1. Introduction

Acute respiratory infection is a common infectious disease in children. Rapid and accurate identification of the pathogen and early targeted treatment are essential to improve the prognosis of children.[1] The mortality rate of lower respiratory tract infections (LRTI) is among the highest among infectious diseases. Early and accurate identification of its etiological diagnosis is essential for the implementation of pathogen-specific treatments. At present, the routine clinical diagnosis method of LRTI is to obtain bronchoalveolar lavage fluid (BALF) by bronchoscopy and to study the characteristics of pathogenic microorganisms of LRTI by culture, but this method has its own limitations.[2,3] The reason is that some microorganisms in the human body cannot be cultured, or the result is false negative due to the failure of anaerobic culture. Due to the limitations of existing microbiological detection methods (low culture sensitivity, long time required; limited number of microorganisms detected by serology and polymerase chain reaction), its diagnosis is still challenging.[4,5] In addition, the lungs are not aseptic and there are microbial colonies in both healthy and diseased conditions. How to distinguish pathogens from colonized bacteria is the main challenge in pathogen diagnosis of LRTI. In critically ill patients, noninfectious inflammatory syndrome like LRTI complicates pathogen diagnosis. In recent years, a variety of new respiratory pathogen detection techniques provide strong support for disease treatment, epidemiological investigation, and epidemic control with the development of biotechnology.

High throughput sequencing, also known as Next Generation Sequencing (NGS) technology, is a technique for defining classification. Compared with Sanger sequencing, which has become the standard method of clinical DNA sequencing, NGS technology is more efficient and can obtain a large amount of information at a lower cost in a short time.[6,7] At present, there are 3 typical sequencing technologies in NGS field, including 454 from Roche Company (Switzerland), Solexa from Illumina Company (USA), and SOLID from ABI Company (USA), and sequencing technologies such as IonTorrent, Heliscope, Nanopore, SMRT, IonPGM and GeXp.[8] Among them, the 454-sequencing technology can read about 700 bp in length, and the sequencing time is 18 to 24 hours. The method adopted is pyrosequencing with an accuracy of 99.9%. The data output is 0.5G and the cost per million bases is US $10, which is featured by high reading length and high speed. The Solexa sequencing technology can read about 300 bp in length and the sequencing time is 8 to 11 days. The method adopted is reversible end-synthesis sequencing with an accuracy of 99.9% and a data output of 4 to 1800 G. The fee is 0.05 to 0.15 US dollars, which is characterized by high throughput and many platforms. The SOLID sequencing technology can read about 75 bp in length, and the sequencing time is 2 to 7 days. The method adopted is ligation sequencing with an accuracy of 99.9% and a data output of 7 to 50 G. The cost per megabase is 0.13 USD, which is characterized by low cost.[9,10] Nowadays, many studies have been performed on the application effect of second-generation sequencing technology. It has been confirmed that clinical application of metagenomic second-generation sequencing technology (mNGS) can noticeably improve the sensitivity of etiological diagnosis of pulmonary infection and enhance people’s understanding of rare and newly discovered pathogens.[11] Interference of human sequences, exogenous microbial contamination, the impact of the detection process, and high sequencing prices have affected or limited the application of mNGS in clinical diseases.[12,13] The viral etiology of respiratory tract infection (RTI) in children remains unclear. The efficiency and feasibility of targeted second-generation sequencing techniques in pediatric RTI require further investigation. It is very necessary to further explore the application effect of targeted second-generation sequencing technology in guiding clinical diagnosis and medication. Therefore, this study will focus on the effect of targeted second-generation sequencing technology to guide clinical diagnosis and medication to treat and diagnose RTI in children.

