Journal of Clinical Microbiology最新文献

Quantitative RT-PCR (qRT-PCR) is essential for monitoring hepatitis delta virus (HDV) RNA, yet assays lack standardization. We aimed to evaluate the performance of three qRT-PCR assays and to assess the impact of a pre-analytical thermal shock procedure. We conducted a comparative study using 206 samples (106 with anti-HDV antibodies and 100 anti-HDV-negative as a control group), which were tested in parallel with three qRT-PCR assays: Vircell, Certest, and Altona. Performance was evaluated for inter-assay agreement (kappa index), quantitative correlation (R²), and bias (Bland-Altman). Altona detected 56 HDV-RNA positive samples, whereas Vircell and Certest detected 55 positive samples. Inter-assay agreement was perfect comparing Vircell vs Certest (agreement = 100%, к = 1.000) and almost perfect comparing Altona with both Vircell and Certest (agreement = 99.5%, к = 0.988). Quantitatively, Vircell and Certest assays showed relevant systematic biases compared to Altona, overestimating viral loads by approximately 0.24 log₁₀ International Units (IU)/mL (Certest) and 0.33 log₁₀ IU/mL (Vircell). The correlation with Altona was strong for Certest (R² = 0.864) and moderate for Vircell (R² = 0.793), while the correlation between Certest and Vircell was weaker (R² = 0.720). Thermal shock improved sensitivity in one case (Certest vs Vircell) but increased quantitative variability, worsening the inter-assay correlation (R² = 0.684). In conclusion, all three assays were highly concordant for the qualitative diagnosis of HDV infection, but their quantitative biases prevent their interchangeable use for treatment monitoring. Thermal shock is not recommended for routine monitoring, as a significant compromise in quantitative accuracy and precision outweighs any potential gains in sensitivity.IMPORTANCEThis study evaluates three hepatitis delta virus (HDV) RNA quantitative real-time PCR (qRT-PCR) assays, crucial for managing the HDV infection, particularly in the setting of new therapies like Bulevirtide, where assessing viral load reduction and accurate monitoring is paramount. We reveal significant quantitative biases among widely used assays, precluding their interchangeable use and risking misinterpretation of treatment response. Furthermore, our systematic assessment of the thermal shock pre-analytical procedure highlights its detrimental impact on quantitative precision, despite modest sensitivity gains. This work provides essential evidence for clinicians and laboratories, guiding assay selection and standardization efforts to optimize HDV diagnosis and patient monitoring.

The resurgence of monkeypox virus (MPXV) has increased demand for validated serological assays to assess exposure and immunity. Cross-reactivity among orthopoxviruses, stemming from high sequence conservation, complicates distinguishing antibody responses from natural MPXV infection versus vaccination or other orthopoxvirus exposures. We validated the Meso Scale Discovery (MSD) V-PLEX Orthopoxvirus Panel 1 (IgG) Kit, which quantifies antibody levels to five MPXV antigens and their vaccinia virus (VACV) orthologs, following Good Clinical Laboratory Practice guidelines. We assessed assay performance using serum from 26 individuals with prior mpox, 52 JYNNEOS vaccine recipients, and 179 unexposed controls. The assay reliably detected antibody responses in all exposed cohorts with peak levels observed 2 months post-vaccination. Antibody levels to specific antigens also correlated with Modified Vaccinia Ankara neutralization titer, particularly for MPXV B6R/VACV B5R, MPXV E8L/VACV D8L, and MPXV M1R/VACV L1. Receiver operating characteristic analysis showed that some individual antigens achieved high sensitivity and specificity for exposure detection (area under the curve [AUC] > 0.96 for VACV D8L, MPXV B6R, VACV B5R); however, individual antigens performed poorly in distinguishing infection from vaccination. In contrast, antibody level ratios between some MPXV and VACV orthologs effectively differentiated MPXV infection from vaccinia vaccination with high sensitivity and specificity (e.g., MPXV A35R/VACV A33R ortholog ratio, AUC = 0.97, sensitivity = 0.97, specificity = 0.96). Our findings validate the MSD assay for clinical research and serosurveillance to assess MPXV immunity and support the utility of ortholog pair ratio analysis as a strategy to discriminate vaccinated and infected individuals.

Importance: Mpox continues to spread around the world, with recent data showing increasing incidence in the United States. While there are multiple Food and Drug Administration (FDA)-authorized real-time PCR tests for diagnostic use, there are no FDA-authorized serological tests and few laboratory-developed serological tests offered. We evaluated the Meso Scale Discovery V-PLEX Orthopoxvirus Panel 1 (IgG) Kit according to Good Clinical Laboratory Practice guidelines and found that the assay reliably detected antibody responses in monkeypox virus (MPXV)- and vaccinia virus (VACV)-exposed cohorts and could distinguish them from unexposed cohorts. Intriguingly, we found that antibody level ratios between certain MPXV and VACV orthologs could distinguish prior mpox infection from vaccinia vaccination. Overall, these data highlight the use of multi-antigen panels in challenging scenarios for serological testing, such as the cross-reactivity presented by orthopoxviruses.

