Journal of Clinical Microbiology最新文献

Community-acquired febrile illness presents a significant diagnostic challenge. The TaqMan Array Card (TAC) enables simultaneous detection of many bacterial, viral, parasitic, and fungal pathogens. This systematic review aimed to characterize the etiologies of community-acquired fever diagnosed by TAC directly on blood specimens. A systematic search of PubMed, Scopus, Embase, and Web of Science (from inception to January 2026 in English) identified studies using TAC to detect pathogens directly in human blood to identify the etiology of community-acquired febrile illness. Eligible studies were appraised using a modified Joanna Briggs Institute (JBI) checklist. Pooled proportions and meta-analyses were conducted using a random-effects model. A total of 16 studies comprising 8,937 participants from 12 African and Asian countries met inclusion criteria. TAC detected at least 1 pathogen in 0.43 (95% CI [0.32; 0.54]), with high between-study heterogeneity (I² = 99%). Parasitic infections were most common (56%), nearly all due to Plasmodium spp., followed by bacterial (26%), viral (22%), and fungal (0.5%) etiologies. Plasmodium spp., Rickettsia spp., dengue virus, Lassa fever virus, Streptococcus pneumoniae, Orientia tsutsugamushi, Mycobacterium tuberculosis complex, and Salmonella spp. were the most frequently identified pathogens. Culture-negative bacteria accounted for 56% of bacterial detections. Positivity with more than 1 organism occurred in 6% of cases. TAC showed strong concordance with blood culture for culturable organisms (positive agreement: 73%, 95%CI [0.53; 0.89]; negative: 98%, 95%CI [0.95; 1.00]). TAC provides culture-independent pathogen detection and reveals overlooked causes of febrile illness, such as culture-negative bacteria. Its application may improve clinical diagnosis and surveillance programs.

Importance: Accurate identification of the causes of community-acquired febrile illness is essential for guiding clinical decision-making, surveillance programs, and public health response. This systematic review and meta-analysis synthesizes data from more than 8,937 patients across Africa and Asia tested with blood-based TaqMan Array Card (TAC) assays. TAC enabled broad, culture-independent detection of bacterial, viral, parasitic, and fungal pathogens-revealing both expected causes, such as Plasmodium spp. and dengue virus, and neglected culture-negative bacteria including Orientia tsutsugamushi, Coxiella burnetii, and Rickettsia spp. By uncovering these underrecognized etiologies, TAC provides valuable pathogen-level data to strengthen fever surveillance, inform outbreak detection, and refine regional disease prioritization frameworks. These findings highlight the potential of high-throughput molecular diagnostics to enhance infectious disease control, particularly in resource-limited settings.

The genus Mycobacterium, including Mycobacterium tuberculosis and over 200 nontuberculous mycobacteria (NTM), shows wide variability in clinical outcomes and drug susceptibility. Although culture-based identification remains the gold standard, slow mycobacterial growth delays diagnosis and treatment. In this study, we evaluated a novel culture-free method for subspecies-level identification directly from sputum. In this single-center prospective cohort study at Osaka Toneyama Medical Center, we analyzed 125 sputum samples from 115 patients with NTM pulmonary disease and 10 with non-NTM respiratory conditions. Samples were decontaminated using N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) or succinic acid. We compared the reference culture method (mycobacterial culture plus whole-genome sequencing) and a culture-free direct target capture sequencing method. Core genome multi-locus sequence typing identified subspecies in both workflows, covering 186 mycobacterial species, including M. tuberculosis. The 115 NTM cohort specimens yielded 57 smear-positive and 93 culture-positive results. The identified subspecies included 48 Mycobacterium avium subsp. hominissuis, 22 Mycobacterium intracellulare subsp. intracellulare, 5 subsp. chimaera, 7 Mycobacterium abscessus subsp. abscessus, 5 subsp. massiliense, 1 M. tuberculosis, and 5 other NTM species. The culture-free method showed a high identification rate for smear-positive specimens (75.4%) but a low identification rate for smear-negative specimens (13.9%). NALC-NaOH pretreatment resulted in higher accuracy (90.5%) than did succinic acid pretreatment (66.7%). Thus, our culture-free subspecies-level identification method achieved high accuracy, especially in alkaline-treated smear-positive sputum samples, achieving rates above 90%. This method is recommended in clinical practice for patients who require rapid diagnosis and timely initiation of appropriate treatment, bypassing time-consuming culture steps.IMPORTANCEAccurate identification of Mycobacterium species and subspecies is crucial for effective treatment, as drug susceptibility and clinical outcomes vary significantly among them. However, conventional diagnosis relies on culture-based methods that can take several weeks, critically delaying appropriate therapy. This study validates a novel culture-free method using target capture sequencing for the comprehensive, subspecies-level identification of over 186 mycobacterial species directly from sputum specimens. Our findings revealed the high accuracy of this approach for smear-positive specimens, especially with alkaline pretreatment. This rapid method is applicable in clinical settings and enables timely and precise treatment decisions, greatly benefiting patients who require urgent intervention.