2. Materials and methods 2.1. General information

During January 2021 to June 2022, a total of 320 children with RTI cured were selected in our hospital as the object of this retrospective study and divided into the control group (n = 160) and the observation group (n = 160) according to different methods that they received. The control group accepted empirical broad-spectrum antibacterial therapy and the observation group accepted targeted second-generation sequencing technique to guide diagnosis and medication. The mean age of the observation group was 2.03 ± 0.55 years, while the mean age of the control group was 2.08 ± 0.61 years. The average course of disease of the control group was 3.14 ± 0.54 days. There were 78 cases of acute bronchitis, 62 cases of pneumonia, and 20 cases of other RTIs. In the observation group, the average course of disease was 3.19 ± 0.57 days. There were 76 cases of acute bronchitis, 66 cases of pneumonia, and 18 cases of other types of respiratory infection. The general data were not statistically noticeable (P > .05). This study was approved by the Ethics Committee of Fujian Children’s Hospital.

Inclusion criteria: all the selected cases were diagnosed as RTI in children in accordance with the relevant diagnostic criteria;[14] the patients with symptoms such as fever, stuffy nose, and runny nose; the patients were diagnosed as RTI by blood routine and immune function tests[15]; parents could actively cooperate with the study and sign informed consent.

Exclusion criteria: those who were not injected with vaccine reagents; those with severe primary respiratory diseases such as primary immunodeficiency disease and congenital respiratory malformation; those with blood system diseases and severe impairment of heart, liver and kidney function; those with mental retardation or mental disorder; those who had used immunosuppressants or immuno-enhancers in the past 3 months.

2.2. Treatment methods

The control group received empirical broad-spectrum antimicrobial therapy: intravenous ceftriaxone (70 mg/kg, qd) for 1 consecutive week + oral azithromycin (10 mg/kg, qd) for 3 days, then stop using 4 days for 1 consecutive week.

Specimen retention: When patients developed RTI, pharyngeal swab, sputum, or alveolar-lavage fluid specimens were collected with sampling tubes, immediately stored at 4°C and sent to the Guangzhou Kingfield Detection Company for NGS testing within 24 h. Sample processing: After centrifugation, 600 µL supernatant was discarded and the remaining fluid in the pipe was blown and mixed. Nonviscous sputum and BALF were centrifuged at 12,000 rpm for 5 minutes. After removing the 600 µL supernatant, the fluid left in the tube was blown and mixed. Samples of viscous expectorations or BALF were classified by degree of viscosity. About 400 µL was collected for particularly viscous samples and 650 µL for general viscous samples and, respectively, added DTT (0.8M) to 1.3 mL. After mixing at ambient temperature, the steps of the nonviscous specimens were repeated. Specific steps: NGS uses quantitative Q-mNGS method for testing. The samples were collected for automatic nucleic acid extraction, nucleic acid fragmentation, terminal repair, terminal adenylate acidification (3’terminal plus A single base), sequencing joint connection, and purification to form a sequencing library. The library was quantified by fluorescence quantitative PCR and sequenced by Illumina Nextseq high throughput sequencing platform. Bioinformatics analysis of the sequence data in the library was performed to screen the sequence data of the human genome (GRCH 38. p13), and the remaining sequence data was compared with the microbial reference database (NCBIGenBank and indoor programming microbial genome data) to determine the microbial species and relative abundance. The compared data were classified and arranged according to bacteria, viruses, and parasites. According to the information on virulence genes and drug resistance genes, antimicrobial therapy was used for 1 week in the observation group.

2.3. Observation index 2.3.1. Therapeutic effect

The therapeutic effect of the children was assessed after 1 week of treatment and the criteria for judging the curative effect.[16] Patients with cough, fever, and other symptoms completely disappeared, normal vital signs are effective. Patients with cough, fever, and other symptoms were improved. Normal vital signs were improved. Patients with cough, fever, and other symptoms were not improved. Abnormal vital signs were invalid. Total effective rate = (number of effective cases + improved cases)/total number of cases.

2.3.2. Improvement time of clinical symptom index

The improvement time of clinical symptoms was compared, including fever, nasal congestion, tonsillar congestion, and cough.

2.3.3. The related indices in laboratory

The levels of serum iron, immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) were compared before treatment and 1 week after treatment. The venous blood was taken in the early morning fasting state. The levels of relevant indicators were checked using a biochemical analyzer and followed up for 3 months after treatment.

