Journal of Clinical Microbiology最新文献

Trypanosoma brucei infections cause African trypanosomiasis, also known as sleeping sickness in humans and nagana in animals, presenting a significant global health and economic burden, especially in sub-Saharan Africa. Animal trypanosomiasis also affects the economic development in Asia and South America. Accurate diagnosis of diseases caused by infection with the parasite of the T. brucei group remains a major challenge due to the persistence of infection-induced antibodies long after parasite clearance, complicating serological discrimination between active and past infections. To address this limitation, we developed a sensitive and specific antigen detection assay targeting Trypanosoma brucei enolase (TbrENO) using a panel of camelid single-domain antibodies (sdAbs also known as nanobodies). Among the candidates, the sdAbsR3-77/sdAbR2-103 sandwich enzyme-linked immunosorbent assay (ELISA) exhibited robust performance in detecting circulating TbrENO in plasma from experimentally infected mice. Additionally, this assay showed strong potential as a "test-of-cure" tool by monitoring real-time antigenemia throughout a chronic T. brucei infection course. We further validated the assay's diagnostic utility in human clinical samples, detecting Trypanosoma brucei rhodesiense infections at both early and advanced stages and Trypanosoma brucei gambiense infections in advanced stage. The sdAbsR3-77H/sdAbR2-103HA ELISA targeting TbrENO shows potential for point-of-care pan-diagnosis of active T. brucei infections (including Trypanosoma brucei brucei, T. b. gambiense, T. b. rhodesiense, Trypanosoma brucei evansi, and Trypanosoma brucei equiperdum) in both animals and humans. Therefore, this assay addresses gaps in current diagnostic capabilities by overcoming the key limitations of antibody-based tests, offering a promising tool for improved disease control.IMPORTANCEAfrican trypanosomiasis, commonly known as sleeping sickness in humans and nagana in animals, is a life-threatening disease that remains a major health and economic concern in many parts of the world. One of the key difficulties in managing this disease is detecting ongoing infections, as existing antibody-based tests cannot reliably distinguish between current and past infections. In this study, we developed a novel laboratory test that detects a specific protein released by the parasite during infection. This test uses special antibodies derived from camels, known for their exceptional stability and precision, to accurately identify infections caused by multiple Trypanosoma brucei subspecies. Our approach not only enables accurate diagnosis but also offers a way to monitor treatment success. This work provides a valuable tool for disease control efforts and could help improve the health of both humans and animals in regions where trypanosomiasis is endemic.

The article by S. J. Oyeniran, A. L. Leber and H. Wang (J Clin Microbiol 63:e00986-25, 2025, https://doi.org/10.1128/jcm.00986-25) describes a novel laboratory-developed diagnostic PCR assay that amplifies the gene encoding Kingella kingae's major outer membrane protein. The test confirmed the prime role of the organism causing skeletal system infections in preschoolers and enabled the administration of targeted antibiotic therapy. It is hoped that a much-needed commercial K. kingae-specific molecular test will soon receive FDA clearance to improve the management of pediatric osteoarticular infections.

Leptospirosis, caused by Leptospira spp. infection, is a globally significant zoonotic disease that affects a wide range of animals. Although renal colonization is well-documented, genital infection by leptospires remained less explored for decades, despite its impact on reproduction. Evidence suggests that genital infection occurs as a primary condition rather than secondary to renal colonization, particularly in cattle suffering from bovine genital leptospirosis (BGL), linked to chronic infections by strains of the Sejroe serogroup. In horses, a similar condition is suggested to be associated with strains of serogroup Australis. Molecular studies confirmed the presence of Leptospira DNA in uterine, follicular, and vaginal samples, strengthening the hypothesis of an independent genital physiopathology. Despite significant advances in molecular diagnostics, the detection of genital carriers remains challenging, requiring refined methodologies beyond standard serology. This review critically examines the historical detection of Leptospira spp. in genital samples of cattle, small ruminants, swine, and equines, emphasizing its relevance to reproductive health. Moreover, we highlight the limitations of current diagnostic approaches, advocating for increased use of genital samples in leptospirosis research of large animals and shedding light on future directions regarding genital leptospirosis in livestock research. Enhanced understanding and diagnosis of genital leptospirosis will contribute to better livestock reproductive management and disease prevention.

