An Evaluation of a Molecular Point‐of‐Care Testing System Designed for Early Diagnosis of Mycoplasma pneumoniae and Respiratory Syncytial Virus Infections

ABSTRACT

Background

Mycoplasma pneumoniae (MP) and respiratory syncytial virus (RSV) contribute to 15%–30% of pneumonia cases in China, yet lack rapid diagnostics. We evaluated a molecular point‐of‐care testing (mPOCT) system for detecting these pathogens versus RT‐qPCR.

Methods

Oropharyngeal swabs from 549 patients (269 MP‐suspected, 280 RSV‐suspected) collected prospectively (Dec 2023‐Aug 2024) were tested by RT‐qPCR and mPOCT (EasyNAT). RT‐qPCR positivity was defined as Ct ≤ 40, as per the kit manufacturer's instructions. Diagnostic accuracy was assessed via sensitivity, specificity, and Cohen's kappa.

Results

For MP detection (n = 269), mPOCT showed 97.25% sensitivity (95% CI: 91.57%–99.29%) and 98.75% specificity (95% CI: 95.09%–99.78%) with kappa = 0.96. For RSV (n = 280), sensitivity was 96.72% (95% CI: 91.31%–98.94%) and specificity 100% (95% CI: 97.04%–100%) with kappa = 0.97. Age‐stratified analysis (pediatric/adult) maintained kappa > 0.90 for both pathogens. Co‐infections occurred in 12.8% (14/109) of MP‐positive and 17.2% (21/122) of RSV‐positive samples, with > 95% mPOCT concordance except one influenza A‐discordant case.

Conclusions

The mPOCT system demonstrated high diagnostic accuracy comparable to RT‐qPCR for MP/RSV detection, with rapid results (< 30 min). Its reliability across age groups and complex co‐infections supports clinical utility for timely treatment decisions and antibiotic stewardship.

On the basis of RT‐qPCR results, 549 oropharyngeal swabs were classified into four groups: MP‐positive (n = 109), MP‐negative (n = 160), RSV‐positive (n = 122), and RSV‐negative (n = 158). Using these as the reference standard, we evaluated a molecular point‐of‐care testing (mPOCT) system (EasyNAT) for simultaneous MP and RSV detection. The mPOCT demonstrated near‐perfect concordance with RT‐qPCR, with kappa values of 0.96 (95% CI: 0.92–0.99) for MP and 0.97 (95% CI: 0.93–1.00) for RSV. Sensitivity and specificity were 97.25% and 98.75% for MP, and 96.72% and 100% for RSV, respectively. Diagnostic reliability remained consistent across pediatric and adult populations (kappa > 0.90 for both pathogens). In polymicrobial co‐infections, the system maintained high accuracy: 92.9% (13/14) for MP and 95.2% (20/21) for RSV. With a turnaround time under 30 min, this mPOCT system offers a rapid, reliable alternative to RT‐qPCR for early diagnosis of MP and RSV infections, supporting timely clinical decision‐making and antimicrobial stewardship.

Abbreviations

AdV

adenovirus

COVID‐19

coronavirus disease 2019

CRISPR

clustered regularly interspaced short palindromic repeats

DNA

deoxyribonucleic acid

Flu A

influenza A virus

Flu B

influenza B virus

GAPDH

glyceraldehyde‐3‐phosphate dehydrogenase

HRV

human rhinovirus

MP

Mycoplasma Pneumoniae

mPOCT

molecular point‐of‐care testing

PCR

polymerase chain reaction

RNA

ribonucleic acid

RSV

respiratory syncytial virus

RT‐qPCR

reverse transcription‐quantitative real‐time polymerase chain reaction

1. Introduction

Acute respiratory infections are a significant global burden, as evidenced by rising morbidity rates [1]. In China, MP and RSV have been identified as two predominant etiological agents of community‐acquired respiratory infections, ranking third in prevalence after Streptococcus pneumoniae and influenza viruses on the basis of recent epidemiological surveillance data. Notably, MP serves as the primary causative agent of respiratory tract infections among school‐aged children (5–15 years), with studies indicating its association with prolonged outbreaks in closed populations such as schools. Conversely, RSV is a globally recognized driver of severe lower respiratory tract infections (LRTIs), contributing substantially to hospitalization rates and morbidity in two high‐risk populations: children under 5 years of age (particularly infants < 1 year) and adults aged ≥ 65 years [2, 3].