2.3.4. The level of inflammatory factors

The fasting blood 3 mL was collected before and 1 week after treatment and was centrifuged at the speed of 3000 rpm for 10 minutes. The blood samples were separated and placed in a refrigerator at −20°C. The levels of IL-4, IL-10, and IFN-γ were detected by Elisa assay, followed up for 3 months after treatment.

2.3.5. The incidence of complications

All children were followed up for 3 months after treatment. The complications of the 2 groups during and after treatment were counted. The common complications included otitis media, febrile convulsion, sinusitis, diarrhea and so on. The incidence of complications = (number of complications/total number of cases) × 100%.

2.3.6. Comparison of treatment satisfaction

After 1 week of treatment, the parents’ satisfaction with the treatment was evaluated by questionnaire. The total score of the questionnaire was 100 points, ≤60 as dissatisfaction, 60 to 85 as general satisfaction, ≥85 as very satisfactory. Treatment satisfaction = (number of very satisfactory cases + general satisfactory cases)/total number of cases × 100%. The reliability and validity were ideal. The stability and reliability were high.

2.4. Statistical analysis

SPSS 21.0 statistical software was used to analyze data. According to a normal distribution, using a t-test, and a Wilcoxon signed rank sum test for nonnormally distributed data. Independent sample t-tests were used for comparison. Paired t-tests were used for within-group comparisons. Count data were expressed as a number of cases (%) and the χ2 test was used. Differences were statistically significant (P < .05).

3. Results 3.1. Comparison of therapeutic effects

The total effective rate for the observation group was 91.25% (including 90 valid and 56 improved, Fig. 1). The total effective rate for the control group was 72.5% (66 valid and 50 improved), suggesting that using targeted second-generation sequencing technique to guide clinical diagnosis can improve the efficacy of the treatment (χ2 = 9.476, P = .002, Fig. 1).

Figure 1.:

Comparison of therapeutic effects between the 2 groups. The blue bar represents observation group and the orange bar represents the control group.

3.2. Comparison of improvement time of clinical index

The time needed to improve the symptoms of fever (P = .012), nasal congestion (P < .001), tonsil congestion (P = .005), and cough (P = .007) in the observation group was shorter. The current data suggested that using targeted second-generation sequencing technique to guide clinical diagnosis could reduce the improvement time (Table 1).

Table 1 - Improvement time (weeks) of clinical indices (n/%). Group N Fever Nasal congestion Tonsil hyperemia Cough Control group 160 1.98 ± 0.35 2.89 ± 0.83 1.98 ± 0.73 2.48 ± 0.81 Observation group 160 1.02 ± 0.14 1.53 ± 0.51 0.89 ± 0.48 1.35 ± 0.52 t 22.778 12.486 11.158 10.5 P 0.012 <0.001 0.005 0.007
3.3. Comparison of related indices in laboratory

After treatment, the levels of serum iron (P = .015), IgA (P = .003), IgG (P < .001), and IgM (P = .019) in the observation group were noticeably higher. The current data suggested that using targeted second-generation sequencing technique to guide clinical diagnosis could enhance the immune functions (Table 2).

Table 2 - Laboratory-related indices of patients. Group N Serum iron (umol/L) Immunoglobulin A (g/L) Immunoglobulin G (g/L) Immunoglobulin M (g/L) Before treatment After treatment Before treatment After treatment Before treatment After treatment Before treatment After treatment Control group 160 13.11 ± 3.05 16.04 ± 3.25* 0.30 ± 0.12 0.47 ± 0.75* 5.81 ± 1.23 7.33 ± 1.56* 1.13 ± 0.30 1.35 ± 0.56* Observation group 160 13.06 ± 3.09 18.79 ± 3.67* 0.31 ± 0.11 1.34 ± 1.04* 5.79 ± 1.19 9.21 ± 1.74* 1.10 ± 0.33 1.89 ± 0.72* T 0.103 5.017 0.549 6.068 0.105 7.195 0.602 5.295 P .918 .015 .584 .003 .917 <.001 .548 .019

*Compared with before treatment, P < .05.

