Journal of Clinical Microbiology最新文献

The transition from MIRU-VNTR-based epidemiology studies in tuberculosis (TB) to genomic epidemiology has transformed how we track transmission. However, short-read sequencing is poor at analyzing repetitive regions such as the MIRU-VNTR loci. This causes a gap between the new genomic data and the large amount of information stored in historical databases. Long-read sequencing could bridge this knowledge gap by allowing analysis of repetitive regions. However, the feasibility of extracting MIRU-VNTRs from long reads and linking them to historical data has not been evaluated. In our study, an in silico arm, consisting of inference of MIRU patterns from long-read sequences (using MIRUReader program), was compared with an experimental arm, involving standard amplification and fragment sizing. We analyzed overall performance on 39 isolates from South Africa and confirmed reproducibility in a sample enriched with 62 clustered cases from Spain. Finally, we ran 25 consecutive incident cases, demonstrating the feasibility of correctly assigning new clustered/orphan cases by linking data inferred from genomic analysis to MIRU-VNTR databases. Of the 3,024 loci analyzed, only 11 discrepancies (0.36%) were found between the two arms: three attributed to experimental error and eight to misassigned alleles from long-read sequencing. A second round of analysis of these discrepancies resulted in agreement between the experimental and in silico arms in all but one locus. Adjusting the MIRUReader program code allowed us to flag potential in silico misassignments due to suboptimal coverage or unfixed double alleles. Our study indicates that long-read sequencing could help address potential chronological and geographical gaps arising from the transition from molecular to genomic epidemiology of tuberculosis.

Importance: The transition from molecular epidemiology in tuberculosis (TB), based on the analysis of repetitive regions (VNTR-based genotyping), to genomic epidemiology transforms in the precision with which we track transmission. However, short-read sequencing, the most common method for performing genomic analysis, is poor at analyzing repetitive regions. This means that we face a gap between the new genomic data and the large amount of information stored in historical databases, which is also an obstacle to cross-national surveillance involving settings where only molecular data are available. Long-read sequencing could help bridge this knowledge gap by allowing analysis of repetitive regions. Our study demonstrates that MIRU-VNTR patterns can be successfully inferred from long-read sequences, allowing the correct assignment of new cases as clustered/orphan by linking new data extracted from genomic analysis to historical MIRU-VNTR databases. Our data may provide a starting point for bridging the knowledge gap between the molecular and genomic eras in tuberculosis epidemiology.

This study evaluates the growth of mycobacteria in samples from cystic fibrosis (CF) patients and tissue samples using the mycobacteria growth indicator tube (MGIT) incubated at 30°C in comparison to conventional MGIT cultures incubated at 37°C in a BACTEC MGIT 960 device and solid media incubated at 36°C and 30°C. A total of 1,549 samples were analyzed, of which 202 mycobacterial isolates were cultured from 197 positive specimens, including five mixed cultures. The highest detection rate was achieved from MGIT at 30°C, with 84.2% of mycobacterial isolates (170 of 202), which was significantly higher than any other culture condition (P < 0.0001 for any condition). MGIT at 37°C yielded 61.4% (124 of 202) of the recovered isolates, whereas Löwenstein Jensen (LJ) and Stonebrink at 36°C, and LJ and Stonebrink at 30°C retrieved 47.0% (95), 49.5% (100), 50.0% (101), and 53.0% (107) of the isolates, respectively. Of the 53 isolates that were grown exclusively under one culture condition, the highest number of isolates (36) was recovered from MGIT incubated at 30°C. MGIT at 37°C recovered eight of the 53 isolates, whereas LJ incubated at 30°C and Stonebrink incubated at 30°C and 36°C recovered five, three, and one isolate, respectively. No isolates were grown exclusively from LJ incubated at 36°C. In CF patients and tissue samples, MGIT cultivated at 30°C for 8 weeks increases the performance of mycobacterial culture.

