The prevalence of Mycoplasma pneumoniae and study of co-infection with COVID-19 from a laboratory perspective, before and after the relaxation of COVID-19 control measures

Abstract

Objective

Describe the prevalence of Mycoplasma pneumoniae (MP) in 2023, and investigate the influence of the relaxaion of COVID-19 pandemic control measures on MP prevalence. Analyze the positivity rates of MP in three distinct testing panels, assess clinical preferences for selecting these packages. Describe the co-infection status of MP with other respiratory pathogens, along with the laboratory characteristics of MP co-infection with COVID-19. Analyse the disparities in data between co-infected patients, healthy controls, and MP-only positive patients in terms of laboratory results, and speculate on its potential clinical implications.

Method and results

We retrospectively collected and analyzed MP epidemiological and clinical data from 2022-2023 using three MP-related tests. The results showed that a total of 37,017 MP-related tests were conducted, with an overall positivity rate of 2.03%. Among the positive cases, 83.36% were aged 0-15 years. In 2023, there were 628 tests for MP and Chlamydia pneumoniae (CP) Nucleic Acid Test, with a positivity rate of 19.11%, surpassing the 347 cases in 2022, which had a positivity rate of 5.76%. Influenza A virus was the most commonly co-infected respiratory pathogen with MP. The laboratory results of the control, single-infection, and co-infection groups showed that in the complete blood count (CBC), COVID-19 may have altered the immune process of MP. The main reason for the abnormal biochemical results in both single and double-positive groups was insufficient nutrition, leading to reduced synthesis. The urinalysis results indicated that the double-positive group had higher renal pressure than the single-positive group.

Conclusion

MP entered the epidemic period in July following the relaxation of COVID-19 pandemic control measures. Additionally, COVID-19 may have altered the immune process of MP, impacting certain liver, heart, and kidney functions, as indicated by the laboratory results. Therefore, this study provides significant reference data for the clinical diagnosis and treatment of MP infections and co-infections with COVID-19.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12879-025-11675-y.

Introduction

Mycoplasma pneumoniae (MP) is the smallest self-replicating organism, lacking a cell wall and growing slowly (generation time: approximately 6 h). It requires close contact for transmission and typically leads to an epidemic every 2–7 years. Its epidemiological characteristics are influenced by factors such as age, gender, and region, also showing seasonal trends [1, 2]. MP often causes acute upper and lower respiratory tract infections, being one of the pathogens responsible for community-acquired pneumonia, primarily infecting children and adolescents [3, 4]. Although MP pneumonia is generally considered a self-limiting disease, it can easily lead to pulmonary and extrapulmonary complications, progressing to refractory pneumonia, necrotizing pneumonia, bronchiolitis obliterans, or even fatal pneumonia [5]. Studies show that around 12% of pediatric MP patients require intensive care [6], with severe mycoplasma pneumonia mortality reaching 3.9% [7]. Moreover, the recent COVID-19 pandemic has significantly impacted respiratory virus epidemiology, including MP. Therefore, it is crucial to monitor the epidemiological trends of MP in real-time for effective clinical disease prevention and control.

On December 5, 2022, China lifted its COVID-19 pandemic control measures, including the elimination of mask-wearing, quarantine, nucleic acid testing, and venue QR code scanning. Following this change, Hangzhou experienced a COVID-19 outbreak, which also influenced the transmission and progression of other respiratory pathogens. Unlike prior studies that used the start of the pandemic as a reference point, this paper innovatively uses the relaxation of control measures as a benchmark to explore the changes in MP prevalence before and after the restrictions were lifted, as well as patterns of co-infection with other pathogens, paying particular attention to laboratory findings in patients co-infected with MP and COVID-19.

Focusing on MP, the paper elucidates its epidemiological trends in Hangzhou in 2023, comparing them with the trends observed in 2022 before the control measures were relaxed, and analyzes the impact of lifting COVID-19 restrictions on MP prevalence. Subsequently, by comparing the MP positivity rates and the proportion of outpatient versus inpatient cases across three clinical test packages, along with the co-infection rates of MP and other respiratory pathogens, we analyze clinical preferences in test package selection and the potential reasons for the differences in co-infections. Finally, for the first time, we compare the results of three routine laboratory tests between MP-COVID-19 co-infected group, MP infection group, and a healthy control group. By identifying differences in the data, we aim to characterize MP-only infections and MP-Covid-19 co-infections from a laboratory perspective, providing data to support early diagnosis and subsequent treatment.