3.4. Comparison of the level of inflammatory factors

After treatment, the levels of IL-4 (P < .001) and IL-10 (P = .005) in the observation group were noticeably lower and the levels of IFN-γ (P = .003) were noticeably higher. The current data suggested that using targeted second-generation sequencing technique to guide clinical diagnosis could improve the levels of IL-4 and IL-10. All results are shown in Table 3.

Table 3 - The levels of inflammatory factors. Group N IL-4 IL-10 IFN-γ Before treatment After treatment Before treatment After treatment Before treatment After treatment Control group 160 349.25 ± 24.49 215.56 ± 14.01* 21.27 ± 7.55 11.23 ± 2.66* 80.25 ± 6.22 114.26 ± 8.21* Observation group 160 348.12 ± 24.10 278.13 ± 14.92* 20.96 ± 7.59 15.30 ± 2.80* 81.06 ± 6.40 92.26 ± 7.34* T 0.294 27.344 0.259 9.426 0.812 17.868 P 0.769 <0.001 0.796 0.005 0.418 0.003

*Compared with before treatment, P < .05.

3.5. Comparison of the incidence of complications

In the observation group, there were 6 people with otitis media, 10 people with febrile convulsion, 8 people with sinusitis, and 10 people with diarrhea, with an incidence of 21.25%. In the control group, there were 2 people with otitis media, 6 people with febrile convulsion, 4 people with sinusitis, and 2 people with diarrhea, with an incidence of 8.75%. The current data suggested that using targeted second-generation sequencing technique to guide clinical diagnosis could reduce the incidence of complications (χ2 = 6.704, P = .009) (Table 4).

Table 4 - The incidence of complications (n/%). Group N Otitis media Febrile convulsion Sinusitis Diarrhea Incidence rate Control group 160 6 (3.75) 10 (6.25) 8 (5.00) 10 (6.25) 21.25% Observation group 160 2 (1.25) 6 (3.75) 4 (2.5) 2 (1.25) 8.75% χ2 6.704 P 0.009
3.6. Comparison of treatment satisfaction

In this study, a total of 320 treatment satisfaction questionnaires were distributed and 320 were recovered, all of which were valid questionnaires with a recovery rate of 100%. A total of 94 people in the observation group were very pleased, 54 people were generally pleased, and 12 people were displeased. The treatment satisfaction rate was 92.5%. In the control group, 56 cases were very pleased, 70 people were generally pleased, and 34 people were displeased. The treatment satisfaction rate was 66.25%. Treatment satisfaction of the observation group was higher, suggesting using a targeted second-generation sequencing technique to guide clinical diagnosis could increase the treatment satisfaction (χ2 = 6.144, P = .013) (Fig. 2).

Figure 2.:

Comparison of treatment satisfaction. The blue bar represents observation group and orange bar represents control group.

4. Discussion

RTI is an obvious clinical disease and a major fatal disease. According to data released by the World Health Organization in 2018, LRTI caused 3 million deaths worldwide in 2016. The pathogens of RTIs are complex. In addition to common bacteria, there are also atypical pathogens in a broad sense, such as viruses, fungi, mycoplasma, chlamydia, and Legionella.[17,18] Traditional atypical pathogen detection techniques include smear microscopy and culture, which require professionals to isolate and identify for a long time. The culture conditions of atypical pathogens are often harsh, resulting in a low detection rate.[19,20] Currently, there are no clear diagnostic criteria for refractory LRTI. Refractory LRTI does not improve after a period of anti-infective treatment with standard antibiotics.[21,22] The occurrence of the disease is often related to low immunity, poor sputum drainage, bacterial drug resistance, and difficulty in judging pathogens. It is particularly vital to accurately judge the pathogens of LRTI.[23] The second-generation sequencing technology with higher flux, higher sensitivity, fast speed, and low cost has been widely used in the study of microbiology and gradually combined with clinical etiological diagnosis. It provides a new idea for the detection of respiratory tract atypical pathogens. Through NGS technology, children’s cerebrospinal fluid samples were collected for the first clinical application of NGS technology when diagnosing infectious diseases.[24,25] Molecular biology techniques such as NGS can improve the sensitivity of pathogen detection and shorten the detection time, especially for the diagnosis of rare and uncommon pathogens.[26]

