Clinical diagnostic application of metagenomic next-generation sequencing in children with severe pneumonia

Background: Pneumonia is one of the most important causes of morbidity and mortality in children. Identification and characterization of pathogens that cause infections are crucial for accurate treatment and accelerated recovery of the patients. However, in most cases the causative agent cannot be identified partly due to the limited spectrum covered by current diagnostics based on nucleic acid amplification. Therefore, in this study we explored the application of metagenomic next-generation sequencing (mNGS) for the diagnosis of children with severe pneumonia. Methods: From April to July 2017, 32 children were hospitalized with severe pneumonia in Shenzhen Children’s Hospital. Blood tests were conducted immediately after hospitalization to assess infection, oropharygeal swabs were collected to identify common pathogens. After bronchoscopy, bronchoalveolar lavage fluids (BALFs) were collected for further pathogen identification using standardized laboratory and mNGS. Results: Blood tests were normal in 3 of the 32 children. In oropharygeal swabs from 5 patients Mycoplasma pneumoniae by qPCR, 27 cases showed negative results for common pathogens. In BALFs we detected 6 cases with Mycoplasma pneumoniae with qPCR, 9 patients with adenovirus by using a Direct Immunofluorescence Assay (DFA) and 4 patients with bacterial infections, as determined by culture, In 3 of the cases a co-infection was detected. In 15 cases no common pathogens were found in BALF samples, using the current diagnostics, while in all the 32 BALFS pathogens were identified using mNGS, including adenovirus, Mycoplasma pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, cytomegalovirus andbocavirus. Conclusions: mNGS can increase the sensitivity of detection of the causative pathogens in children with severe pneumonia. In addition, mNGS will give more strain specific information, will help to identify new pathogens and could potentially help to trace and control outbreaks. In this study we have shown that it is feasible to have the

3 results within 24 hours, making the application of mNGS feasible for clinical diagnostics.

Background
Pneumonia is one of the most important causes of morbidity and mortality in children [ 1 ]. A multitude of pathogens have been identified as potential causes making it challenging to determine the microbial etiology of pneumonia. Identification and characterization of micro-organisms that cause infections are crucial for targeted treatment to enable fast recovery of the patients. Culture-based techniques, nucleic acid amplification tests (NAATs) and immunological assays target only a fraction of the currently known pathogens [ 2 ]. In addition, culture-based tests require two days and even longer to identify the causative pathogen while immunological assays lack sensitivity and are therefore generally prone to false negative results. This often leads to empirical treatment solely based on clinical examination potentially resulting in misuse or overuse of antibiotics. Metagenomic next-generation sequencing (mNGS) technologies have previously shown to be a promising for the identification of the causative in a given sample [ 3 ]. The advent of rapid and low-cost mNGS technologies has improved their applications from laboratory research to clinical diagnostics of pathogens [4][5][6][7]. mNGS has been mainly used to diagnose emerging pathogens and rare infectious diseases [ 8 , 9 ]. In this study, we simultaneously used mNGS to detect pathogens in bronchoalveolar lavage fluid (BALF) and clinical examination to compare the differences in diagnostic outcome, to finally adjust the treatment option and observe the recovery of the patients.

Samples collection and information
From April to July 2017, the Department of Respiratory Diseases in Shenzhen Children's Hospital received a cluster of pediatric patients with persistent fever, wheezing and coughing for at least 7 days. All patients were diagnosed with standardized radiography to measure abnormalities associated with pneumonia. Blood tests were conducted immediately after hospitalization to assess the total numbers of leukocytes, neutrophils and lymphocytes counts and to measure the concentration of C-reactive protein (CRP) and procalcitonin (PCT). Meanwhile, oropharyngeal swabs (155C, COPAN, Murrieta, California, USA) were collected to identify the common pathogens, as mentioned here below.
Bronchoscopy was required for the disease condition of these patients, allowing the collection of BALF for further pathogen identification by mNGS analysis and for standardized assays to facilitate the clinical decision for treatment, as described here

Sequencing and data analysis
In mNGS analysis, DNA was extracted from BALF using the TIANamp Micro DNA Kit (DP316, Tiangen Biotech) in accordance with the manufacturer's standard protocols. Agilent 2100 was used for quality control of the DNA libraries, which were sequenced on BGISEQ-100 platform [10]. Processed by removing low-quality and short (length < 35 bp) reads, sequencing data then were aligned with the human reference genome (hg19) to remove human-derived sequences using Burrows-Wheeler Alignment. The remaining data were classified by simultaneously aligning to four Microbial Genome Databases, consisting of 2,686 viruses, 1,492 bacteria, 60 species of fungi, and 33 parasites [8]. If there were more than 50 unique reads found it was regarded as likely causative pathogen and further verified by Sanger sequencing.

Clinical diagnosis and treatment
The 32 cases were diagnosed as severe pneumonia based on diffused lung consolidation, as well as atelectasis or pleural effusion. Of these patients, aged from 5 months to 8 years and 7 months, the median age was 21.5 months, 18 were males and 14 were females.
Total leukocyte counts ranged from 2.34 -21.9×10 9 /L, while total number lymphocyte and neutrophil counted were 0.58 -10.6 and 1.17 -22.1×10 9 /L, respectively. Normal values for total leukocytes, lymphocytes and neutrophils were found in 18, 20 and 23 cases, respectively ( Table 1). The concentration of serum CRP was higher than 10.0mg/L in 18 cases, and for PCT higher than 0.5µg/L in 22 cases (Table 1). Empirical antibiotic treatment was applied in all cases to and bronchoscopy was used for diagnostic purposes.
Steroid therapy was applied in 20 cases, based on patients' conditions.  In particular, after receiving the patient result of CMV, we confirmed that the patient was indeed infected by CMV using real time PCR. Bocavirus has not be confirmed by qPCR.

Unique reads of mNGS used in distinguishing co-infection
In patients co-infected with multiple pathogens, we can distinguish the main pathogens according to the number of unique reads by mNGS, then provided reference for treatment.
In this study, we identified 2 patients co-infected with adenovirus and M. pneumoniae  (Table 3).

Table 3. Pathogen detection based on >50 unique reads of mNGS in 4 co-infection cases.
Depth: the ratio of the total amount of the microbial base detected to the detected length of the microbial genome sequence, one of the indicators for evaluating the amount of sequencing.

Discussion
In this study, we present a proof of principle study using clinical samples obtained from pediatric patients with severe pneumonia.
Limitations of the current diagnostic practice is the delay in results to be able to take the right medical decision [11]. In addition, there is high proportion of pneumonia cases with unknown microbial etiology [ 12 ], leading to an increase in mortality and to empirical treatment with antibiotics. Using mNGS directly on clinical samples broadens the range of pathogen detection. In this study, we have shown the feasibility of a turnaround of 24 hours, which enables re-direction of the initial treatment, potentially accelerating the recovery and improving the outcome of disease.
With standard molecular diagnostics, M. pneumoniae was detected in BALFs samples of 6 patients by qPCR. This was confirmed in 5/6 samples by mNGS. Thus, sensitivity of the mNGS approach was at least similar to the sensitivity of the diagnostic PCR in this set of samples. The sensitivity of mNGS was higher than that of DFA used for the detection of adenovirus as only 9 were detected with DFA while 25 were found with mNGS.

Consent for publication
Not applicable.

Availability of data and material
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.  Table 3. Pathogen detection based on >50 unique reads of mNGS in 4 co-infection cases.
Depth: the ratio of the total amount of the microbial base detected to the detected length of the microbial genome sequence, one of the indicators for evaluating the amount of sequencing.