The use of carbapenem antibiotics is threatened by the global spread of carbapenemase-producing Enterobacterales (CPE), bacterial pathogens which hydrolyze these last-resort antimicrobials. Rapid detection of CPE is vital to ensure timely administration of antimicrobial therapy to infected patients, as well as the implementation of infection control measures to prevent outbreaks in healthcare settings. In this study, we report the development of resaCPE, a rapid, low-cost CPE detection method that couples the inactivation of an imipenem disk with a cell viability assay employing a carbapenem-hypersusceptible Escherichia coli strain. Results are interpreted by a simple colorimetric readout in which CPE-positive samples turn pink, while CPE-negative samples remain purple. The assay was validated with a panel of 116 CPE and non-CPE isolates, and its performance was compared with two widely used CPE detection strategies: the modified carbapenem inactivation method (mCIM) and the CARBA-NP test. The resaCPE test demonstrated a sensitivity of 98.7%, outperforming the CARBA-NP (87.8% sensitivity) and the mCIM (96.3% sensitivity) assays. The resaCPE test provides results within 3.5 h, significantly faster than the mCIM, and demonstrated a high degree of specificity (94.1%). Due to the rapid turnaround time, minimal setup requirements, and low cost (~$1 USD/sample), the resaCPE test is a potentially attractive option for primary CPE screening, particularly in lower resource settings.IMPORTANCECarbapenem antibiotics serve as a last line of defense against severe bacterial infections. However, these antibiotics are endangered by bacterial pathogens which produce carbapenemases, enzymes that inactivate the carbapenem antibiotics, rendering them ineffective. Infections caused by carbapenemase-producing bacteria are associated with high mortality and pose a significant threat to global health. Rapid and reliable detection of these pathogens is critical to ensure timely treatment and prevent their spread in healthcare settings. Current detection methods suffer from long turnaround times, high cost, or the requirement of specialized laboratory equipment. In this study, we developed resaCPE, a simple, inexpensive, and rapid colorimetric test that identifies carbapenemase-producing bacteria in 3.5 h. This test demonstrated excellent performance when compared to currently employed assays. Due to the rapid turnaround time, low cost, and minimal equipment requirements, the resaCPE test could be a promising alternative for routine carbapenemase screening, particularly in laboratories with limited resources.

Shigella is a leading cause of diarrhea and dysentery in children younger than 5 years of age in low-resource settings, and several vaccines are in development. Due to its fastidious nature, Shigella can be difficult to culture, and eventual vaccine trials will need to optimize the isolation of Shigella to ensure efficient sample sizes. In the recently concluded Enterics for Global Health (EFGH) Shigella Surveillance study, we compared Shigella culture isolation rates between rectal swab vs. whole stool, between two swabs vs. one, and between Cary Blair (CB) vs. modified-Buffered Glycerol Saline (mBGS) transport media to identify the optimal methods for Shigella recovery by microbiologic culture. Among 9,476 children aged 6-35 months enrolled in the EFGH study from seven country sites, Shigella isolation rates did not differ significantly between CB (7.8%) and mBGS (7.9%) (P > 0.99). Using two swabs improved the detection rates (9.3%), compared with one swab (7.9%) (P < 0.001). Among the 2,048 children from Bangladesh and The Gambia, where both rectal swabs and whole stool were collected from the same children, rectal swabs were found to be non-inferior to whole stool for Shigella culture (12.4% and 12.7%, respectively, with a difference of -0.29% [95% CI: -0.83% to 0.24%]). To optimize Shigella recovery for future multi-country vaccine trials, we recommend collecting two flocked rectal swabs in CB or mBGS media with strict adherence to transit conditions-an approach proven feasible across EFGH sites.Clinical TrialsThis study is registered with ClinicalTrials.gov as NCT06047821.IMPORTANCEShigella is difficult to isolate by culture, making optimized sampling and transport essential for microbiologic confirmation. As Shigella vaccines are tested for efficacy and eventual licensure, it is critical that the laboratory methods are optimized to avoid missing children with shigellosis. In the multi-country Enterics for Global Health (EFGH) study, which enrolled over 9,000 children with diarrhea, rectal swabs and whole stool recovered a similar number of Shigella isolates, with two swabs improving detection over a single swab. Cary Blair (CB) and modified-Buffered Glycerol Saline (mBGS) transport media also resulted in similar isolation rates. To maximize outcome ascertainment in future vaccine trials, two flocked rectal swabs with transport in either medium are recommended to balance sensitivity, feasibility, and scalability.

