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Detection of Nocardia by 16S Ribosomal RNA Gene PCR and Metagenomic Next-Generation Sequencing (mNGS).

ABSTRACT: In this study, the aim was to investigate the discriminatory power of molecular diagnostics based on mNGS and traditional 16S ribosomal RNA PCR among Nocardia species. A total of fourteen clinical isolates from patients with positive Nocardia cultures and clinical evidence were included between January 2017 and June 2020 in HeNan Provincial People's Hospital. DNA extraction and 16S rRNA PCR were performed on positive cultures, and pathogens were detected by mNGS in these same samples directly. Among the 14 Nocardia isolates, four species were identified, and N. cyriacigeorgica (8 cases) is the most common species. Twelve of the 14 Nocardia spp. isolates were identified by the two methods, while two strains of N. cyriacigeorgica were not identified by mNGS. All tested isolates showed susceptibility to trimethoprim-sulfamethoxazole (SXT), amikacin and linezolid. Apart from Nocardia species, other pathogens such as Acinetobacter baumannii, Klebsiella pneumonia, Aspergillus, Enterococcus faecalis, Human herpesvirus, etc., were detected from the same clinical samples by mNGS. However, these different pathogens were considered as colonization or contamination. We found that it is essential to accurately identify species for determining antibiotic sensitivity and, consequently, choosing antibiotic treatment. 16S rRNA PCR was useful for identification of nocardial infection among species, while this technique needs the clinicians to make the pre-considerations of nocardiosis. However, mNGS may be a putative tool for rapid and accurate detection and identification of Nocardia, beneficial for applications of antimicrobial drugs and timely adjustments of medication.

SUBMITTER: Ding J

PROVIDER: S-EPMC8779735 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMetagenomic next-generation sequencing (mNGS) is a promising technology that allows unbiased pathogen detection and is increasingly being used for clinical diagnoses. However, its application in urinary tract infection (UTI) is still scarce.MethodsThe medical records of 33 patients with suspected UTI who were admitted to the Second Hospital of Tianjin Medical University from March 2021 to July 2022 and received urine mNGS were retrospectively analyzed. The performance of mNGS and conventional urine culture in diagnosing infection and identifying causative organisms was compared, and the treatment effects were evaluated in terms of changes in urinalyses and urinary symptoms.ResultsIn the detection of bacteria and fungi, mNGS detected at least one pathogen in 29 (87.9%) cases, including 19 (57.6%) with positive mNGS but negative culture results and 10 (30.3%) with both mNGS and culture positive results. The remaining 4 (12.1%) patients were negative by both tests. Overall, mNGS performed better than culture (87.9% vs. 30.3%, P < 0.001). Within the 10 double-positive patients, mNGS matched culture results exactly in 5 cases, partially in 4 cases, and not at all in 1 case. In addition, mNGS detected a broader pathogen spectrum, detecting 26 species compared to only 5 species found in culture. The most abundant bacteria detected by mNGS was Escherichia coli, detected in 9 (27.2%) patients. All anaerobic bacteria, Mycobacterium Tuberculosis and all mixed pathogens were detected by mNGS. The final clinical diagnosis of UTI was made in 25 cases, and the sensitivity of mNGS was significantly higher than culture (100.0% vs 40.0%; P < 0.001) when using the diagnosis as a reference standard; the positive predictive value, negative predictive value and specificity were 86.2%, 100% and 50.0%, respectively. Importantly, targeted antibiotic therapy based on mNGS resulted in significant improvement in urinalyses and urinary symptoms in patients.ConclusionsmNGS is a technology that has shown clear advantages over culture, particularly in the context of mixed infections and UTIs that are difficult to diagnose and treat. It helps to improve the detection of pathogens, guide changes in treatment strategies, and is an effective complement to urine culture.

Project description:BackgroundThe SARS-CoV-2 Omicron variant, with the main subtypes BA.5.2 and BF.7 in China, led to off-target effects on the S and N genes from December 1, 2022, to January 31, 2023. The kits used for studying and developing these agents were not adequately and independently evaluated. It is important to verify the performance of commercial Real-Time quantitative PCR (RT-qPCR) tests.ObjectiveWe conducted a clinical evaluation of two Real Time SARS-CoV-2 Omicron assays to verify their performance using various detection reagents and clinical specimens.MethodsWe performed clinical evaluations of two existing Chinese SARS-CoV-2 Omicron RT-qPCR kits 2019-nCoV nucleic acid diagnostic kits (Fosun Biotechnology, National instrument registration 20203400299, Shanghai, China) and COVID-19 nucleic acid detection kits (eDiagnosis Biomedicine, National instrument registration 20203400212, Wuhan, China) and using BSD (Bondson) (Guangzhou Bondson Biotechnology Co. Ltd, batch number 2022101), quality controls provided by the inspection center and a large number of clinically confirmed specimens.ResultsThe concordance rates for the Fosun and eDiagnosis kits were 95% and 100%, respectively. The detection limit for the Fosun and eDiagnosis kits was verified to be 300 copies/mL and 500 copies/mL. The Fosun assay exhibited the largest coefficient of variation (CV) for ORF1ab and N gene at the detection limit concentration (4.80%, 3.49%), whereas eDiagnosis showed a smaller CV (0.93%, 1.10%). In the reference product from the Hangzhou Clinical Laboratory Center test, it was found that Fosun had the lowest sensitivity of 93.47% and a specificity of 100%, while eDiagnosis exhibited 100% for both sensitivity and specificity. The lowest single target gene detection rate of Fosun reagents was 68.7% for the ORF1ab gene and 87.5% for the N gene, while eDiagnosis detection rate was 100%. Among the clinical group S specimens, the missed detection rate of the Fosun reagent was 10.9%, which was higher than the 3.9% of eDiagnosis. However, there was no significant difference in the clinical diagnostic efficiency of the two reagents.ConclusionsThe ORF1ab and N assays of SARS-CoV-2 Omicron on the eDiagnosis platform yielded higher values compared to those on the Fosun platform. Consequently, the eDiagnosis kit has also been used as standard detection reagents. Considering that the Fosun reagent has a relatively low detection limit and targets three single genes, it is more advantageous as a confirmatory reagent for the new museum.

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Detection of Nocardia by 16S Ribosomal RNA Gene PCR and Metagenomic Next-Generation Sequencing (mNGS).

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