Targeting RNA with Next‐ and Third‐Generation Sequencing Improves Pathogen Identification in Clinical Samples

Zhao, Na; Cao, Jiabao; Xu, Jiayue; Liu, Beibei; Liu, Bin; Chen, Dingqiang; Xia, Baojia; Chen, Liang; Zhang, Wenhui; Zhang, Yuqing; Zhang, Xuan; Duan, Zhimei; Wang, Kaifei; Xie, Fei; Xiao, Kun; Yan, Wei; Xie, Lei; Zhou, Hongwei; Wang, Jun

doi:10.1002/advs.202102593

Cited by 40 publications

(22 citation statements)

References 35 publications

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“…This was probably because of the development of diagnostic techniques such as pathogenic metagenomic technology in recent years [15]. However, these techniques have limitations in clinical application due to various factors, such as cost [16].…”

Section: Discussionmentioning

confidence: 99%

Timing of bronchoscopy and application of scoring tools in children with severe pneumonia

Sang

et al. 2023

Ital J Pediatr

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Background There is still a lack of effective scoring criteria for assessing the severity of pulmonary infection associated with changes in the endobronchial lining of the bronchus in children. This study aimed to ascertain the timing and value of endoscopic scoring of fibreoptic bronchoscopy (FOB) and bronchoalveolar lavage (BAL) in children with severe pneumonia. Method The clinical data of 229 children with severe pneumonia treated with BAL in the Pediatric Intensive Care Unit of the First Affiliated Hospital of Xinxiang Medical University between November 2018 and December 2021 were collected. According to the severity of the disease, patients were divided into an invasive ventilation group and a non-invasive ventilation group, as well as an early BAL group (receiving BAL within 1 day of admission) and a late BAL group (receiving BAL 2 days after admission). A Student’s t-test, Chi-square test, receiver operating characteristic (ROC) curve and survival curve were used to analyse the bronchitis score, aetiology of BAL fluid and survival data. Results The scores of endoscopic mucosal oedema, erythema and pallor and the total score in the invasive ventilation group were higher than those in the non-invasive ventilation group (P < 0.05), and they were consistent with the Sequential Organ Failure Assessment (SOFA) scores. The secretion colour score was lower in the early BAL group than in the late BAL group (P < 0.05). On the bronchitis scores, which were evaluated using a ROC curve, the difference in the mucosal erythema, pallor, oedema and total score of the invasive and non-invasive groups was statistically significant (P < 0.05), which was consistent with the area under the ROC of the SOFA scores. Acute Physiology and Chronic Health Assessment II and SOFA scores after FOB were lower than those before treatment (P < 0.05). In terms of ICU hospitalisation days and total hospitalisation days, the time of the early FOB patients was shorter than that of the late FOB patients (P < 0.05). A total of 22 patients (9.61%) died. The Kaplan–Meier analysis and log-rank test showed that the survival rate of the non-invasive ventilation group was higher than that of the invasive ventilation group (P < 0.05). Conclusion This study found that FOB combined with BAL is an important method for the diagnosis and treatment of severe pneumonia. Early BAL can reduce hospitalisation and ICU time; however, it cannot improve the survival rate. The endoscopic score has a certain role to play in assessing the severity of pulmonary inflammation, but studies with a large sample are still needed to confirm this.

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Section: Discussionmentioning

confidence: 99%

Timing of bronchoscopy and application of scoring tools in children with severe pneumonia

Sang

et al. 2023

Ital J Pediatr

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“…In this study, we show that a tNGS-based kit, RPIP, can be used for pathogen microbial and ARG detection in BALF specimens from children. tNGS is a new workflow that uses nucleic acid probes to enrich for specific pathogens and other genes of interest [30]. RPIP is a commercial kit that applies this technology to identify respiratory pathogens and ARGs.…”

Section: Discussionmentioning

confidence: 99%

Performance of targeted next-generation sequencing in the detection of respiratory pathogens and antimicrobial resistance genes for children

