Journal of Clinical Microbiology最新文献

Cervical cancer screening using the Pap smear is limited by invasive sampling and low participation among select populations. As human papillomavirus (HPV) is the leading cause of cervical cancer, urine-based testing offers a non-invasive alternative with the potential to increase overall screening participation. We developed and evaluated the performance of the Phase HPV Urine Test, a qualitative real-time PCR assay for the detection of 14 high-risk HPV (HR-HPV) subtypes in first-void urine samples. The assay utilizes a proprietary, room-temperature-stable sample collection system and a novel PHASiFY DNA extraction system optimized for large-volume (40 mL) urine processing. The analytical performance was evaluated to determine accuracy, precision, limit of detection (LOD), cross-reactivity, interference, and sample stability. Analytical performance was also assessed using 128 paired urine and clinician-collected cervical specimens (cervical broom in BD SurePath) tested with the Roche cobas 4800 HPV Test as the reference assay. The assay demonstrated 82.3% initial overall percent agreement with the cobas 4800 HPV test, improving to 93% after adjudication with HPV next-generation sequencing. The assay exhibited high precision (≥97.7%), a low LOD (15-30 viral copies/mL), interference with C. albicans and T. vaginalis, whole blood and peripheral blood mononuclear cells. Urine samples remained stable at room temperature for 10 days, with 95% concordance in results. The Phase HPV Urine Test provides accurate, reproducible, and non-invasive detection of HR-HPV from self-collected urine samples, supporting its potential for expanding access to cervical cancer screening.IMPORTANCEUrine-based testing for high-risk human papillomavirus (HPV) provides a non-invasive, easily accessible alternative that may improve cervical cancer screening participation, especially among under-screened and underserved populations. The Phase HPV Urine Test combines a proprietary urine collection device with a novel large-volume DNA extraction system to enhance the sensitivity and reliability of HPV detection. Our study demonstrates that this approach yields high concordance with standard-of-care testing and maintains sample stability at ambient temperature, enabling flexible, cost-effective transport. By simplifying and standardizing urine collection and by expanding access beyond traditional clinical settings, this technology has the potential to increase uptake of HPV screening programs by overcoming barriers associated with invasive sampling methods and thereby reduce inequities. More importantly, these methods can contribute to earlier detection and prevention of cervical cancer.

Lefamulin is a novel pleuromutilin antibiotic with potent activity against methicillin-resistant Staphylococcus aureus (MRSA). While broth microdilution (BMD) is the reference method for susceptibility testing, simpler alternatives such as MIC Test Strip (gradient diffusion strip [GDS]) and disk diffusion (DD) are urgently needed for routine laboratories. We evaluated the accuracy of GDS and DD compared to BMD for lefamulin against S. aureus. A total of 422 non-duplicate clinical isolates of S. aureus (256 methicillin-susceptible S. aureus [MSSA] and 166 MRSA) collected between December 2023 and December 2024 at the Affiliated Tai'an Central Hospital of Qingdao University were tested by BMD, GDS, and DD. Interpretation followed CLSI M100-S34 breakpoints (MIC ≤ 0.25 mg/L or zone diameter ≥ 23 mm = susceptible). BMD served as the reference to calculate categorical agreement (CA), essential agreement (EA), major errors (ME), and very major errors (VME). Lefamulin exhibited potent activity against S. aureus, with 98.6% (416/422) of isolates classified as susceptible by BMD; six isolates (four MSSA and two MRSA) were non-susceptible. Compared with BMD, GDS showed 100% CA and 92.2% EA and yielded no VME and ME, and MICs were 0.49 log_₂ lower than BMD. Disk diffusion with the 20 µg lefamulin disk achieved 99.5% CA (421/422), with a VME rate of 16.7% (1/6) and an ME rate of 0.2% (1/416). Lefamulin GDS fulfilled CLSI criteria but systematically underestimated MICs; BMD confirmation is advised for GDS MIC = 0.25 mg/L. Disk diffusion showed excellent agreement, yet its VME rate surpassed the CLSI threshold owing to scarce non-susceptible isolates; any non-susceptible result by disk should be verified by BMD.

Importance: Lefamulin is a new antibiotic active against methicillin-resistant Staphylococcus aureus, but simple susceptibility tests are lacking. We compared two easy-to-perform methods with reference broth microdilution for 422 S. aureus clinical isolates. Both showed high accuracy after on-site confirmation of borderline results and can be immediately implemented in routine laboratories to guide appropriate lefamulin therapy and help contain resistance.

While South African guidelines recommend a lumbar puncture (LP) to exclude cryptococcal meningitis (CM) among all people with a newly positive cryptococcal antigen (CrAg) test, irrespective of CM symptoms, this is not always feasible. High blood CrAg lateral flow assay (LFA) titers are associated with concurrent CM and increased mortality. Single-strip CrAg semi-quantitative (SQ) tests could risk-stratify people with antigenemia. Consecutive fresh LFA-positive remnant plasma samples from a CD4 laboratory network collected between April and July 2021 were retested. We described LFA titers, CrAgSQ scores, and the proportion with cerebrospinal fluid (CSF) collected 28 days before or after a positive CrAg screening test. Of 2,240 re-tested plasma samples from unique patients, 2,166 (97%) were confirmed LFA-positive. The median LFA titer was 640 (IQR, 40-5,120), 63% (1,354/2,166) had a titer of ≥160, and 52% (1,124/2,166) had SQ scores of ≥3+. Only 31% (662/2,166) had a CSF sample collected 28 days before or after a CrAg LFA-positive test; 60% (398/662) had confirmed CM. More than half of the people with cryptococcal antigenemia had a blood CrAg titer of ≥160 or a CrAgSQ score of ≥3+, both previously shown to confer a high risk of concurrent CM. Of 3 in 10 who had an LP, most had CM, suggesting that meningitis symptoms prompted LP. Healthcare worker support/training is required to improve adherence to the universal LP recommendation. When immediate LP is not feasible, blood CrAgSQ testing can rapidly identify people at the highest risk of CM who require urgent referral for LP.

Importance: A majority of patients with HIV-associated cryptococcal antigenemia identified through a large screening program in South Africa had high cryptococcal antigen titers and thus an elevated risk of concurrent meningitis and death. Despite this, a relatively small proportion had a lumbar puncture to definitively exclude meningitis. Routine CrAg semi-quantification can help to stratify patients at higher risk for meningitis and guide clinicians' management, but performing a full range of titers for all CrAg-positive blood samples increases costs and is labor-intensive. An alternative approach is to use a single test strip, which yields a semi-quantitative score.

For cervical cancer screening, testing for high-risk human papillomavirus (hrHPV) detects more high-grade precancerous lesions, has a higher negative predictive value, and requires fewer lifetime screenings compared to cytology. As a result, both the US Preventive Services Task Force and the World Health Organization have endorsed hrHPV testing as the preferred screening method. Despite the utility, there are significant implementation challenges to adopting hrHPV primary screening across patients, providers, and institutions that must be addressed to ensure its widespread effectiveness. Here, we take a laboratory-centric approach to reviewing hrHPV primary screening, including discussion of specimen types and collection methods. For user experience, clinical and analytical validations, we focused on self-collected vaginal swab specimens stored dry during transport. Our analysis indicates that clinical laboratories should do their part to engage with institutional and clinical leadership to validate and promote the use of vaginal self-sampling for cervical cancer screening options within and outside the clinic. This work highlights the multiple studies that have validated dry swab collection as a simplified and high-quality method for hrHPV detection.

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.