The higher effective rate of the observation group is mainly because the application of targeted second-generation sequencing technology can accurately diagnose the condition of children and provide scientific guidance for the formulation of treatment plans. It made the treatment work more scientific and targeted and its clinical efficacy is noticeably improved. The time needed to improve the symptoms of fever, nasal congestion, tonsil congestion, and cough in the observation group was shorter. Children’s symptoms improve faster because treatment is more efficient in targeted second-generation sequencing technology that can be tailored to the child’s pathogen type. The improvement of children’s immune function is mainly due to the elimination of pathogens after scientific medication and the gradual relief of immune damage to children. This showed that the application of targeted second-generation sequencing technology had positive guidance and significance to treat children. Systematic treatment can be carried out from the cause of the disease. The inflammatory reaction can be effectively reduced after medication according to the specific condition of the children. The reduction of complications was mainly due to the excellent clinical effect of the treatment plan developed under the guidance of a targeted second-generation sequencing technique. It can fundamentally improve the condition of children and reduce the risk of complications. The treatment satisfaction of the parents of the study group was higher, indicating that the parents of the children had a high degree of recognition of this treatment plan. This treatment plan had a broad application prospect.

At present, it is believed that the lower respiratory tract of healthy people is a dynamic and stable ecosystem composed of a variety of microorganisms, including potential pathogenic bacteria. The microorganisms in the lower respiratory tract mainly come from the microinhalation of the oropharynx, the inhalation of the surrounding air, and the direct dispersion of the mucosal surface.[27] The pulmonary microbiome is close to the oral microbiome, but the oral microinhalation reaches the peak at night, weakens gradually during the day, and the clearance effect is enhanced.[28] This diurnal variation led to differences in the lung microbiome from the oral microbiome. The respiratory microbiome develops dynamically in early life and is related to birth patterns, feeding patterns, antibiotic use, and living environment.[29] There are slight regional changes in physiological parameters such as oxygen partial pressure, PH value, and temperature in the lungs of healthy people, but no noticeable differences in microorganisms have been found in the lungs of healthy people. People have gradually found that there are many kinds of microorganisms in the “sterile lungs” of healthy people. If the microbial diversity in the lungs decreases or the abundance of some microorganisms increases abnormally, it will break the dynamic balance and immune homeostasis of microorganisms and lead to pneumonia. In recent years, NGS has been more and more widely used in lung diseases, in which the application of mNGS in LRTI shows a great advantage.[30]

Using NGS technology to detect pathogens can identify pathogens faster than traditional culture techniques. One patient with primary abdominal sepsis developed a pulmonary infection. After BALF was taken by bronchoscopy, bacterial culture and NGS detection were performed at the same time. Six high-quality DNA sequence readings (909–8288 bp) were revealed. Each base pair was well aligned with the genome of Staphylococcus aureus. The next day, the microbiological laboratory reported Staphylococcus aureus growth in the same sample.[31] NGS technology can optimize the diagnosis and management of such diseases in hospitals. It is well known that H3N2 influenza virus has high morbidity and mortality, but its severity varies with different seasons. The genomes of 176 cases of H3N2 influenza were sequenced by NGS. The amino acid fragments NAV263I and NS1K196E related to severity were found. The higher the content of these fragments in the host, the more serious the disease. Therefore, NGS can be used as an effective tool to explore the genomic characteristics of severe influenza. Further studies showed that due to the heterogeneity of HRV target region, HRV-positive samples may be missed by conventional molecular diagnostic methods. Therefore, NGS can improve the positive rate of etiological diagnosis of respiratory tract virus infection. Mycoplasma pneumoniae pneumonia accounts for 10% to 20% of community-acquired pneumonia in hospitalized children. At present, nucleic acid and serological tests (specific test and nonspecific test) are common to effectively diagnose mycoplasma pneumoniae infection. The analysis and comparison of the gene-phenotype of mycoplasma by NGS is helpful to deepen the understanding of its biological and epidemiological characteristics. NGS detection of BALF is helpful to confirm whether there is a mycoplasma pneumoniae infection in the clinic. Moreover, NGS provides the direction and theoretical support for the empirical treatment of refractory mycoplasma infection. The pathogens in BALF were detected by NGS and culture. Only 18 cases were found to be infected by mycoplasma and the results of NGS were consistent with those of culture. It is suggested that high levels of C-reactive protein may be mainly caused by strong immune reactions. This provides a basis for the treatment and curative effect of glucocorticoid in children with RMPP.[32] In addition, it was reported that an infant with community-acquired pneumonia who could not detect any pathogen by routine methods was detected by NGS that Chlamydia trachomatis invasive pulmonary aspergillosis was a life-threatening opportunistic fungal infection, mainly in patients with immunodeficiency or hematological malignant tumors. Early diagnosis and treatment can greatly reduce mortality.[33] Chest CT of a patient with bronchial asthma showed scattered nodules in the lungs and negative sputum culture. The effect of antibiotics was not obvious. Finally, the NGS test of BALF showed Aspergillus fumigatus infection.[34]