Current detection of MP and RSV nucleic acids relies on advanced technologies, including multiplex polymerase chain reaction (PCR), sequencing, clustered regularly interspaced short palindromic repeats (CRISPR) detection platforms, among others [4, 5, 6, 7]. However, these methods are limited by their high costs, operational complexity, complex workflows, and operator dependency, making them unable to meet the clinical demand for rapid detection of respiratory pathogens. Delayed diagnosis of MP risks macrolide resistance and extrapulmonary complications, whereas RSV delay increases transmission in vulnerable populations such as infants and the elderly, potentially leading to severe lower respiratory tract infections and hospitalization. As a result, mPOCT systems that involve automated cell lysis, extraction of nucleic acids, amplification, and analysis of end‐products have received significant attention because of their rapidity, automated nature, and reduced likelihood of cross‐contamination [8]. For this reason, the present study aimed to evaluate the diagnostic performance and potential clinical implications of mPOCT systems in detecting MP and RSV nucleic acids.

2. Methods

2.1. Study Design and Patient Enrollment

This cross‐sectional study enrolled 269 patients with suspected MP infection and 281 patients with suspected RSV infection presenting to the First Affiliated Hospital of Kunming Medical University between December 2023 and August 2024. The MP and RSV cohorts were enrolled independently and were mutually exclusive. After excluding one invalid specimen from the RSV cohort (because of insufficient volume for both tests), 549 valid oropharyngeal swab samples were included for final analysis. Participants were consecutively enrolled during the study period to minimize selection bias.

2.2. Inclusion Criteria

This diagnostic accuracy study defined specific clinical criteria for enrollment into each target pathogen cohort.

2.2.1. MP Cohort

Patients were enrolled in the MP‐suspected cohort if they met all of the following criteria: (1) clinical indication: the attending physician suspected MP infection on the basis of clinical presentation (e.g., persistent dry cough, characteristic pulmonary imaging findings); (2) core symptoms: presentation with documented fever (axillary temperature ≥ 38.0°C) accompanied by at least one respiratory symptom (e.g., cough, pharyngalgia, headache, or fatigue); (3) case definition: clinical manifestations met World Health Organization (WHO) case definitions for community‐acquired pneumonia (CAP).

2.2.2. RSV Cohort

Patients were enrolled in the RSV‐suspected cohort if they met all of the following criteria: (1) clinical syndrome: patients meeting WHO criteria for suspected RSV infection, which included acute bronchiolitis or acute lower respiratory tract infection with wheezing in children < 2 years, or exacerbations of COPD/asthma in adults ≥ 65 years; (2) core symptoms: presentation with documented fever (axillary temperature ≥ 38.0°C) accompanied by at least one respiratory symptom; (3) case definition: Clinical manifestations met the WHO case definition for CAP.

The study included both inpatients and outpatients from pediatric and adult departments. Immunocompromised patients or those with chronic pulmonary disease were not explicitly excluded.

2.3. Instruments and Reagents

Nucleic acid extraction was performed using the GeneRotex 96 Automated System (Xi'an Tianlong Science and Technology Co. Ltd.). Molecular detection was conducted on two independent platforms: the UC0108 Nucleic Acid Amplification Analyzer (Hangzhou Ustar Biotech Co. Ltd.) and the SLAN‐96P Real‐time PCR System (Shanghai Hongshi Medical Technology Co. Ltd.). For pathogen identification, two commercial diagnostic kits were employed: the Six Respiratory Pathogens Multiplex PCR Kit (Sansure Biotech Inc., CFDA registration no. 20213400256) covering MP, RSV, and other common pathogens, alongside MP/RSV‐specific isothermal amplification kits (Ustar Biotech, batch no. 20240302/20240402). All procedures included negative controls and positive controls in each batch.