Materials and methods

Study subjects and methods

This retrospective study collected data from three MP-related testing panels conducted at the Zhaohui and Wangjiangshan campuses of Zhejiang Provincial People’s Hospital between January 1, 2022, and December 31, 2023. The Respiratory Pathogen Nucleic Acid Test (hereafter “Nucleic Acid Test”) panel included nine pathogens: Influenza A/B viruses (IVA/B), adenovirus (ADV), respiratory syncytial virus (RSV), parainfluenza virus types 1 and 3 (PIV1/3), Mycoplasma pneumoniae (MP), Chlamydia pneumoniae (CP), and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The Respiratory Pathogen Antigen Test (hereafter “Antigen Test”) panel detected five pathogens: IVA/B, ADV, RSV, and MP. The MP and Chlamydia pneumoniae Nucleic Acid Test (hereafter “MPCP Test”) focused on two pathogens: MP and CP. A total of 97,387 case records were analyzed and grouped according to gender, age, outpatient or inpatient status, department, and primary symptoms. Simultaneously, seven cases of MP and COVID-19 co-infection were identified among the collected cases. For comparative purposes, based on three test panels CBC + C-reaction protein (CRP), biochemical analysis, and urinalysis, 14 age-matched and gender-matched individuals with MP monoinfection and normal health check-ups (normal controls) were collected in a 1:2 ratio according to case-control study criteria. Controls were age-matched within a one-year range. Due to the absence of urinalysis in one co-infected patient, the urinalysis cases were as follows: six double-positive patients, twelve single-positive patients, and twelve normal controls.Exclusion criteria for normal controls: concurrent diagnosis with other diseases, pre-existing conditions, and incomplete clinical data.

Instruments and reagents

The Nucleic Acid Test was performed using three kits to detect nine pathogens: the Beijing ZC Bio-Tech respiratory virus nucleic acid detection kit for IVA/B, ADV, RSV, PIV1/3; the MD-BioTech 2019-nCoV nucleic acid detection kit for SARS-CoV-2; the Shanghai ZJ Bio-Tech Mycoplasma pneumoniae and Chlamydia pneumoniae nucleic acid joint detection kit for MP and CP, which is the same as MPCP Test. All detections were performed using fluorescent quantitative PCR.

The Antigen Test employed the GENESIS antigen detection kit (colloidal gold method), using antigen-antibody binding reactions to detect five pathogens: IVA/B, ADV, RSV, and MP.

All fluorescent quantitative PCR assays were conducted on the ABI 7500 Real-Time PCR System. The colloidal gold method was manually operated.

Statistical methods

Data analysis was performed using SPSS version 25.0. Normally distributed continuous variables are presented as mean ± SD, and intergroup comparisons are conducted using the t-test. Non-normally distributed continuous variables are expressed as the median (M) with interquartile ranges (P25, P75), and comparisons between groups are performed using the Mann-Whitney U test (M-W U test) and the Kruskal-Wallis test (K-W test) for independent samples. Ranked ordinal data is analyzed by calculating ranks, followed by M-W U test and K-W test. Categorical variables are expressed as frequencies and percentages (%), with intergroup comparisons conducted using the chi-square test and U test. Comparisons between two proportions are made using the chi-square or corrected chi-square test, while multiple proportion comparisons are carried out using the chi-square partition method. A two-tailed P value of less than 0.05 is considered statistically significant.

