Pathogen identification is crucial in VAP diagnosis and treatment, building a time-saving method friendly to clinical department is the key point to reduce severe and death rate. Nanopore technology had been used in several epidemiological diagnosis occasions [12, 16–22]. However, there were no unified procedure about how to deal with the respiratory samples, and the efficiency of different methods to different pathogens is not clear. Here we compared different methods, and provided a theoretical basis for the choice of methodology.
This study included 83 ETA samples from patients with suspected VAP who had been intubated for more than 48hrs. Like previously indicated by other researchers [17, 23], our pilot also found that DNA extracted directly from ETA were dominated by host genome, leading to a poor sequencing performance with no pathogen detected. Therefore, the removal of human genome has become a necessary measure of sample processing. Human genomes were removed from all 12 samples after depletion, and the genomic abundance of pathogens and sequence time was significantly improved after depletion, suggesting that the depletion operation of saponins was of great significance for the optimization of sequencing results.
However, there is no clear conclusion on the effect of saponin depletion on the pathogen genome. After comparing the effects of depletion on the 7 ATCC pathogens involved in this study, the results showed that depletion did not significantly affect the abundance of 6 pathogens except S.pneumoniae. This result is similar to the research result of Charalampous et al. from the UK, which may be due to the simultaneous lysis of S.pneumoniae genes during the process of lysis of human genome [17, 24]. In this study, however, among 83 cases, 7 cases involving S.pneumoniae (S14, S16, S23, S36, S68, S78, S80) showed that the clinical culture were negative and sequencing results were positive, and 6 samples besides S16 also suggest the existence of S.pneumoniae from qRT-PCR results. Although DNA extraction from human sources may damage pathogen, as S.pneumoniae is difficult to be cultured in clinical practice and has a low positive rate, sequencing is still a better choice for detection of S.pneumoniae.
Whether to conduct PCR amplification during library build process is also one of the issues that needed to be discussed. One of the advantages of Nanopore sequencing is that the DNA sequence information can be obtained without PCR amplification, thus preserving the methylation and other modification information on the DNA, which is conducive to further data mining and processing. In this study, the differences in pathogen detection between PCR amplification and non-PCR amplification in 29 samples were compared, and for most pathogens, adequate and effective pathogen information could still be obtained without PCR amplification and sequencing.
Some samples showed positive from culture and qRT-PCR results but negative from sequencing results (K.pneumoniae: S11, S23; S.aureus: S27; P.aeruginosa: S40, S55, S63; S.maltophilia: S59; A.baumannii: S40, S44, S45), which may due to the high content of human or oropharyngeal pathogen genome content who covered up the pathogen information. Some samples showed positive from clinical culture results but no positive results from sequencing and qRT-PCR (K.pneumoniae: S43; A.baumannii: S44, S45), which may due to sampling error. Repeated sampling, proper protection and cleaning of oropharynx during sampling, and adequate saponin mixing of samples can appropriately avoid the occurrence of such phenomena.
For patients with tracheal intubation for 48hrs or more, the types of pathogens in the lower respiratory tract decreased with the extension of intubation time, but the abundance of individual pathogens increased with the extension of intubation time [25]. For patients with newly intubated trachea and suspected VAP, the sequencing results often present a mixed form of multiple pathogens. In addition, the number of reads of different pathogens may vary greatly in the sequencing results of the same sample. For such samples, it is still necessary for clinicians to judge the specific pathogenic bacteria types and precise drug use in combination with the clinical manifestations of patients.