Current diagnostic approaches for mucormycosis are often limited by low sensitivity and prolonged turnaround times, which result in delayed treatment and poor clinical outcomes. We developed a novel diagnostic method utilizing a colorimetric loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of mucormycosis. The assay incorporates specifically designed primers capable of detecting as low as 0.001 picograms (pg) of spiked genomic DNA from Mucorales fungi. This LAMP assay demonstrated a high sensitivity of 98% and a 100% specificity of detecting fungal ribosomal DNA (rDNA) in bronchoalveolar lavage (BAL) samples collected from mice infected with Mucorales fungi (n = 48) or from an uninfected control group (n = 15). To align the assay with clinical antifungal therapy, a subset of infected mice was treated with either liposomal amphotericin B (LAMB) or a combination of LAMB and a humanized monoclonal antibody (VX-01) targeting the Mucorales-specific surface protein CotH3. Consistent with the treatment efficacy, the LAMP assay detected significantly lower fungal burdens in BAL samples from mice receiving the combination therapy compared to those treated with LAMB alone or placebo. Further validation was conducted using BAL samples from patients diagnosed with mucormycosis (n = 24) or aspergillosis (n = 17). The assay demonstrated a sensitivity of 79% and a specificity of 94%. These findings highlight the diagnostic potential of this LAMP-based assay as a point-of-care. Its high sensitivity, specificity, and rapid turnaround time position this assay as a promising tool for early and accurate detection of mucormycosis, with the potential to improve patient management and clinical outcomes.IMPORTANCEMucormycosis is a rapidly progressive and fatal fungal infection. Timely diagnosis is critical for effective treatment, yet current diagnostic tools are slow, insensitive, or require complex laboratory procedures. In this study, we developed and validated a colorimetric loop-mediated isothermal amplification (LAMP) assay that enables rapid and reliable detection of Mucorales DNA directly from bronchoalveolar lavage (BAL) specimens. The assay demonstrated high sensitivity and specificity in both experimental mouse models and clinical samples, producing results within 1 h without the need for sophisticated equipment. This simple, robust, and cost-effective molecular diagnostic tool holds great potential for early detection of mucormycosis, facilitating prompt antifungal therapy and improving patient survival.

Tuberculosis (TB) remains the leading cause of mortality caused by a single infectious agent. Targeted next-generation sequencing (tNGS) has become a promising molecular method for TB diagnosis, but its accuracy and application for non-sputum specimens still remain unexplored. In this multicenter prospective lab-developed assay, 701 participants from five designated TB hospitals from different provinces in China were recruited. Non-sputum specimens were collected for tNGS, Xpert Mycobacterium tuberculosis (MTB)/RIF (Xpert), and culture tests. The diagnostic accuracy of tNGS for TB patients was evaluated compared with the microbiological reference standard (MRS). Phenotypic drug susceptibility tests (DSTs) were conducted using culture-positive isolates and employed to assess the accuracy of tNGS for detecting drug resistance. We found that the tNGS assay exhibited high diagnostic accuracy with sensitivity and specificity of 93.4% (95% CI, 91.5%-95.2%) and 93.2% (95% CI, 91.3%-95.0%) when utilizing MRS as the gold standard. The diagnostic performance of tNGS was robust regardless of specimen types and clinical symptoms. tNGS also showed a detection potential for other coinfecting respiratory pathogens. More than 90% of tNGS-positive individuals gained drug susceptibility results, which were mainly dependent on bacterial loads. Compared with phenotypical DSTs, tNGS had high sensitivities and specificities for the detection of rifampicin, isoniazid, streptomycin, ethambutol, and levofloxacin resistance. Our findings illustrated that tNGS assay is a rapid and highly sensitive test for TB diagnosis and simultaneous detection of drug resistance in non-sputum specimens. Its accuracy gain compared with conventional methods is most remarkable in TB patients with low bacterial loads.IMPORTANCETuberculosis (TB) diagnosis still remains challenging, especially in non-sputum patients. Nanopore-based targeted next-generation sequencing (tNGS) is a promising technology for the detection of TB cases and drug resistance, which has capacities to provide a panel of drug resistance profiles. The purpose of this study is to explore the diagnostic performance of tNGS for non-sputum specimens from five designated TB hospitals from different regions in China. Compared with the microbiological reference standard (MRS), tNGS exhibited high sensitivity and specificity, which are associated with bacterial loads in samples. Meanwhile, tNGS has capacities to detect coinfecting respiratory pathogens and produce drug-resistant profiles. Therefore, our findings suggested that tNGS is an alternative method for the diagnosis of non-sputum TB patients.CLINICAL TRIALSThis study is registered with Chinese Clinical Trial Registry as ChiCTR2400088518.