2.4. Detection

Samples Were Processed and Tested Within 2 h of Collection for Both RT‐qPCR and mPOCT to Prevent Nucleic Acid Degradation.

2.4.1. RT‐qPCR Detection

Essential PCR reagents were prepared, and nucleic acids were extracted. Then, samples were loaded for PCR amplification following the specific instructions provided in the kits. The detection was completed in less than 3 h. For every sample detected, the glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) gene, which serves as a human housekeeping gene, was monitored to confirm that amplification was normal and therefore facilitated the extraction and PCR amplification of the samples. A cycle threshold (Ct) value of ≤ 40.0 was defined as positive, as specified by the manufacturer of the RT‐qPCR kits.

2.4.2. EasyNAT MP Assay Detection

The procedures for amplification and detection were executed according to the guidelines provided for the MP Nucleic Acid Detection Kit. After fully mixing, MP‐DNA extraction solution was loaded into a tube prepared for an MP detection. Then, 500 μL of a tested sample was added, sealed, mixed, and then scanned before being put into the UC0108 Nucleic Acid Amplification Detection Analyzer. The detection process was conducted for a period of 25 min before tests were automatically generated by the device. Moreover, this kit had an internal control system that was built on a competitive basis. Such included an internal control template together with related specific amplification primers and fluorescent probes which were meant to assess whether sample collection, nucleic acid extraction, as well as purification and amplification reactions were effective.

2.4.3. EasyNAT RSV Assay Detection

As per the guidelines mentioned in the particular kit for the detection of nucleic acids related to RSV. The fully mixed RSV‐RNA extraction solution was transferred into a tube that had been prepared for RSV detection. Subsequently, 500 μL of the tested sample was added, sealed, mixed, and scanned before being placed in the UC0108 Nucleic Acid Amplification Detection Analyzer. The total processing time was 25 min, during which the system automatically generated identification results and made an assessment. The internal control measure is similar to the MP detection process.

2.5. Statistical Analysis

The data were processed and analyzed using SPSS 27.0 statistical software. Categorical data were expressed as frequencies or as percentages. Comparisons between the mPOCT and RT‐qPCR results were conducted using McNemar's test for paired nominal data. The concordance between the two detection methods was evaluated using Cohen's Kappa statistic. Kappa values were interpreted as follows: values below 0.00 indicated poor agreement; 0.00–0.20, slight agreement; 0.21–0.40, fair agreement; 0.41–0.60, moderate agreement; 0.61–0.80, substantial agreement; and 0.81–1.00, almost perfect agreement [9, 10]. A p‐value less than 0.05 was considered statistically significant.

3. Results

3.1. Detection Results

On the basis of the RT‐qPCR detection results, all enrolled samples were divided into four groups: MP‐positive, MP‐negative, RSV‐positive, and RSV‐negative. The number of samples included in each group is shown in Figure 1.

FIGURE 1.

Demographic characteristics and sample sizes of the study groups on the basis of RT‐qPCR results. (A) MP‐positive and MP‐negative groups: Age and sex distributions, and number of samples; (B) RSV‐positive and RSV‐negative groups: Age and sex distributions, and number of samples.

Using RT‐qPCR as the reference standard, the performance of the mPOCT system was evaluated for both pathogens.

For the MP cohort, the positive detection rates of RT‐qPCR and the EasyNAT MP Assay were 40.52% (109/269) and 40.15% (108/269), respectively, indicating a high level of concordance between the two methods. A total of 106 samples were positive by both assays, and 158 were negative by both, yielding an overall agreement rate of 98.1% (264/269). McNemar's test revealed no statistically significant difference between the two methods (p = 1.000), and the kappa coefficient was 0.96 (95% CI: 0.92–0.99), indicating almost perfect agreement. The detailed comparison is presented in Table 1.

