Next-Generation DNA Sequencing Refines Pneumonia Diagnosis

Applying advanced next-generation sequencing (NGS) of DNA from samples taken from intubated patients with suspected pneumonia has the potential for providing physicians with rapid, precise, culture-independent identification of bacterial, fungal, and viral pathogens and their antimicrobial sensitivity profiles.

Accurate and rapid identification of the microbial pathogens in patients with pulmonary infections could lead to targeted antimicrobial therapy with potentially less adverse effects and lower costs. Toward this end, investigators at George Washington University (Washington DC, USA) combined an NGS approach with data interpretation based on the "PathoScope" bioinformatics software package to analyze bronchial aspirates from 61 intubated patients with suspected pneumonia.

Pathoscope capitalizes on a Bayesian statistical framework that accommodates information on sequence and mapping quality and provides probabilities of matches to a known database of reference genomes. This approach incorporates the possibility that multiple species can be present in the sample or that the target strain is not even contained within the reference database. It also accurately discriminates between very closely related strains of the same species with much less than one time coverage of the genome and without the need for sequence assembly or complex preprocessing of the database or taxonomy. No other method so far described in the literature has been shown to identify species or substrains in such a direct and automatic manner and without the need for large numbers of reads.

The present study used NGS of essentially full-length PCR-amplified 16S ribosomal DNA from the bronchial aspirates. The results from the 61 patients demonstrated that sufficient DNA could be obtained from 72% of samples, 44% of which (27 samples) yielded PCR amplimers suitable for NGS. Out of 27 sequenced samples, only 20 had bacterial culture growth, while microbiological and NGS identification of bacteria coincided in 17 (85%) of these samples. Despite the lack of bacterial growth in seven samples that yielded amplimers and were sequenced, the NGS identified a number of bacterial species in these samples.

Overall, a significant diversity of bacterial species was identified from the same genus as the predominant cultured pathogens. The number of NGS-identifiable bacterial genera was consistently higher than identified by standard microbiological methods.

“Currently, patients who develop pneumonia after entering the ICU are subjected to broad-spectrum antibiotics, which adds costs, potentially increases the risk of development of antimicrobial resistance, and creates a greater likelihood of an adverse effect attributable to the antibiotics,” said senior author Dr. Gary Simon, professor of medicine at George Washington University. “In our paper, we show these methods could improve if we establish a more precise microbiologic cause.”

The study was published in the August 20, 2014, online edition of the Journal of Clinical Microbiology.

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