In the neonatal period, infectious diseases associated with high morbidity (e.g., neonatal sepsis and meningitis) are preliminarily assessed using indicators like C-reactive protein (CRP) and procalcitonin, but definitive diagnosis relies on pathogen detection through methods such as blood culture, which is time-consuming and has low sensitivity. To improve diagnostic efficiency, metagenomic next-generation sequencing (mNGS) is increasingly utilized, offering three testing modalities: DNA-only, RNA-only, and combined DNA+RNA channels. This retrospective study analyzed 894 clinical samples (peripheral blood, sputum, bronchoalveolar lavage fluid) to compare detection rates across channels. The overall mNGS positivity rate was 51.9% (464/894), with no significant differences among DNA-only (50.8%), RNA-only (55.7%), and combined channels (49.6%) (p＞0.05).Notably, bronchoalveolar lavage fluid samples exhibited the highest positivity rate (84.57%, 148/175), reaching 97.33% (73/75) with dual-channel testing. Sputum samples showed a 53.7% positivity rate (87/162), increasing to 82.35% (14/17) with dual-channel detection. Conversely, peripheral blood had an overall positivity rate of 43.14% (132/306), with the DNA-only channel outperforming RNA-only and dual channels (45.34% vs. 43.00% and 34.21%). These findings underscore the importance of channel selection based on sample type to optimize diagnostic accuracy and cost-effectiveness. 【keywords】neonate, infectious diseases, mNGS, channelNeonatal death remains the leading cause of mortality in children under five years of age, with infectious diseases accounting for a significant proportion of these deaths 1. Newborns are particularly vulnerable to infectious diseases due to their immature immune systems and potential vertical transmission of pathogens from the mother 2. In China, preterm and very low birth weight infants (VLBW) represent approximately 10% of live births 3. These infants face elevated risks of sepsis due to immunological immaturity and are prone to intracranial infections owing to a weakened blood-brain barrier. Such infections are associated with high mortality rates and poor long-term neurodevelopmental outcomes 45 6. Current diagnostic methods for neonatal infections primarily rely on nonspecific biomarkers, such as white blood cell count, CRP, and procalcitonin, which only indicate the possibility of infection 78 9. Furthermore, obtaining adequate clinical specimens from newborns for pathogen detection is challenging, particularly for blood volume-dependent tests like blood culture-the current gold standard for sepsis diagnosis. However, blood cultures are time-consuming and exhibit low sensitivity in neonatal sepsis 10, specially given their limited blood volume, which further reduces the detection rate compared to older children and adults 11. mNGS is a laboratory diagnostic technology based on high-throughput sequencing technology to sequence the whole biological genome in a variety of clinical samples. By simultaneously detecting millions of DNA/RNA fragments, mNGS offers rapid and efficient pathogen detection, leading to its increasing adoption in clinical settings 12 1314 15. Its clinical utility is particularly evident in identifying pathogens undetectable by conventional methods, though cost remains a limiting factor. When mNGS testing is selected, three sequencing modalities must be chosen: DNA-only, RNA-only, or combined DNA/RNA. The diagnostic performance of mNGS, as reflected by positivity rates, depends critically on this sequencing modalities selection. While multiple studies have validated mNGS efficacy in neonates 1617 18 and Chinese guidelines outline its use in infant infections 19, real-world data on sequencing modality selection patterns and their impact on pathogen detection rates in this population remain rare. To address this gap, we conducted a multicenter, cross-sectional study to assess mNGS sequencing modality selection practices and their correlation with positivity rates and pathogen profiles in neonates.This multicenter cross-sectional study analyzed real-world data from five hospitals in China: Nanfang Hospital of Southern Medical University, Dongguan Women and Children Hospital, Guangdong Maternal and Child Health Hospital, Jiangmen Women and Children Hospital, and Lianjiang People's Hospital. All participating institutions met the following criteria: 1) annual delivery volume ≥ 5,000 births, and 2) availability of Level IV neonatal intensive care units (NICUs). We retrospectively analyzed neonates undergoing mNGS testing between January 2020 and December 2022. Testing indications followed national clinical guidelines 19. mNGS employs a standardized clinical laboratory workflow comprising specimen processing; nucleic acid extraction (simultaneous DNA/RNA isolation); library preparation through fragmentation and adapter ligation; high-throughput sequencing on platforms; and bioinformatic analysis via alignment to microbial reference genomes and clinical pathogen databases (All nucleic acid samples were processed under a standardized protocol across all participating hospitals to ensure consistency and reproducibility. Specifically, RNA preservation was prioritized immediately after sample collection using RNase inhibitors and appropriate storage conditions to prevent degradation. The integrity of RNA was quantitatively confirmed prior to analysis via spectrophotometry or automated electrophoresis) 20.Clinical specimens were collected from all enrolled patients for mNGS, with testing modality allocation (DNA-only, RNA-only, or combined DNA/RNA) determined by clinical indications. All samples underwent standardized collection and processing protocols to ensure analytical consistency.Statistical Analysis ： All data in the study were statistically analyzed by SPSS 25.0. Count data is expressed as composition ratios or percentages (%), while measurement data is represented as mean ± standard deviation (mean ± SD). The differences in proportions across multiple groups were analyzed using Pearson's chi-square test. Results were considered statistically significant if p value was ＜ 0.05.A total of five hospitals participated in the study, including 509 patients (refer to Table 1). The total number of samples sent for testing was 894, with 464 positive cases detected, resulting in an overall positive rate of 51.90% (464/894).Statememt: The clinical diagnosis was the primary one in this patient RDS：Respiratory distress syndromeA total of 894 clinical specimens were analyzed, with sequencing modalities distribution as follows (Figure 1): DNA-only (44.52%, 398/894), RNA-only (29.53%, 264/894), and combined DNA/RNA (25.95%, 232/894). The overall pathogen detection rate was 51.90% (464/894). Positivity rates varied marginally across modalities: RNA-only (55.68%, 147/264), DNA-only (50.75%, 202/398), and combined DNA/RNA (49.56%, 115/232). No statistically significant differences in positivity rates were observed among the three sequencing modalities (p > 0.05).A total of 894 clinical specimens were analyzed (Figure 2 3).We further compared pathogen detection rates across sequencing modalities for four specimen types (Figure 4). Among peripheral blood specimens (n=306), DNA-only sequencing modality was predominantly utilized (56.86%, 161/306), demonstrating positivity rates of 45.34% (73/161) for DNA-only, 43.00% (46/107) for RNA-only, and 34.21% (13/38) for combined sequencing modalities, with no statistically significant inter-sequencing modalities differences (p = 0.460). In contrast, BALF specimens (n=175) exhibited markedly enhanced diagnostic performance with combined DNA/RNA sequencing modalities, achieving a 97.33% positivity rate (73/75) compared to 77.05% (47/61) for DNA-only and 71.80% (28/39) for RNA-only sequencing modality (p 0.05). Sample-specific performance varied markedly (p < 0.05). Peripheral blood, the most frequently submitted specimen type (34.23%, 306/894), demonstrated limited diagnostic yield (43.14%, 132/306). In contrast, BALF achieved exceptional sensitivity (84.57%, 145/175), establishing it as the optimal choice for suspected respiratory infections. Combined DNA/RNA testing further enhanced BALF detection (97.33%, 73/75) and sputum analysis (82.35%, and what samples should be tested under different clinical manifestations 19.
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