Results

General overview of MP infections in 2023

In 2023, there were a total of 37,017 MP-related tests conducted, with an overall positive rate of 2.03%. Of those testing positive, 415 were male (55.26%) and 336 were female (44.74%). As illustrated in Fig. 1a, MP infections predominantly occurred in children and adolescents aged 0–15, representing 83.36% of all cases, with the highest concentration in the 6–8 year age group. MP positivity cases gradually increases from July, and positivity rates was peaking in July of 10.09% (Fig. 1b). The distribution of positive cases varied across clinical departments (Fig. 2a). Pediatrics (605, 80.56%), Respiratory Medicine (69, 9.19%), Fever Clinic (16, 2.13%), and Emergency (9, 1.20%) accounted for 93.08% of all positive cases. Clinically (Fig. 2b), pulmonary manifestations were the most prominent feature, with pneumonia and bronchitis having the highest prevalence (415, 55.26%). Other common symptoms included fever (145, 19.31%), upper respiratory tract infection (86, 11.45%), and cough (21, 2.80%).

Fig. 1.

a Gender and age distribution of MP positive cases in 2023. b Positive cases and positive rates of MP in 2023. MP, Mycoplasma pneumoniae

Fig. 2.

a Total positive cases varied across clinical departments in 2023. b Total positive cases distributed in clinical Manifestations in 2023

Comparison between different MP testing panels

Positivity rate varies in three panels in 2023

The MPCP Test had the highest positivity rate of 19.11% in three panels. The Nucleic Acid Test came second, giving a positivity rate of 9.38%. The Antigen Test was in last place with a positivity rate of 0.72%. Significant differences were observed among the three panels (X² = 2402.1, P < 0.001). The findings were listed in Table 1.

Table 1.

Comparison of positive rates in three panels

Tests	Total cases	Negative	Positive	χ2 test
		Cases(%)	Cases(%)	χ2 value	P value
MPCP	628	508(80.89%)	120(19.11%)	2402.1	< 0,001
Nueleic Acid	4383	3972(90.62%)	411(9.38%)a
Antigen	32,006	31,786(99.31%)	220(0.69%)bc

^acompared to MPCP Test, P < 0.017

^bcompared to MPCP Test, P < 0.017

^ccompared to Nucleic Acid Test, P < 0.017

As shown in Fig. 3, both the Nucleic Acid and Antigen panels experienced a rise in the total number of tests conducted in March, while the MPCP Test reached its peak in November. In the second half of the year, positive cases and rates in the Nucleic Acid Test and MPCP Test saw a noticeable increase compared to the first half, which wasn’t as evident in the Antigen Test. The positivity rates for the Nucleic Acid Test and MPCP Test were higher from August to December and lower from March to May.

Fig. 3.

Total cases, positive cases and positive rates distributed respectively in 3 tests in 2023. a in the Nucleic Acid Test. b in the Antigen Test. c in the MPCP Test. The nucleic acid test, the respiratory pathogen nucleic acid test; the antigen test, the respiratory pathogen antigen test; the MPCP test, the Mycoplasma pneumoniae and Chlamydia pneumoniae test

Comparison of MP positivity rates and outpatient/inpatient categories across the three testing panels

In 2023, the MPCP Test had a total of 628 cases with a positivity rate of 19.11%. The Nucleic Acid Test had 4,383 cases, of which 411 tested positive, giving a positivity rate of 9.38%. The Antigen Test conducted 33,437 tests, with 241 positive cases, resulting in a positivity rate of 0.72%. The findings were listed in Table 1.

Analyzing the personnel categories, the proportions of outpatient (including emergency) and inpatient numbers varied across the three detection items (Table 2). Among outpatients, the Antigen Test had the highest proportion at 92.69%, while in inpatients, the MPCP Test had the highest proportion at 98.73%. Further analysis of the personnel categories among MP-positive patients revealed that in the outpatient categories (Table 3), the positivity rate of Antigen Test was the highest at 87.73%. While among inpatients, proportion was the highest positivity rate was settled on the Nucleic Acid Test at 80.29%.

Table 2.

Comparison of personnel categories in three panels

Tests	Total cases	Outpatient Cases(%)	Inpatient Cases(%)	χ2 test
				χ2 value	P value
MPCP	628	404(64.33%)	224(35.67%	18347.3	< 0.001
Nueleic Acid	4383	437(9.97%)	3946(90.03%)a
Antigen	32,006	29,667(92.69%)	2339(7.31%)bc

^acompared to MPCP Test, P < 0.017

^bcompared to MPCP Test, P < 0.017

^ccompared to Nucleic Acid Test, P < 0.017

Table 3.

