Dataset Information

Identification of Leptotrichia goodfellowii infective endocarditis by next-generation sequencing of 16S rDNA amplicons.

ABSTRACT: The oral aerotolerant anaerobe Leptotrichia goodfellowii is an unusual cause of endocarditis and is amenable to treatment with ?-lactam antibiotics. Because this organism is difficult to identify by conventional methods, molecular detection is a key diagnostic modality. Broad-range 16S rDNA PCR followed by Sanger sequencing constitute the first-line molecular approach, yet poor DNA quality, contaminating DNA, or low template quantity make identification challenging. Here we report a case of culture-negative, aortic and mitral valve endocarditis in a 66-yr-old woman with a history of cardiomyopathy, atrial fibrillation with intracardiac pacer, poor dentition, and recent tooth infection. In this case, 16S rDNA amplicon Sanger sequencing was not sufficient for pathogen identification because of interfering DNA, but deconvolution of the clinical sample using reflexive next-generation amplicon sequencing enabled confident identification of a single pathogenic organism, L. goodfellowii The patient developed a sigmoid colon perforation and died despite additional surgical treatment. Most Leptotrichia endocarditis cases have been subacute and have been successfully treated with antibiotics, with or without valve replacement. This case highlights both an unusual etiologic agent of endocarditis, as well as the rational utilization of advanced molecular diagnostics tools for characterizing serious infections.

SUBMITTER: Lieberman JA

PROVIDER: S-EPMC7903886 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Identification of <i>Leptotrichia goodfellowii</i> infective endocarditis by next-generation sequencing of 16S rDNA amplicons.

Lieberman Joshua A JA Kurosawa Kyoko K SenGupta Dhruba D Cookson Brad T BT Salipante Stephen J SJ Busch David D

Cold Spring Harbor molecular case studies 20210219 1

The oral aerotolerant anaerobe <i>Leptotrichia goodfellowii</i> is an unusual cause of endocarditis and is amenable to treatment with β-lactam antibiotics. Because this organism is difficult to identify by conventional methods, molecular detection is a key diagnostic modality. Broad-range 16S rDNA PCR followed by Sanger sequencing constitute the first-line molecular approach, yet poor DNA quality, contaminating DNA, or low template quantity make identification challenging. Here we report a case ...[more]

PMID: 33288524

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Project description:ObjectivesMetagenomic next-generation sequencing (mNGS) technology is helpful for the early diagnosis of infective endocarditis, especially culture-negative infective endocarditis, which may guide clinical treatment. The purpose of this study was to compare the presence of culture-negative infective endocarditis pathogens versus culture-positive ones, and whether mNGS test results could influence treatment regimens for patients with routine culture-negative infective endocarditis.MethodsThe present study enrolled patients diagnosed with infective endocarditis and tested for mNGS in the First Affiliated Hospital of Zhengzhou University from February 2019 to February 2022 continuously. According to the culture results, patients were divided into culture-negative group (Group CN, n=18) and culture-positive group (Group CP, n=32). The baseline characteristics, clinical data, pathogens, 30 day mortality and treatment regimen of 50 patients with infective endocarditis were recorded and analyzed.ResultsExcept for higher levels of PCT in the Group CN [0.33 (0.16-2.74) ng/ml vs. 0.23 (0.12-0.49) ng/ml, P=0.042], there were no significant differences in the basic clinical data and laboratory examinations between the two groups (all P>0.05). The aortic valve and mitral valve were the most involved valves in patients with infective endocarditis (aortic valve involved: Group CN 10, Group CP 16; mitral valve involved: Group CN 8, Group CP 21; P>0.05) while 9 patients had multiple valves involved (Group CN 2, Group CP 7; P>0.05). The detection rate of non-streptococci infections in the Group CN was significantly higher than that in the Group CP (9/18 vs. 3/32, P=0.004). There was no significant difference in patients with heart failure hospitalization and all-cause death at 30 days after discharge (3 in Group CN vs. 4 in Group CP, P>0.05). It is worth noting that 10 patients with culture-negative infective endocarditis had their antibiotic regimen optimized after the blood mNGS.ConclusionsCulture-negative infective endocarditis should be tested for mNGS for early diagnosis and to guide clinical antibiotic regimen.

Project description:The analysis of HIV-1 sequences has helped understand the viral molecular epidemiology, monitor the development of antiretroviral drug resistance, and design candidate vaccines. The introduction of single genome amplification (SGA) has been a major advancement in the field, allowing for the characterization of multiple sequences per patient while preserving linkage among polymorphisms in the same viral genome copy. Sequencing of SGA amplicons is performed by capillary Sanger sequencing, which presents low throughput, requires a high amount of template, and is highly sensitive to template/primer mismatching. In order to meet the increasing demand for HIV-1 SGA amplicon sequencing, we have developed a platform based on benchtop next-generation sequencing (NGS) (IonTorrent) accompanied by a bioinformatics pipeline capable of running on computer resources commonly available at research laboratories. During assay validation, the NGS-based sequencing of 10 HIV-1 env SGA amplicons was fully concordant with Sanger sequencing. The field test was conducted on plasma samples from 10 US Navy and Marine service members with recent HIV-1 infection (sampling interval: 2005-2010; plasma viral load: 5,884-194,984 copies/ml). The NGS analysis of 101 SGA amplicons (median: 10 amplicons/individual) showed within-individual viral sequence profiles expected in individuals at this disease stage, including individuals with highly homogeneous quasispecies, individuals with two highly homogeneous viral lineages, and individuals with heterogeneous viral populations. In a scalability assessment using the Ion Chef automated system, 41/43 tested env SGA amplicons (95%) multiplexed on a single Ion 318 chip showed consistent gene-wide coverage >50×. With lower sample requirements and higher throughput, this approach is suitable to support the increasing demand for high-quality and cost-effective HIV-1 sequences in fields such as molecular epidemiology, and development of preventive and therapeutic strategies.

Dataset Information

Identification of Leptotrichia goodfellowii infective endocarditis by next-generation sequencing of 16S rDNA amplicons.

Publications

Identification of <i>Leptotrichia goodfellowii</i> infective endocarditis by next-generation sequencing of 16S rDNA amplicons.

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