Lin,

Xing,

Liu

et al. 2023

Journal of Medical Microbiology

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Introduction. Respiratory tract infection, which is associated with high morbidity and mortality, occurs frequently in children. At present, the main diagnostic method is culture. However, the low pathogen detection rate of the culture approach prevents timely and accurate diagnosis. Fortunately, next-generation sequencing (NGS) can compensate for the deficiency of culture, and its application in clinical diagnostics has become increasingly available. Gap Statement. Targeted NGS (tNGS) is a platform that can select and enrich specific regions before data enter the NGS pipeline. However, the performance of tNGS in the detection of respiratory pathogens and antimicrobial resistance genes (ARGs) in infections in children is unclear. Aim and methodology. In this study, we estimated the performance of tNGS in the detection of respiratory pathogens and ARGs in 47 bronchoalveolar lavage fluid (BALF) specimens from children using conventional culture and antimicrobial susceptibility testing (AST) as the gold standard. Results. RPIP (Respiratory Pathogen ID/AMR enrichment) sequencing generated almost 500 000 reads for each specimen. In the detection of pathogens, RPIP sequencing showed targeted superiority in detecting difficult-to-culture bacteria, including Mycoplasma pneumoniae . Compared with the results of culture, the sensitivity and specificity of RPIP were 84.4 % (confidence interval 70.5–93.5 %) and 97.7 % (95.9 –98.8%), respectively. Moreover, RPIP results showed that a single infection was detected in 10 of the 47 BALF specimens, and multiple infections were detected in 34, with the largest number of bacterial/viral coinfections. Nevertheless, there were also three specimens where no pathogen was detected. Furthermore, we analysed the drug resistance genes of specimens containing Streptococcus pneumoniae , which was detected in 25 out of 47 specimens in the study. A total of 58 ARGs associated with tetracycline, macrolide-lincosamide-streptogramin, beta-lactams, sulfonamide and aminoglycosides were identified by RPIP in 19 of 25 patients. Using the results of AST as a standard, the coincidence rates of erythromycin, tetracycline, penicillin and sulfonamides were 89.5, 79.0, 36.8 and 42.1 %, respectively. Conclusion. These results demonstrated the superiority of RPIP in pathogen detection, particularly for multiple and difficult-to-culture pathogens, as well as in predicting resistance to erythromycin and tetracycline, which has significance for the accurate diagnosis of pathogenic infection and in the guidance of clinical treatment.

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“…The length of DNA fragments from the samples is an important factor affecting the enrichment effect of adaptive sequencing, and libraries with longer fragment lengths showed better enrichment performance ( Zhao et al, 2021 ; Martin et al, 2022 ). In this study, the RPNAS workflow, which generated a longer length of library fragments, also showed better enrichment performance than that of the LNAS workflow.…”

Section: Discussionmentioning

confidence: 99%

Rapid PCR-Based Nanopore Adaptive Sequencing Improves Sensitivity and Timeliness of Viral Clinical Detection and Genome Surveillance

et al. 2022

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Nanopore sequencing has been widely used for the real-time detection and surveillance of pathogens with portable MinION. Nanopore adaptive sequencing can enrich on-target sequences without additional pretreatment. In this study, the performance of adaptive sequencing was evaluated for viral genome enrichment of clinical respiratory samples. Ligation-based nanopore adaptive sequencing (LNAS) and rapid PCR-based nanopore adaptive sequencing (RPNAS) workflows were performed to assess the effects of enrichment on nasopharyngeal swab samples from human adenovirus (HAdV) outbreaks. RPNAS was further applied for the enrichment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from nasopharyngeal swab samples to evaluate sensitivity and timeliness. The RPNAS increased both the relative abundance (7.87–12.86-fold) and data yield (1.27–2.15-fold) of HAdV samples, whereas the LNAS increased only the relative abundance but had no obvious enrichment on the data yield. Compared with standard nanopore sequencing, RPNAS detected the SARS-CoV-2 reads from two low-abundance samples, increased the coverage of SARS-CoV-2 by 36.68–98.92%, and reduced the time to achieve the same coverage. Our study highlights the utility of RPNAS for virus enrichment directly from clinical samples, with more on-target data and a shorter sequencing time to recover viral genomes. These findings promise to improve the sensitivity and timeliness of rapid identification and genomic surveillance of infectious diseases.

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Targeting RNA with Next‐ and Third‐Generation Sequencing Improves Pathogen Identification in Clinical Samples

Cited by 40 publications

References 35 publications

Timing of bronchoscopy and application of scoring tools in children with severe pneumonia

Timing of bronchoscopy and application of scoring tools in children with severe pneumonia

Performance of targeted next-generation sequencing in the detection of respiratory pathogens and antimicrobial resistance genes for children

Rapid PCR-Based Nanopore Adaptive Sequencing Improves Sensitivity and Timeliness of Viral Clinical Detection and Genome Surveillance

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