Comparison of personnel categories in three panels among positive cases

Tests	Positive cases	Outpatient Cases(%)	Inpatient Cases(%)	χ2 test
				χ2 value	P value
MPCP	120	87(72.50%)	33(27.50%)	299.7	< 0.001
Nueleic Acid	411	81(19.71%)	330(80.29%)a
Antigen	220	193(87.73%)	27(12/27%)bc

^acompared to MPCP Test, P < 0.017

^bcompared to MPCP Test, P < 0.017

^ccompared to Nucleic Acid Test, P < 0.017

Comparison of MP infection rates before and after COVID-19 pandemic control relaxation

In 2022, a comprehensive analysis of MP testing revealed a total of 2,869 assays performed, yielding 30 positive results, corresponding to an overall positivity rate of 1.05%. Within the cohort of positive cases, gender distribution was even, with 50% Males and 50% females. The Majority of these positives, accounting for 73.33%, comprised hospitalized patients. The primary clinical departments involved were Pediatrics (23 cases, accounting for 76.67%) and Respiratory Medicine (5 cases, 16.67%). Pneumonia (19 cases, 63.33%) and acute upper respiratory tract infections (6 cases, 20%) were most common diagnoses.

As shown in Fig. 4, a comparison of the MPCP Test data from 2022 to 2023 reveals notable trends. In 2023, there were 628 cases, compared to 347 in 2022. From January to July, 2022 had more tests than 2023, but starting in August, 2023 had more tests than 2022, with the gap widening and reaching its peak in November. In 2023, there were 120 positive cases, with an overall positivity rate of 19.11%, showing a gradual increase in positive cases in the latter half of the year. In contrast, 2022 saw only 20 positive cases, with a positivity rate of 5.76%. All positive cases in 2022 were hospitalized (100%), whereas in 2023, 33 positive cases (27.50%) were hospitalized, and 87 (72.50%) were outpatients.

Fig. 4.

Total cases, positive cases and positive rates distributed in the MPCP test compared between 2022 and 2023. The MPCP test, the Mycoplasma pneumoniae and Chlamydia pneumoniae test

Comparing data from the Antigen Test between two periods: August 17 to December 31, 2022, and August 17 to December 31, 2023 (Fig. 5a). In 2023, the total number of cases was 22,414, significantly higher than the 2,522 cases in 2022. The Antigen Test shares the same trends of total cases as the MPCP Test. The proportion of outpatients in 2023 (97.1%) was significantly higher than in 2022 (86.0%). The number of MP-positive cases in the 2023 group increased monthly and was significantly higher than in the same period of 2022 (Fig. 5b).

Fig. 5.

a Total cases and positive rates distributed in the antigen test compared between 2022 and 2023. b Positive cases distriuted in the antigen test compared to 2022 and 2023. The antigen test, the Mycoplasma pneumoniae and Chlamydia pneumoniae test

Co-infection of MP with other respiratory pathogens

Analysis of co-infection with MP in the Nucleic Acid Test and Antigen Test in 2023. Of the 411 MP-positive cases from the Nucleic Acid panel, 47 were co-infected with other 8 pathogens, representing 11.43% of the total. The rates of co-infection with various viruses are presented in Fig. 6. Among the 220 positive cases from the Antigen panel, 124 showed co-infection (56.36%). A comparative analysis of co-infection positivity rates for IVA, IVB, ADV, RSV with MP using two methods reveals statistically significant differences (Table 4). The Antigen Test identified 30 patients (24.19%) with mixed infections involving three or more pathogens. Of these, 4 patients (3.23%) tested positive for four pathogens, while 3 (2.42%) tested positive for all pathogens. No cases of triple or higher co-infections were detected using the Nucleic Acid Test.

Fig. 6.

a MP co-infection rates in the antigen test. b MP co-infection rates in the nucleic test. The antigen test, the respiratory pathogen antigen test; the nucleic test, the respiratory pathogen nucleic acid test; MP, Mycoplasma pneumoniae; IVA, Influenza A virus; PIV3, parainfluenza virus types 3; COV, the severe acute respiratory syndrome coronavirus 2; RSV, respiratory syncytial virus; CP, Chlamydia pneumoniae; ADV, adenovirus; IVB, Influenza B viruses; PIV1, parainfluenza virus types 1

Table 4.