TABLE 1.

Consistency analysis of EasyNAT MP Assay and RT‐qPCR detection results (n).

	RT‐qPCR		McNemar's test (p)	Kappa coefficient (95% CI)
	Positive	Negative
EasyNAT MP
Positive	106	2	1.000	0.96 (0.92–0.99)
Negative	3	158
Total	109	160

For the RSV cohort, the positive detection rates of RT‐qPCR and the EasyNAT RSV Assay were 43.57% (122/280) and 42.14% (118/280), respectively, indicating a high level of concordance between the two methods. A total of 118 samples were positive by both assays, and 158 were negative by both, yielding an overall agreement rate of 98.6% (276/280). McNemar's test revealed no statistically significant difference between the two methods (p = 0.125), and the kappa coefficient was 0.97 (95% CI: 0.93–1.00), indicating almost perfect agreement. The detailed comparison is presented in Table 2.

TABLE 2.

Consistency analysis of EasyNAT RSV Assay and RT‐qPCR detection results (n).

	RT‐qPCR		McNemar's test (P)	Kappa coefficient (95% CI)
	Positive	Negative
EasyNAT RSV
Positive	118	0	0.125	0.97 (0.93–1.00)
Negative	4	158
Total	122	158

3.2. Detection Performance of EasyNAT MP Assay

Using RT‐qPCR results as the standard, the sensitivity, specificity, positive predictive value, and negative predictive value of the EasyNAT MP Assay for MP detection were 97.25% (95% CI: 91.57%–99.29%), 98.75% (95% CI: 95.09%–99.78%), 98.15% (95% CI: 92.81%–99.68%), and 98.14% (95% CI: 94.22%–99.52%), respectively. The overall agreement rate with RT‐qPCR results was 98.14% (95% CI: 95.47%–99.31%).

3.3. Detection Performance of EasyNAT RSV Assay

Using RT‐qPCR results as the standard, the sensitivity, specificity, positive predictive value, and negative predictive value of EasyNAT RSV Assay for RSV detection were 96.72% (95% CI: 91.31%–98.94%), 100.00% (95% CI: 97.04%–100.00%), 100.00% (95% CI: 96.07%–100.00%), and 97.53% (95% CI: 93.40%–99.20%), respectively. The overall concordance rate with RT‐qPCR detection results was 98.57% (95% CI: 96.14%–99.54%).

3.4. Age‐Stratified Analysis

Diagnostic performance was further evaluated after stratifying the study population by age. Because of sample size constraints, aggregate analysis was performed on the adult cohort aged ≥ 18 years.

For MP, in the < 18 years age group, the positive detection rates of RT‐qPCR and the EasyNAT MP Assay were 55.43% (97/175) and 53.71% (94/175), respectively, indicating a high level of concordance between the two methods. A total of 94 samples were positive by both assays, and 78 were negative by both, yielding an overall agreement rate of 98.3% (172/175). McNemar's test revealed no statistically significant difference (p = 0.250), and the kappa coefficient was 0.97 (95% CI: 0.92–1.00), indicating almost perfect agreement. In the ≥ 18 years age group, the positive detection rates of RT‐qPCR and the EasyNAT MP Assay were 12.77% (12/94) and 14.89% (14/94), respectively. A total of 12 samples were positive by both assays, and 80 were negative by both, corresponding to an overall agreement rate of 97.9% (92/94). McNemar's test showed no significant difference (p = 0.500), and the kappa coefficient was 0.91 (95% CI: 0.76–1.00), also indicating almost perfect agreement. The kappa values in both age groups exceeded 0.9, and the difference between them was not statistically significant (Z = 0.829, p > 0.05), suggesting that the EasyNAT MP Assay maintains consistently high agreement with RT‐qPCR across age groups. Details are presented in Table 3.

TABLE 3.

Consistency analysis of EasyNAT MP Assay and RT‐qPCR detection results by age group (n).