Ceftobiprole is an advanced-generation cephalosporin active against methicillin-resistant Staphylococcus aureus and other clinically relevant bacteria. Ceftobiprole was recently (April 2024) approved for clinical use in the United States, but susceptibility testing is still not available on commercial automated susceptibility testing systems. We evaluated the accuracy and error rates when using susceptibility to ceftaroline to predict susceptibility to ceftobiprole. A total of 42,363 clinical isolates were collected through the SENTRY Antimicrobial Surveillance Program from 34 US medical centers in 2016-2020 and tested against ceftobiprole and ceftaroline by broth microdilution method. The accuracy of the surrogate test (susceptibility to ceftaroline) was defined as the percentage of isolates susceptible to ceftobiprole among ceftaroline-susceptible isolates. Minor error rate was then defined as the percentage of ceftobiprole-intermediate among ceftaroline-susceptible isolates, and very major error was defined as the percentage of ceftobiprole-resistant isolates among ceftaroline-susceptible isolates. The accuracy of the surrogate test to predict susceptibility to ceftobiprole was >99% for S. aureus (99.98%), including methicillin-resistant S. aureus (99.95%), Streptococcus pneumoniae (99.48%), β-hemolytic streptococci (100.00%), Escherichia coli (99.97%), and Klebsiella pneumoniae (99.91%). Accuracy was also extremely high for Haemophilus spp. (98.86%) and Enterobacterales (98.73%). Minor error rates were <1% for all organism groups evaluated, and very major errors were only observed with S. pneumoniae (0.06%), Haemophilus spp. (0.31%), Enterobacterales (0.40%), and K. pneumoniae (0.03%). In conclusion, the results of the comparative analyses of ceftobiprole and ceftaroline MIC values show that susceptibility to ceftobiprole can be inferred by susceptibility to ceftaroline with very high accuracy.

Importance: Ceftobiprole was recently (April 2024) approved for clinical use in the United States but susceptibility testing is still not available on commercial automated susceptibility testing systems and may not be available for some time. In the interim, one possible strategy to assess ceftobiprole susceptibility would be to apply the susceptibility results of ceftaroline as a surrogate marker of susceptibility. This strategy could facilitate rapid clinical use of ceftobiprole in the United States pending the more necessary availability of commercial susceptibility testing devices to direct testing ceftobiprole.

Accurate identification of recent HIV-1 infections is critical for real-time epidemic monitoring. However, conventional Limiting Antigen Avidity Enzyme Immunoassays (LAg-EIAs) are restricted to laboratory settings. A novel rapid recency test based on the limiting antigen avidity principle was developed for point-of-care use. We evaluated the performance of the rapid HIV-1 recency test using 500 longitudinal plasma specimens from 107 seroconverters. The mean duration of recent infection (MDRI) was estimated via binomial regression with maximum likelihood modeling. The false recent rate (FRR) was assessed using samples from individuals with long-term infection, including those on antiretroviral therapy (ART). Concordance was compared with two commercial LAg-EIA kits (Maxim and KingHawk). The rapid assay yielded an MDRI of 123 days (95% CI: 87-138), shorter than Maxim (152 days, 95% CI: 137-172) and KingHawk (131 days, 95% CI: 107-140). Among ART-naïve individuals infected for over 1 year, FRR was 5.3%, similar to Maxim (5.9%) and slightly higher than KingHawk (2.7%). High concordance was observed with Maxim (93.1%, kappa = 0.743) and KingHawk (89.4%, kappa = 0.558). In ART-treated individuals, FRR was significantly higher in the early ART group (69.9%) compared to the late ART group (20.2%, P < 0.001). The novel rapid HIV-1 recency assay demonstrates acceptable MDRI and FRR and strong agreement with commercial LAg-EIA kits. Its simplicity and rapid turnaround make it a promising tool for decentralized surveillance and targeted HIV interventions, especially in resource-limited settings.IMPORTANCERapid detection of recent HIV-1 infections is essential for monitoring ongoing transmission and guiding targeted prevention efforts. However, currently used laboratory-based recency assays require specialized facilities and trained personnel, limiting their use in decentralized or resource-limited settings. In this study, we evaluated a newly developed rapid test that identifies recent HIV-1 infections within minutes using a simple, instrument-free format. The test showed strong agreement with two widely used laboratory assays and demonstrated performance suitable for surveillance applications. Its ease of use, rapid turnaround, and minimal infrastructure requirements make this rapid test a practical tool for expanding real-time HIV monitoring and improving the efficiency of public health responses.