Comparison of co-infection rate in the nucleic acid test and the antigen test

Co-infection pathogens	Nucleic Acid Test Cases(%)	Antigen Test Cases(%)	χ2 test
			χ2 value	P value
IVA	16(3.89%)	73(33.18%)	101.464	< 0.001
IVB	3(0.73%)	11(5.00%)	10.156	0.001
ADV	3(0.73%)	68(30.91%)	123.49	< 0.001
RSV	5(1.22%)	12(5.45%)	13.783	< 0.001
Total	27(6.57%)	124(56.36%)	195.18	< 0.001

Comparison of clinical laboratory data among MP-COVID-19 co-infection, MP-only infection, and healthy controls

The initial CBC + CRP, biochemistry, and urinalysis results were collected from seven patients co-infected with MP and COVID-19 (double positive group, group 3). In a 1:2 ratio, 14 MP-positive patients of the same gender and age (age ± 1) (single positive group, group 2) and healthy individuals (control group, group 1) were matched. As shown in Table S1, in the CBC + CRP tests, the main differences were observed in leukocytes and CRP. The CRP values in positive groups (MP single positive group and MP-Covid-19 double positive group) were significantly higher than in the control group. The single positive group had higher neutrophil count, monocyte count, NLR, and lower lymphocyte count, basophil count compared to the control group. In the biochemical tests (Table S2), the positive groups had lower levels than the control group in the following parameters: albumin, prealbumin, cholinesterase, α-L-fucosidase, alkaline phosphatase, calcium, phosphorus, high-density lipoprotein cholesterol, apolipoprotein A1, and total amylase. The total bile acid, APOB/APOA, and lactate dehydrogenase levels were higher in positive groups compared to the control group. The glucose level in the double positive group was significantly higher than that in the control group. The single positive group had significantly lower levels of total bilirubin, direct bilirubin, urea, potassium, and sodium, and higher levels of magnesium compared to the control group. As illustrated in Table S3, in the urinalysis, albumin was the only indicator showing a significant difference between the double positive and single positive groups. Additionally, the double positive group had significantly higher levels of urinary protein, urinary albumin, and the ratio of urinary albumin to creatinine compared to the control group.

Discussion

MP outbreaks typically occur more frequently during the summer, fall, and winter seasons [2, 8, 9], with seasonal variations dependent on the circumstances each year [10]. In this study, the number of positive MP cases gradually increased in the latter half of the year, peaking in July with a positivity rate of 10.09%, and reaching its highest count in November. Among the 751 MP-positive cases in our research, the gender ratio was 55.26% Male and 44.74% female, fluctuating within the 50 ± 10% range in other areas of china [8, 11, 12], which may primarily be due to the local school-age gender distribution. Some studies exhibit that patients are concentrated in infants and young children [11, 13]; some researches demonstrate that school aged children and adolescents are the main targets of infection [8, 14, 15]. In this article, MP primarily affected children and adolescents under 15, particularly school-aged children between 5 and 10 years old. MP commonly attacks the lower respiratory tract, rashes and vasculitis are occasionally seen [16]. Children, who are more susceptible to initial MP infection might due to weaker immunity compared to adults, often contract the illness at younger ages. Close-contact environments like kindergartens and schools facilitate MP transmission, leading to outbreaks among school-aged children.

According to global testing data, the incidence of MP was 8.61% before COVID-19 (2017–2020) and dropped to 1.69% [17] post-COVID-19 (2020–2021) due to non-pharmaceutical interventions. This demonstrates that non-pharmaceutical measures such as wearing masks, maintaining social distancing, and practicing good hand hygiene can effectively disrupt MP transmission and help curb its spread. In our study, the positive cases and positivity rates of Antigen Test and MPCP Test significantly increased after easing COVID-19 control measures, especially in outpatient and emergency departments, indicating the widespread resurgence of MP in the population. Globally, MP has undergone a resurgence following an extended period of minimal infection. Although China’s re-emergence aligns more tightly with the relaxation of COVID-19 mitigation measures, it’s crucial to note that MP incidence was similarly low and re-emerged roughly concurrently (in 2023) in areas where mitigation measures had been Lifted for a period spanning over 1–2 years. During this period, less restrictive non-pharmaceutical interventions (NPIs) like Personal Protective Equipment (PPE) and social distancing may have partially curbed MP transmission. MP’s slow growth (6 h) and long incubation (1–3 weeks) could also contribute to its slower establishment in populations.