The diagnosis of congenital toxoplasmosis or disseminated toxoplasmosis in immunocompromised patients nowadays relies on molecular tools, in particular real-time PCR. There are many reagents on the market, and their evaluation by independent experts provides valuable information to medical biologists who are looking for a high-performance kit among the different references. Under the aegis of the French National Reference Center for Toxoplasmosis, we report here a multicenter evaluation of the analytical and clinical performances of the "quanty TOXO (RH region)" PCR assay manufactured by Clonit. The kit showed good analytical performance, as indicated by the results of serial dilution tests and external quality control samples. PCR efficiencies varied from 95% to 105%; linearity zone extended over four log units (R² >0.99), and limit of detection varied from <1 parasite/mL to between 1 and 5 parasites/mL, i.e., from <0.08 parasite/PCR to between 0.2 and 1 parasite/PCR, depending on the center. Based on 141 cryopreserved DNAs from a large range of clinical specimens, we determined a clinical sensitivity of 94.7% (71/75; 95% confidence interval [CI]: 87.1%-97.9%) and a clinical specificity of 100% (66/66; 95% CI: 94.5%-100%). Four false negative results were detected despite amplification carried out in duplicate. Overall, the "quanty TOXO (RH region)" PCR assay demonstrated satisfactory analytical and clinical performances for the diagnosis of toxoplasmosis, even using extraction and amplification techniques or biological matrices not validated by the manufacturer.IMPORTANCEDue to its speed and accuracy, PCR is now the gold standard for diagnosing congenital and disseminated toxoplasmosis. High-performance molecular testing is essential, especially for immunocompromised patients and congenital infections, to initiate early treatment. This diagnostic approach increasingly relies on commercial assays. However, commercially available kits do not guarantee performance. In this study, conducted by the French National Reference Center for Toxoplasmosis, we performed an independent multicenter evaluation of the "quanty TOXO (RH region)" PCR assay manufactured by Clonit. Our results showed that this kit delivered satisfactory results for routine diagnostic use. However, among the 141 clinical samples tested, four false negative results were noted, corresponding to specimens with low parasitic load.

Left-ventricular assist devices (LVADs) are increasingly used as a bridge to heart transplantation and destination therapy. These devices, especially the driveline, are susceptible to difficult-to-treat infections, associated with high morbidity and mortality rates. Staphylococcus aureus (S. aureus) is a major causative pathogen of LVAD infections. Antibiotic resistance and biofilm formation can complicate the treatment of these infections. A novel in vitro assay was developed to study the antibiotic susceptibility of S. aureus biofilm grown on LVAD drivelines. Besides antibiotic monotherapy, the effect of various antibiotics combined with rifampicin was studied. Additionally, we explored the efficacy of four individual phages and phage-antibiotic combinations as potential treatment strategies. Our data showed a decrease of susceptibility of the S. aureus biofilms to antibiotic monotherapy compared to planktonic S. aureus. With only rifampicin and erythromycin monotherapy resulting in full bacterial clearance. Combining antibiotics with rifampicin showed similar antimicrobial efficacy against S. aureus biofilms as rifampicin monotherapy. While both individual phages and a phage cocktail were effective against planktonic bacteria, phage efficacy was limited against S. aureus in biofilm. Combining phages with antibiotics did not clearly improve treatment efficacy, compared to antibiotic monotherapy. Contrarily, it even increased bacterial growth when phage administration preceded antibiotic treatment. Here, both antibiotic- and phage monotherapy showed reduced efficacy on LVAD-driveline biofilms. Additionally, phages did not show an additive value to antibiotic treatment of LVAD driveline infections. Further studies are needed to elucidate optimal treatment strategies for LVAD-driveline infections.IMPORTANCECurrent treatment strategies for S. aureus LVAD-driveline infections are based on in vitro antibiotic susceptibility of planktonic bacteria. However, LVAD infections are most often biofilm-related, which decreases antibiotic susceptibility significantly, resulting in discrepancies between in vitro antibiotic susceptibility and in vivo treatment success. Here, we have developed a novel in vitro assay to determine antibiotic susceptibility of S. aureus biofilm, grown in conditions relevant to LVAD-driveline infections. Next to antibiotic susceptibility, the susceptibility of this biofilm to bacteriophage mono- and combination treatment with antibiotics was evaluated as an alternative treatment strategy. In the future, this assay can be used to provide a better insight in in vivo antibiotic- and bacteriophage susceptibility of LVAD-driveline biofilms. Thereby improving in vivo treatment strategies for LVAD-driveline infections.