	RT‐qPCR		McNemar's test (P)	Kappa coefficient (95% CI)	Z
	Positive	Negative
< 18					0.829
EasyNAT MP
Positive	94	0	0.250	0.97 (0.92–1.00)
Negative	3	78
Total	97	78
≥ 18
EasyNAT MP
Positive	12	2	0.500	0.91 (0.76–1.00)
Negative	0	80
Total	12	82

For RSV, in the < 18 years age group, the positive detection rates of RT‐qPCR and the EasyNAT RSV Assay were 58.62% (102/174) and 57.47% (100/174), respectively, indicating a high level of concordance between the two methods. A total of 100 samples were positive by both assays, and 72 were negative by both, yielding an overall agreement rate of 98.9% (172/174). McNemar's test revealed no statistically significant difference (p = 0.500), and the kappa coefficient was 0.98 (95% CI: 0.94–1.00), indicating almost perfect agreement. In the ≥ 18 years age group, the positive detection rates of RT‐qPCR and the EasyNAT RSV Assay were 18.87% (20/106) and 16.98% (18/106), respectively. A total of 18 samples were positive by both assays, and 86 were negative by both, corresponding to an overall agreement rate of 98.1% (104/106). McNemar's test showed no significant difference (p = 0.500), and the kappa coefficient was 0.94 (95% CI: 0.83–1.00), also indicating almost perfect agreement. The kappa values in both age groups exceeded 0.9, and the difference between them was not statistically significant (Z = 0.832, p > 0.05), suggesting that the EasyNAT RSV Assay maintains consistently high agreement with RT‐qPCR across age groups. Details are presented in Table 4.

TABLE 4.

Consistency analysis of EasyNAT RSV Assay and RT‐qPCR detection results by age group (n).

	RT‐qPCR		McNemar's test (P)	Kappa coefficient (95% CI)	Z
	Positive	Negative
< 18					0.832
EasyNAT RSV
Positive	100	0	0.500	0.98 (0.94–1.00)
Negative	2	72
Total	102	72
≥ 18
EasyNAT RSV
Positive	18	0	0.500	0.94 (0.83–1.00)
Negative	2	86
Total	20	86

3.5. Comparison of EasyNAT MP and EasyNAT RSV Assays With RT‐qPCR in the Context of Co‐Infections

3.5.1. Comparison of EasyNAT MP Assay and RT‐qPCR Results in Detecting MP Co‐Infections

Among the 109 RT‐qPCR MP‐positive group samples, 14 (12.8%, 14/109) had co‐infections with other respiratory pathogens: 12 cases were positive for human rhinovirus (HRV), 1 case for RSV, and 1 case for influenza B virus (Flu B). Of the 14 samples tested by mPOCT, 1 that was positive for HRV yielded a negative result for MP, whereas the remaining 13 samples tested positive for MP.

Among the 160 RT‐qPCR MP‐negative group samples, 32 (20.0%, 32/160) had co‐infections with other respiratory pathogens: 20 cases were positive for HRV, 6 cases for AdV, 3 cases for RSV, 2 cases for both RSV and HRV, and 1 case for both AdV and HRV. All 32 samples tested by mPOCT were found to be negative for MP.

In summary, the mPOCT system maintained a high detection accuracy (13/14, 92.9%) for MP in the presence of co‐infections and showed perfect specificity (32/32, 100%) in samples infected solely with other respiratory pathogens. Detailed results are presented in Table 5.

TABLE 5.

Comparison of RT‐qPCR and EasyNAT results for MP co‐infections.