In this study, significant differences were observed in the categories of patients tested across the three different packages. Antigen Test had the highest proportion of outpatient and emergency cases (92.96%), while Nucleic Acid Test had the highest proportion of hospitalized cases (90.03%). With the shortest turnaround time and a moderate price, the Antigen Test is suitable for initial screening of outpatient and emergency patients, allowing diagnosis and medication on the same day. Therefore, outpatient and emergency clinicians prefer the Antigen Test, resulting in the highest proportion of outpatient cases. The Nucleic Acid Test covers nine respiratory pathogens and provides results within 24 h. Meanwhile, it is the most expensive one. Considering the complexity of hospital infection scenarios and the higher insurance reimbursement rate compared to outpatients, the clinicians prefer the more accurate nucleic acid testing in inpatient cases. The MPCP Test has the fewest detection targets (MP and CP) and lowest cost, delivers results within 48 h. Its focused scope provides the highest positivity rate (19.11%). Pediatrics (68.79%) and department of respiratory medicine department (17.52%) are the most likely to order this test. During MP outbreaks, clinicians may prescribe empirical medication based on the patient’s clinical symptoms to prevent disease progression without an accurate pathogen test. As a result, this test is ordered the least among the three packages.

MP frequently co-infects with other pathogens. For instance, Zhang et al. [18] provided a co-infection rate of 27.02% with bacteria and viruses in Suzhou during 2011–2016. Shin et al. [4] reported that the co-infection rate with bacteria and viruses reached 88.49% in South Korea from 2017 to 2022. In our study, the total co-infection rate was 21.36% with other 8 pathogens in the Nucleic Acid Test, 6.60% with IVA/B, RSV, and ADV among them. In the Antigen Test, this rate was 56.36%. Interestingly, in Antigen-positive patients, there were 30 individuals (24.19%) with co-infections involving three or more pathogens, which is 0 in the Nucleic Acid Test. This discrepancy May stem from two factors. First, the methodologies differ in sensitivity. PCR is more sensitive than colloidal gold immunoassays, allowing nucleic acid-positive patients to be detected earlier in the disease course. Antigen-positive patients, requiring higher pathogen concentrations, are often in more advanced disease stages and thus have more time to contract additional pathogens before presenting to the clinic. Second, the methodologies vary in specificity. Respiratory antigen testing relies on antigen-antibody reactions, which have a lower specificity than PCR and might yield false positives, leading to inflated co-infection rates. Both methods identified influenza A as the most frequent co-infecting pathogen. The distribution of co-infecting pathogens Likely related to the prevalence of other respiratory pathogens in 2023. Further research could explore the clinical symptoms and treatment differences between patients with MP co-infections and those with isolated MP infections.

In our study, we observed significant differences in C-reactive protein (CRP) levels between the positive groups (single positive and double positive for Mycoplasma pneumoniae [MP] and COVID-19) and the control group, with both positive groups exhibiting notably higher CRP levels (P < 0.001). However, in contrast to a study by Zha et al. [19], which found a lower trend in CRP in the COVID-19 + MP double-positive group compared to the COVID-19-only group, our research did not detect a significant difference between the two positive groups. The discrepancy in CRP levels could be attributed to regional and ethnic differences, variations in CRP values over the course of the disease, and the waning severity of the inflammatory response in later stages of COVID-19 infections compared to the early pandemic period, as noted in studies by Choubey [20] and Gamay [21].