Importance: Our study shows that the addition of mycobacteria growth indicator tube (MGIT) liquid culture incubated at 30°C improves the detection of mycobacteria from CF and tissue samples. MGIT incubated at 30°C recovered significantly more mycobacterial isolates than MGIT incubated at 37°C and significantly more isolates than either Lowenstein Jensen or Stonebrink solid media incubated at either 36°C or 30°C. Of 202 mycobacterial isolates recovered from 1,549 specimens, 170 were recovered from MGIT incubated at 30°C, followed by MGIT incubated at 37°C with 124 isolates and solid media culture conditions that recovered between 95 and 107 mycobacterial isolates. All conventional culture conditions combined without MGIT incubated at 30°C recovered 166 isolates. MGIT incubated at 30°C recovered the highest number of isolates detected exclusively by a single culture condition and recovered mycobacterial isolates of highly relevant mycobacterial species, including Mycobacterium abscessus and Mycobacterium tuberculosis.

Bloodstream HSV-1 and HSV-2 infections can cause devastating outcomes with high morbidity and mortality, especially in neonates or immunocompromised individuals. Proper patient management for herpes simplex virus (HSV) bloodstream infections is time-sensitive and requires a rapid, accurate, and definitive diagnosis. The absence of the U.S. Food and Drug Administration (FDA)-approved molecular assays for HSV detection in blood, coupled with a lack of consensus on the optimal sample type, underscores the unmet need for improved diagnostics. We prospectively compared the cycle threshold values in paired samples including whole blood (WB), plasma, serum, and peripheral blood mononuclear cells (PBMCs) from patients with bloodstream HSV infections. This analysis employed a modified use of the FDA-cleared Simplexa HSV-1 & 2 Direct assay. The clinical performance in serum was assessed by comparing the results of 247 remnant specimens on this sample-to-answer platform to established laboratory-developed tests in a blinded fashion. Serum samples exhibited significantly lower cycle thresholds than whole blood samples [2.6 cycle threshold (Ct) bias, P < 0.001]. The modified Simplexa assay demonstrated 100% positive percent agreement for the detection of HSV-1 and HSV-2 DNA in serum samples and yielded an overall agreement of 95% (95% CI, 0.92 to 0.97), with a κ statistic of 0.75 (95% CI, 0.62 to 0.86) compared to the composite reference method. Discordance rates were 5.20% for HSV-1 and 0.81% for HSV-2. This investigation demonstrates that serum is an optimal specimen type for HSV detection when compared to several blood compartments. Serum offers a promising sample type for rapid and accurate diagnosis of HSV bloodstream infections using the modified Simplexa assay.

Importance: Rapid, accurate, and definitive diagnosis of herpes simplex virus (HSV) infections is crucial in clinical settings for patient management. The absence of FDA-authorized molecular assays for HSV-1/2 detection in blood, coupled with a lack of consensus on the optimal sample type, underscores the need for improved diagnostic methods. Furthermore, rapid diagnosis of HSV bloodstream infections enables timely administration of antiviral treatment, influences patient management decisions for those at high risk, and can contribute to shorter hospital stays, thereby reducing healthcare costs.

Since their discovery in 1953, research on human adenoviruses (HAdVs) has had diverse foci, resulted in groundbreaking discoveries, such as gene splicing, and generated powerful oncolytic constructs and expression vectors for vaccine development and gene therapy. In contrast, virologists working in this field have made relatively little progress toward the prevention and treatment of the wide spectrum of HAdV-associated diseases. The understanding of species-specific features of viral pathogenesis, or of the mechanisms underlying the establishment of latency and reactivation, is still limited. This group of viruses currently comprises 7 species, 51 serotypes, and 116 unique genotypes. This complexity manifests with a challenging pathophenotypic diversity. Some types are highly virulent, and others do not seem to cause disease in immunocompetent hosts. The assessment of viral load in blood and respiratory specimens has well-acknowledged clinical utility, but the lack of virus typing capabilities easily implementable in clinical laboratories represents a lingering major limitation to the interpretation of positive tests. Some HAdV infections do have severe consequences for both immunocompetent and immunocompromised patients, and the understanding of why this is the case will require more research. Clinical isolates and collections of positive specimens can provide unique resources to investigate the molecular bases of viral virulence and fitness and also help gather information of spatial-temporal patterns of viral circulation in susceptible communities, but they are extremely scarce. Clinical laboratories are underutilized interfaces between patients and academic scientists and have, therefore, a high potential to become valuable collaborators in research moving forward.