EasyNAT MP	RT‐qPCR		Total
	Positive	Negative
MP + HRV
Positive	11	0	11
Negative	1	20	21
Total	12	20	32
MP + RSV
Positive	1	0	1
Negative	0	3	3
Total	1	3	4
MP + Flu B
Positive	1	0	1
Negative	0	0	0
Total	1	0	1
MP + AdV
Positive	0	0	0
Negative	0	6	6
Total	0	6	6
MP + RSV + HRV
Positive	0	0	0
Negative	0	2	2
Total	0	2	2
MP + AdV + HRV
Positive	0	0	0
Negative	0	1	1
Total	0	1	1

3.5.2. Comparison of EasyNAT RSV Assay and RT‐qPCR Results in Detecting RSV Co‐Infections

Among the 122 RT‐qPCR RSV‐negative group samples, 21 (17.2%, 21/122) had co‐infections with other respiratory pathogens: 12 cases were positive for HRV, 4 cases for AdV, 1 case for both HRV and AdV, 1 case for influenza A virus (Flu A), and 3 cases for MP. Of the 21 samples tested by mPOCT, one Flu A‐positive sample yielded a negative result for RSV, whereas the remaining 20 samples were positive for RSV.

Among the 158 RT‐qPCR RSV‐negative group samples, 31 (19.6%, 31/158) had co‐infections with other respiratory pathogens: 25 cases were positive for HRV, 3 cases for AdV, and 3 cases for MP. All 31 samples tested by mPOCT were found to be negative for RSV.

In summary, the mPOCT system demonstrated high detection accuracy (20/21, 95.2%) for RSV in co‐infected samples and perfect specificity (31/31, 100%) in samples infected with other respiratory pathogens alone. Detailed results are provided in Table 6.

TABLE 6.

Comparison of RT‐qPCR and EasyNAT results for RSV co‐infections.

EasyNAT RSV	RT‐qPCR		Total
	Positive	Negative
RSV + HRV
Positive	12	0	12
Negative	0	25	25
Total	12	25	37
RSV + AdV
Positive	4	0	4
Negative	0	3	3
Total	4	3	7
RSV + HRV + AdV
Positive	1	0	1
Negative	0	0	0
Total	1	0	1
RSV + MP
Positive	3	0	3
Negative	0	3	3
Total	3	3	6
RSV + Flu
Positive	0	0	0
Negative	1	0	1
Total	1	0	1

The mPOCT system maintained > 95% concordance with RT‐qPCR in co‐infected samples except one influenza A‐discordant case (Table 6), suggesting minimal cross‐reactivity.

4. Discussion

MP has long been recognized as a major cause of community‐acquired pneumonia in children. It defines infection and, afterward, colonization of the respiratory tract of the pediatric subject, further causing inflammation at various anatomic levels, from the areas of bronchial and bronchiolar grade up to alveolar structures and into pulmonary interstitial spaces. In view of this fact, MP infections in children are characterized by variable severity and often show a non‐uniform spectrum of respiratory manifestations that may span from mild to severe. Recent studies have shown that refractory MP is related to long duration of recurrent infection, increasing complication rates, and the emergence of macrolide‐resistant strains [11, 12]. Hence, early detection of MP will be highly useful for the early diagnosis of mycoplasma pneumonia, prevention from the occurrence of the disease, and its complications associated with the disease [13].

These unique infection patterns of RSV would therefore be seasonally related [14]. However, great changes in RSV epidemiological trends have indeed been noticed since the onset of the coronavirus disease 2019 (COVID‐19) pandemic. These indeed include disrupted seasonality, longer durations of epidemics, and increased diversity among RSV genotypes [15, 16, 17, 18]. Although in 2017 and again in 2021, the World Health Organization recommended RSV vaccines and monoclonal antibodies for passive immunization against RSV disease, until now, no specific antiviral agents have been found against RSV infections [19]. Hence, early and accurate identification of RSV remains very important in preventing outbreaks and addressing the progression of disease.