In terms of complete blood count (CBC) changes, our focus was on the leukocyte series, particularly the neutrophil-to-lymphocyte ratio (NLR). Consistent with previous research by Che-Morales JL [22], Cui et al. [23] and Lai et al. [24] we found a significant correlation between NLR and MP infection severity, with higher NLR values indicating more severe infections. Notably, the NLR was significantly higher in both positive groups compared to the control group (P < 0.001), and the double-positive group showed a trend towards a lower NLR compared to the single-positive group. This suggests that the inclusion of COVID-19 may influence the immune response elicited by MP infection. Similarly, monocytes play a crucial role in immune defense and antigen presentation [25], and the monocytes-to-neutrophil ratio (MNR) can also reflect immune status [26]. While the MNR did not show significant differences among the three groups, the double-positive group exhibited a tendency towards lower MNR values, further supporting the notion that COVID-19 alters the immune response to MP.

Among biochemical blood parameters, we observed a significant reduction in high-density lipoprotein cholesterol (HDL-C) levels in both positive groups compared to the control group, with the double-positive group showing even lower levels. Previous studies have linked reductions in HDL-C to pathogen infections, such as HIV-1 [27, 28], and COVID-19 [29]. However, the mechanisms behind HDL-C changes in MP and COVID-19 co-infections remain unclear. Similarly, Zhu et al. [30] identified apolipoprotein A1 (ApoA1) as a strong predictor of COVID-19 severity among numerous biochemical markers. ApoA1, the primary component of HDL-C, was significantly higher in the positive groups compared to the control group, suggesting that changes in HDL-C and related markers may be more indicative of inflammatory responses than lipid metabolic abnormalities.

Additionally, we found elevated total bile acids and lactate dehydrogenase levels in the positive groups, indicating possible liver or myocardial cell damage and partial impairment of liver and heart function. Many liver function indicators were also reduced in the positive groups compared to the control group, potentially due to nutritional deficiency and slower metabolic synthesis caused by reduced food intake [31]. Glucose levels were significantly higher in the double-positive group compared to the control group, possibly related to glucose administration during hospitalization.

Urinalysis revealed that double-positive patients might have faced greater renal stress and damage, with more significant differences in urinary protein, albumin, and the albumin/creatinine ratio compared to normal controls. In Zhao et al.‘s [32] study, proteinuria was observed in patients with MP. In our research, Albumin was the only indicator with a significant difference between the double-positive and single-positive groups (P < 0.05), being higher in the double-positive group.

Regarding patient outcomes, our findings contrast with previous studies reporting high mortality rates among MP + COVID-19 co-infected patients [20, 21, 32, 33]. None of the seven double-positive patients in our study experienced severe illness or adverse outcomes. We hypothesize that the higher incidence of severe illness and death in previous studies might be linked to the greater virulence of COVID-19 during the initial pandemic wave, leading to more severe clinical responses. Alternatively, the lack of severe outcomes in our study could be attributed to the small sample size, which might have introduced bias. Further research is needed to better understand the clinical outcomes of MP and COVID-19 co-infections.

Conclusion

MP entered the epidemic period in July after the COVID-19 pandemic control mesures was released. When facing panel selection, clinicians prioritize speed when testing outpatients but prioritize accuracy for hospitalized patients. IVA is the most common respiratory pathogen co-infected with MP. The laboratory analysis suggests that the inclusion of COVID-19 might alter the immune response to MP. MP positive patients are in a nutritionally deficient state, affecting some liver and cardiac functions. The co-infection group faces a higher likelihood of renal stress and damage. The limited number of co-infection samples might have affected the accuracy of significance analyses. Expanding the sample size in future studies could yield more precise results.

Authors’ contributions

W. Wang and Y. Yu conceived and designed the experiments; R. Zhang performed the experiments; R. Zhang and M. Xu analyzed the data; R. Zhang wrote the paper; R. Zhang, M. Xu, W. Yang, W. Yuan, L. Jiang, W. Wang, Y. Yu revised the manuscript. All authors read and approved the final version of the manuscript.

Funding

This work was supported by the Traditional Chinese Medicine Science and Technology Project of Zhejiang Province, China (Grant number 2024ZL253) to Yan Yu, and the Medicine and Health Science and Technology Project of Zhejiang Province, China (Grant number 2021KY475) to Weizhong Wang.

Data availability

All data generated or analyzed during this study are included in this published article.

Declarations

Ethics approval and consent to participate

Not required.

Competing interests

The authors declare no competing interests.