Since the launch of NGS in 2005, its utilization rate has increased greatly, but the challenges it faces should not be underestimated. First, there is still no systematic and complete evaluation process for many clinical samples and quality control implementation. NGS contains many steps. Each step of generated sequence must be strictly controlled, including the contamination of the sample environment and reagents. But now there are more efficient and easier-to-use NGS processes coming out. Secondly, although NGS has changed the development of genetics and is more and more widely used, the sensitivity of NGS virus detection is reduced due to the low nucleic acid abundance of some viruses. Currently, some researchers propose the use of 2 complementary sets of probes to improve the density efficiency of clinical samples, i.e., the first set of probes targets the whole genome and identifies mainly known viruses and whole genome sequences. The second group of probes is designed to target viral or conserved regions, primarily detecting known or unknown viruses in the sample. This probe focuses on the conserved regions of viral families, subfamilies, and gene sequences, which can enrich more diverse viral combinations, such as SARS-like virus MERS-CoV, camel coronavirus HKU23, and Colorado tick fever virus. In addition, there are still many uncertainties about the promotion and effectiveness of the vaccine. The increasing development of NGS technology allows for a deeper and easier understanding of the genomes of pathogens.[35] There are more and more studies applying mNGS to the diagnosis of clinical infectious diseases, among which the research of this technology in respiratory diseases has become a recent research hotspot.[36] New technologies bring hope to overcome the technical bottleneck of mNGS detection timeliness. A newly developed nanopore sequencing method for bacterial LRTI is reported to effectively remove host DNA and accurately identify pathogens and pathogens within 6 hours.[37] There is no doubt that NGS technology has a broad prospect in the detection of pathogens of LRTI, especially in the diagnosis and differentiation of unknown infections. It is important for improving the accuracy of diagnosis of LRTI pathogens, differentiating nasal cross-infection, studying drug resistance, and developing vaccines. The present study had a limited sample size and all subjects were from the same study center, further large-scale multi-center clinical studies are needed in the future.

To sum up, the application of targeted second-generation sequencing technology to guide clinical diagnosis and drug use can improve the treatment of childhood RTIs and improve the prognosis of children. Targeted second-generation sequencing can achieve precise treatment, reduce drug resistance of drug-resistant strains, and improve the efficacy. It has high promotion and application value in the clinic.

Author contributions

Conceptualization: Di Lian, Qiuyu Tang.

Data curation: Di Lian.

Formal analysis: Di Lian.

Funding acquisition: Di Lian.

Investigation: Di Lian.

Methodology: Di Lian, Qiuyu Tang, Ling Wu.

Project administration: Di Lian.

Resources: Di Lian, Ling Wu.

Software: Di Lian, Qiuyu Tang, Xing Liao.

Supervision: Di Lian, Ling Wu, Xing Liao.

Validation: Di Lian, Xing Liao.

Visualization: Ling Wu, Xing Liao.

Writing—original draft: Xing Liao.

Writing—review & editing: Xing Liao.

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