to standard-of-care testing for diagnosis of infections throughout the body. Here, we report the analytical and clinical validation of a novel application of mcfDNA sequencing, the non-invasive detection of seven common antimicrobial resistance (AMR) genetic markers in 18 important pathogens. The AMR markers include SCCmec, mecA, mecC, vanA, vanB, bla_CTX-M, and bla_KPC. The AMR markers were computationally linked to the pathogens detected. Analytical validation showed high reproducibility (100%), inclusivity (54 to 100%), and exclusivity (100%). Clinical accuracy was assessed with 114 unique plasma samples from patients at seven study sites with concordant culture results for target bacteria from a variety of specimen types and correlated with available phenotypic antimicrobial susceptibility test results and genotypic results. The positive percent agreement (PPA), negative percent agreement (NPA), and diagnostic yield (DY) were estimated for each AMR marker. DY was defined as the percentage of tests that yielded an actionable result of either detected or not detected. The results for the combination of SCCmec and mecA for staphylococci were PPA 19/20 (95.0%), NPA 21/22 (95.4%), DY 42/60 (70.0%); vanA for enterococci were PPA 3/3 (100%), NPA 2/2 (100%), DY 5/6 (83.3%); bla_CTX-M for gram-negative bacilli were PPA 5/6 (83.3%), NPA 29/29 (100%), DY 35/49 (71.4%); and bla_KPC for gram-negative bacilli were PPA 0/2 (0%), NPA: 23/23 (100%), DY 25/44 (56.8%). The addition of AMR capability to plasma mcfDNA sequencing should provide clinicians with an effective new culture-independent tool for optimization of therapy.

Importance: This manuscript is ideally suited for the Innovative Diagnostic Methods sections as it reports the analytical and clinical validation of a novel application of plasma microbial cell-free DNA sequencing for direct detection of seven selected antimicrobial resistance markers in 18 target pathogens. Clearly, it has potential clinical utility in optimizing therapy and was incorporated into the Karius test workflow in September 2023. In addition, the workflow could readily be adapted to expand the number of target bacteria and antimicrobial resistance markers as needed.

Feral swine are invasive in the United States and a reservoir for infectious diseases. The increase in feral swine population and the geographic range are a concern for the spread of zoonotic diseases to humans and livestock. Feral swine could contribute to the spread of Coxiella burnetii, the causative agent of human Q fever. In this study, we characterized the seroprevalence of C. burnetii in feral swine populations of Hawai'i and Texas, which have low and high rates of human Q fever, respectively. Seropositivity rates were as high as 0.19% and 6.03% in Hawai'i and Texas, respectively, indicating that feral swine cannot be ruled out as a potential reservoir for disease transmission and spread. In Texas, we identified the overlap between seropositivity of feral swine and human Q fever incidence. These results indicate that there is a potentially low but detectable risk of C. burnetii exposure associated with feral swine populations in Hawai'i and Texas.