Many emergency departments (ED) use rapid human immunodeficiency virus (HIV) antibody tests as screening tools, despite limited sensitivity for detecting acute HIV infections. In a 4-year retrospective analysis of 1,192 patients, we evaluated the performance of a third-generation rapid HIV antibody assay tested at point-of-care (POC, Chembio Sure Check HIV 1/2) against in-lab fourth-generation screening (Abbott Architect Ag/Ab Combo). Compared to complete algorithmic testing, the POC test demonstrated a 92.5% sensitivity (95% CI = 84.6-96.5), 98.1% specificity (95% CI = 97.1-98.8), 99.5% negative predictive value (NPV; 95% CI = 98.8-99.8), and a 77.9% positive predictive value (PPV; 95% CI = 68.6-85.1). Notably, the POC test failed to detect 100% (3/3) of acute HIV infections (defined as Fiebig stage 2) and 3.8% (2/52) established HIV infections, where viral loads were 5.9, 6.7, and >7 log₁₀ copies/mL. Symptoms such as fever, nausea/vomiting, malaise, headache, and photophobia were significantly associated with acute HIV infections diagnosed in the ED. The rapid HIV antibody test demonstrated high sensitivity, specificity, and NPV in our study population, reaffirming its effectiveness as a valuable screening tool. However, the low PPV and 100% failure to detect acute HIV infections underscore the importance of prioritizing in-lab fourth-generation HIV antigen/antibody combination immunoassays in cases of suspected acute HIV infection to ensure a timely and accurate diagnosis.

Nanopore sequencing has shown the potential to democratize genomic pathogen surveillance due to its ease of use and low entry cost. However, recent genotyping studies showed discrepant results compared to gold-standard short-read sequencing. Furthermore, although essential for widespread application, the reproducibility of nanopore-only genotyping remains largely unresolved. In our multicenter performance study involving five laboratories, four public health-relevant bacterial species were sequenced with the latest R10.4.1 flow cells and V14 chemistry. Core genome MLST analysis of over 500 data sets revealed highly strain-specific typing errors in all species in each laboratory. Investigation of the methylation-related errors revealed consistent DNA motifs at error-prone sites across participants at read level. Depending on the frequency of incorrect target reads, this either leads to correct or incorrect typing, whereby only minimal frequency deviations can randomly determine the final result. PCR preamplification, recent basecalling model updates and an optimized polishing strategy notably diminished the non-reproducible typing. Our study highlights the potential for new errors to appear with each newly sequenced strain and lays the foundation for computational approaches to reduce such typing errors. In conclusion, our multicenter study shows the necessity for a new validation concept for nanopore sequencing-based, standardized bacterial typing, where single nucleotide accuracy is critical.

Given the cost and unclear clinical impact of metagenomic next-generation sequencing (mNGS), laboratory stewardship may improve utilization. This retrospective observational study examines mNGS results from two academic medical centers employing different stewardship approaches. Eighty mNGS orders [54 cerebrospinal fluid (CSF) and 26 plasma] were identified from 2019 to 2021 at the University of Washington (UW), which requires director-level approval for mNGS orders, and the University of Utah (Utah), which does not restrict ordering. The impact of mNGS results and the relationship to traditional microbiology orders were evaluated. Nineteen percent (10/54) of CSF and 65% (17/26) of plasma studies detected at least one organism. Compared to CSF results, plasma results more frequently identified clinically significant organisms (31% vs 7%) and pathogens not detected by traditional methods (12% vs 0%). Antibiotic management was more frequently impacted by plasma versus CSF results (31% vs 4%). These outcome measures were not statistically different between study sites. The number and cumulative cost of traditional microbiology tests at UW were greater than Utah for CSF mNGS testing (UW: 46 tests, $6,237; Utah: 26 tests, $2,812; P < 0.05) but similar for plasma mNGS (UW: 31 tests, $3,975; Utah: 21 tests, $2,715; P = 0.14). mNGS testing accounted for 30%-50% of the total microbiology costs. Improving the diagnostic performance of mNGS by stewardship remains challenging due to low positivity rates and difficulties assessing clinical impact. From a fiscal perspective, stewardship efforts should focus on reducing testing in low-yield populations given the high costs of mNGS relative to overall microbiology testing expenditures.

Importance: Metagenomic next-generation sequencing (mNGS) stewardship practices remain poorly standardized. This study aims to provide actionable insights for institutions that seek to reduce the unnecessary usage of mNGS. Importantly, we highlight that clinical impact remains challenging to measure without standardized guidelines, and we provide an actual cost estimate of microbiology expenditures on individuals undergoing mNGS.