Providing timely and accurate antimicrobial susceptibility testing (AST) results is a crucial component of clinical microbiology practice. Commercial rapid AST (RAST) is an emerging and quickly expanding area. These phenotypic RAST systems use various novel methods to monitor bacterial growth and replication in order to shorten the duration of time required for testing. Implementation of RAST has the potential to expedite antimicrobial therapeutic optimization, which can improve patient care. This minireview describes the current state of commercial phenotypic RAST including tests designed to report antimicrobial susceptibilities directly from clinical specimens.

The reported human infections with the emerging zoonotic pathogen Streptococcus parasuis are steadily rising. Rapid and standardized genotyping tools specific to S. parasuis are critically needed for epidemiological surveillance and identification of strains with zoonotic potential. This study developed a whole-genome sequence (WGS)-based typing strategy, encompassing average nucleotide identity, a minimum core genome (MCG) typing scheme, and a multilocus sequence typing (MLST) scheme using 255 S. parasuis genomes isolated from eight countries between the 1980s and 2024. The S. parasuis population was categorized into 12 MCG clusters based on 72,172 SNPs in non-recombining regions distributed across an MCG comprising 607 genes, forming two distinct lineages. The rapid MCG typing program accurately assigned 92.5% of S. parasuis genomes to their corresponding MCG clusters by identifying 4,509 cluster/subcluster-specific SNPs. To elucidate the clonal relationships among S. parasuis genomes, an MLST scheme was developed, defining 161 sequence types (STs) based on the allelic profiles of seven housekeeping loci (aroA, cpn60, gki, mutS, sdhA, recA, and thrA). Thirty-two STs that shared identical alleles at 6 loci were assigned to 10 complex clones, whereas 100 STs that shared identical alleles at 4 or more loci were grouped into 9 ST clades. The MCG typing scheme and the MLST scheme demonstrated sufficient discriminatory power, with Simpson's diversity index values of 0.8864 and 0.9821, respectively. This study characterized the S. parasuis population and provided a rapid, reproducible, and expandable WGS-based typing strategy for taxonomic identification, epidemiological surveillance, and evaluation of the zoonotic potential of S. parasuis.IMPORTANCEOur study provides valuable insights for developing effective prevention and control strategies for Streptococcus parasuis infections, by revealing the structural characteristics and phylogenetic relationship of S. parasuis population, by developing a whole-genome sequence-based typing strategy applicable for epidemiological surveillance, transmission investigation, and zoonotic potential evaluation.