Molecular diagnosis has exhibited very high sensitivity and specificity in the detection of respiratory pathogens and therefore is being used more and more in clinical settings [20, 21]. This study was designed to compare an mPOCT system with RT‐qPCR for the detection of MP and RSV. In the current study, the agreement between the mPOCT system and RT‐qPCR was almost perfect (Kappa > 0.90) for both MP and RSV detection. It also showed high concordance in samples with common respiratory pathogen co‐infections, highlighting its potential utility in complex clinical presentations and its possible role in guiding targeted therapy for polymicrobial infections. Although both mPOCT and RT‐PCR are based on the principle of nucleic acid amplification, their operating procedures and detection times differ vastly. The mPOCT system is a monologue, monosample system that applies an integrated whole detection process from nucleic acid extraction to purification, amplification, and its subsequent identification. Therefore, it highly simplifies the procedure flow and reduces TAT to less than 30 min. In comparison, RT‐qPCR is the “gold standard” for detecting infectious agents and requires a niche laboratory setting, educated personnel, and special equipment. The complications of sample preparation and operation of such instruments make most detection times commonly over several hours. This variation is significantly distinct in clinical settings that rely on speed to make decisions, such as in the emergency departments and outpatient clinics, where timely initiation of macrolides for MP is required while avoiding unnecessary antibiotics for viral RSV infections. This may reduce complications (e.g., macrolide resistance) and improve bed management through prompt isolation of RSV‐positive cases.

An important consideration for any nucleic acid test is its clinical detection limit. In this study, the few discordant results (RT‐qPCR+/mPOCT‐) occurred in samples with high Ct values (low viral load). Although the specific Ct values of discordant samples were not systematically analyzed in this cohort, this observation aligns with the expected performance characteristics of a rapid POC test compared to a highly sensitive laboratory‐based RT‐qPCR. RT‐qPCR can detect very low levels of nucleic acids, which may not always correlate with active, clinically significant infection or transmissibility. In contrast, mPOCT may detect viral loads that are more consistently associated with symptomatic disease. Future studies correlating viral loads (Ct values) with clinical symptoms and outcomes are needed to better define the optimal diagnostic threshold for clinical management.

The limitations of this study include: (1) single‐center design may limit generalizability; (2) cost‐effectiveness and impact on patient outcomes (e.g., hospitalization duration, antibiotic use) were not evaluated; (3) follow‐up data on clinical progression are lacking; (4) age‐stratified analysis, particularly for adult RSV, was constrained by sample size. Future multicenter studies with larger, diverse populations are needed to validate these findings and further explore the clinical impact of implementing such mPOCT systems.

5. Conclusions

The evaluated mPOCT system demonstrated high diagnostic accuracy for MP and RSV, providing results within 30 min. This has the potential to streamline clinical workflows, enable rapid therapeutic decisions, and potentially expedite the initiation of targeted antimicrobial therapy for MP while reducing unnecessary antibiotic prescriptions for viral infections like RSV. In resource‐limited settings, such efficiency could alleviate laboratory burden and enhance infection control through timely isolation. This mPOCT system shows promise as a rapid and reliable tool for the early diagnosis of MP and RSV, which may contribute to improved patient management.

Author Contributions

Yang Su and Yanliang Zhang conceived and designed the study. Yang Su, Yongmei Li, Zhirui Zhang, and Jing Yang performed the main sample data collection and analyses under the supervision of Ying Zhao, Yandan Su, and Yanliang Zhang. Qiuyue Xu, Yu Song, Yunjuan Wang, Kai Yang, Yamin Wu, Jinliang Chen, and Xueting Yang helped with the sample testing, analyses, and data organization. Yang Su wrote the draft of the manuscript. Yanliang Zhang reviewed and revised the manuscript. All authors have contributed to, seen, and approved the final, submitted version of the manuscript.

Funding

Supported by Central Guidance for Local Science and Technology Development Fund Project (202507AB040006). Funding Major Science and Technology Projects of Yunnan Province/First Affiliated Hospital of Kunming Medical University (2023zdpy05). Yunnan Province “High level Talent Training Support Plan”–Youth Top Talents Project (YNWR‐QNBJ‐2019‐159). 535 Talent Project of First Affiliated Hospital of Kunming Medical University (2023535D08).

Ethics Statement

This study was approved by the Ethics Committee of the First Affiliated Hospital of Kunming Medical University, and informed consent was obtained from all participants or their legal representatives.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

All data generated or analysed during this study are included in this published article. The data that support the findings of this study are available from the corresponding author upon reasonable request.