Invasive fungal disease (IFD) is a major cause of morbidity and mortality in the immunocompromised population. Early diagnosis is challenging due to the low sensitivity and non-specificity of non-invasive fungal biomarkers, the need for invasive specimen collection, and the limitations of culture and histopathology. Detection of circulating fungal cell-free DNA (cfDNA) in plasma and serum by polymerase chain reaction (PCR) represents a novel testing modality for rapid and accurate diagnosis of IFD. In this review, we summarize the performance characteristics of fungal cfDNA PCR for the diagnosis of invasive aspergillosis, mucormycosis, and Pneumocystis pneumonia. We discuss a testing algorithm that incorporates fungal cfDNA and the added diagnostic value of invasive specimen collection when non-invasive mold cfDNA PCR is performed first. Lastly, we discuss the role of diagnostic stewardship in fungal cfDNA PCR testing.

Blood culture contamination is common, causing diagnostic uncertainty and unnecessary antibiotic use. Analyzing growth patterns within culture sets might offer diagnostic value. We retrospectively analyzed peripheral blood culture sets from 2019 and 2024. Growth pattern (one bottle [discordant] vs both bottles [concordant]) was analyzed according to clinical significance (contaminant vs true pathogen). Overall, 38,216 blood culture sets were analyzed, including 1,491 (3.9%) discordant and 1,938 (5.1%) concordant sets (remaining 34,787 [91.0%] sets were sterile). Discordant sets grew 1,060/1,491 (71.1%) contaminants and 431/1,491 (28.9%) true pathogens. Concordant sets grew 222/1,938 (11.4%) contaminants and 1,716/1,938 (88.5%) true pathogens (P < 0.001). Examining coagulase-negative staphylococci (CoNS) only (2019 data set), 629/642 (98.0%) discordant sets grew contaminants, while 13/642 (2.0%) grew true pathogens. In contrast, Staphylococcus aureus grew in only 82/270 (30.4%) discordant sets. Among 858 first CoNS-positive cultures per patient, 624/636 (98.1%) discordant sets grew contaminants, and 12/636 (1.9%) grew CoNS defined as a true pathogen. The negative predictive value of a discordant first CoNS set to exclude true CoNS bacteremia was 98.1% (95% confidence interval 96.7%-98.9%). Examining aerobic vs anaerobic bottles in 356 discordant sets, contaminants were found more frequently in aerobic bottles (135/356, 37.9% vs 73/356, 20.5%, P = 0.04). The proportion of true pathogens was similar in both (79/356, 22.2% vs 69/356, 19.4%, P = 0.4). Discordant CoNS-positive cultures were strongly associated with contamination. This could assist in interpreting blood culture results and supporting antimicrobial stewardship. Discordance might result from a diversion effect, the aerobic bottle acting as a diversion device for the anaerobic bottle.IMPORTANCERapidly distinguishing blood culture contaminants from true pathogens is essential for optimizing antimicrobial stewardship and avoiding unnecessary antibiotic therapy. In this large, two-period study, we demonstrate that discordant growth of coagulase-negative staphylococci in a two-bottle set has a negative predictive value of 98.1% for true bacteremia. This finding remained robust across both study years and when restricted to first positive cultures, highlighting its reliability. Incorporating simple growth pattern analysis into early blood culture interpretation can provide clinicians with reliable and timely information within 24 hours, supporting more targeted and judicious antibiotic use.

Rapid TB diagnostics are essential for effective TB control. Combining WHO-recommended rapid molecular tests with downstream targeted next-generation sequencing (tNGS) enables faster drug resistance profiling. The objective of this study was to establish a one-day diagnostic platform (ODDP) integrating BD MAX MDR-TB and AmPORE-TB tNGS from a single sample. Pooled sputum samples spiked with 52 pre-characterized Mycobacterium tuberculosis (MTB) strains and 74 MTB-positive clinical samples were tested using BD MAX MDR-TB for TB, isoniazid, and rifampicin resistance. tNGS was performed from 5 µL of purified DNA leftover for each TB-positive sample in the BD MAX strips. IS6110/IS1081 Ct-values served as surrogate markers for TB DNA concentration. A total of 104 spiked and 60 clinical samples tested positive by BD MAX. The average time to the final ODDP result was 8.5 h. For samples with Ct ≤28, tNGS generated antibiotic resistance profiles for ≥12 antibiotics with 85.1% sensitivity in spiked and 73% in clinical samples. Failure rates were 10% and 8.3%, respectively. Resistance profiling most frequently (up to 11.3%) failed for clofazimine, pretomanid, and delamanid. The ODDP enables comprehensive TB diagnosis and resistance profiling from a single sample in 1 day. This platform can significantly accelerate the time to informed drug-resistant (DR)-TB treatment decisions.IMPORTANCEReducing the time to treatment initiation decreases patient drop-out rates, morbidity, the emergence of new drug resistances, and onward transmission of infection. Obtaining the complete resistome from the start is crucial for choosing a fully effective treatment regimen. Until now, diagnosis with full resistance profiling has required at least two sputum samples and 3 to 7 days for the complete workflow, obliging patients to return two to three times, which dramatically increased the risk of loss to follow-up. Our one-day diagnostic platform enables both diagnosis and comprehensive resistance testing from a single sample within 1 day. Patients can remain in a day clinic during testing and receive a fully effective, individualized treatment regimen the same day. This approach is expected to markedly reduce morbidity, drop-out rates, and transmission. The necessary instruments and technologies are already available in many high-prevalence countries and are currently being rapidly scaled up worldwide.