Metabolic profiling of respiratory samples from individuals infected and uninfected with respiratory viral infections may identify biomarker signatures that complement routine clinical diagnostic testing and offer unique insights into pathophysiology. We used liquid chromatography quadrupole time-of-flight mass spectrometry to generate untargeted metabolomic profiles and identified top biomarker signatures differentiating severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) positive from negative samples via machine learning. We then adapted these signatures to liquid chromatography-tandem mass spectrometry for targeted profiling and assessed classification performance, including samples positive for other respiratory viruses and negative for viral testing. A total of 1,226 samples were tested, including 521 positive samples for SARS-CoV-2, 97 for influenza A, 96 for respiratory syncytial virus (RSV), 211 for other respiratory viruses, and 301 negative samples. The top-performing model was the Light Gradient Boosting Model, which showed an area under the receiver operating characteristic curve (AUC) of 0.99 (95% confidence interval [CI], 0.99-1.00), sensitivity of 0.96 (95% CI, 0.91-0.99), and specificity of 0.95 (95% CI, 0.90-0.97). A separate machine learning analysis investigating the performance by viral subtype showed high performance for the identification of influenza A virus with an AUC of 0.97 (95% CI, 0.94-0.99) and RSV with an AUC of 0.99 (95% CI, 0.97-1.00). The two features with the highest ranking were identified as 3-oxo-heneicosanoic acid and 2-(4-hydroxyphenyl) ethanol. These findings extend our understanding of the metabolic impact of respiratory viral infections and support the potential of metabolomics to complement routine clinical diagnostic methods.IMPORTANCEMolecular testing has greatly improved how viruses are diagnosed; however, gaps remain, including limited sensitivity directly from specimens and inability to differentiate active from resolved infection. In this study, we investigated the use of a distinct diagnostic approach, mass spectrometry for detection of metabolites (small molecules) combined with machine learning analysis, for the diagnosis of SARS-CoV-2 and other respiratory viruses. We demonstrated strong performance of this approach directly from upper respiratory swab samples to differentiate SARS-CoV-2-infected versus uninfected individuals. Extension of this approach to influenza and RSV maintained a high level of performance. This research suggests that mass spectrometry-based infectious disease diagnostic testing has clinical potential and that these metabolomic features may reveal novel host-pathogen interactions and therapeutic targets. Applying a similar approach to prospective, multisite cohorts of patients with other infectious diseases carries potential to extend our understanding of the metabolic pathways involved in the host response to infection.

Drug-resistant tuberculosis (TB) remains a primary global health concern. Multidrug-resistant TB is defined by resistance to at least rifampicin (RIF) and isoniazid (INH), the two key drugs used in TB treatment. The BD MAX Multi-Drug Resistant Tuberculosis (BD MAX) assay is a fully automated real-time PCR platform recommended by the World Health Organization for the initial diagnosis of TB and RIF and INH resistance (RIF-R and INH-R) directly from pulmonary clinical samples. This study aimed to assess the off-label performance of BD MAX in clinical M. tuberculosis complex (MTBC) isolates under routine laboratory conditions. The assay was first validated using non-tuberculous mycobacteria (NTM) and MTBC isolates with known mutations. For real-world validation, it was compared to the GenoType MTBDRplus by testing 1,440 clinical isolates prospectively. The BD MAX assay correctly excluded MTBC from all NTM cultures. Among MTBC isolates with known mutations, it identified 19 of 20 RIF-R isolates and 14 of 15 INH-R isolates. In prospective testing, BD MAX achieved 99.6% sensitivity (1,403/1,409), 96.8% specificity (30/31), and 99.5% overall accuracy (1,433/1,440) for MTBC detection. For drug resistance detection, it showed 95.2% (40/42) concordance for RIF, 96.8% (30/31) for INH, and 81.3% (13/16) for MDR when compared to MTBDRplus. Discrepancies between MTBDRplus and BD MAX included heteroresistant cases and unreportable resistance results by BD MAX due to infrequent mutations or low bacterial load. Overall, this study confirms BD MAX as an accurate and reliable tool for MTBC detection and drug resistance profiling in clinical isolates in high-volume TB laboratories.IMPORTANCEThis study highlights the importance of the BD MAX Multi-Drug Resistant Tuberculosis assay (BD MAX) applied in clinical isolates for the detection of multidrug-resistant tuberculosis (MDR-TB), i.e., Mycobacterium tuberculosis resistance to rifampicin and isoniazid. TB is a global health issue, and drug-resistant TB makes treatment more difficult, favoring transmission and disease amplification. The BD MAX platform offers a faster and more automated way to detect TB and drug resistance. The study showed that BD MAX, applied off-label in clinical isolates, accurately identified TB and resistance to rifampicin and isoniazid, with results comparable to those of the widely used line probe assay. This is significant in a high-volume laboratory because it is more straightforward and more rapid than the line probe assay. BD MAX showed some limitations, especially in detecting rare mutations and in cases of low bacterial levels. Overall, this tool could improve TB care, especially in high-volume laboratories.