Antibodies against atypical pathogens and respiratory viruses detected by Pneumoslide IgM test in adults with community‐acquired pneumonia in Guangzhou City

Abstract Background To detect the serum antibodies against respiratory viruses and atypical pathogens in adults with community‐acquired pneumonia (CAP) in Guangzhou City (Guangdong province, China). Methods A retrospective study was carried out with samples from 685 adults who were admitted with CAP and 108 non‐CAP control patients. Atypical pathogens and respiratory viruses in serum were detected using the Pneumoslide IgM test from Vircell, Spain. All patients were divided into 6 groups according to age: 18‐24, 25‐44, 45‐59, 60‐74, 75‐89, and >90. Results The total positive rate of CAP was 35.4%, which was highest in the 18‐24 age group (P < .05). The highest positive rate, 17.11%, was observed for Mycoplasma pneumoniae (MP). The mean age of MP‐infected patients was higher than that of the controls (P < .05). The positive rates for influenza B (INFB), Legionella pneumophila (LP1), Coxiella burnetii (COX), influenza A (INFA), parainfluenza virus (PIV), respiratory syncytial virus (RSV), Chlamydophila pneumoniae (CP), and adenovirus (ADV) were 5.56%, 3.07%, 2.63%, 2.34%, 1.90%, 1.61, 0.88%, and 0.29%, respectively. There were 4.37% of patients with CAP having multiple infections. The main symptoms observed in the 685 CAP patients were cough and sputum production, in 78.4% and 67.4%. Fever was followed by 54% of CAP patients. Dyspnea (39.1%), anorexia (36.8%), increased thirst (26.7%), chills (18.7), headache (14.6%), and nausea (13.1%) were also frequently observed in the CAP patients. Conclusions MP infection was the most common in adult CAP patients in Guangzhou City with the highest positive rate in the 18‐24 age groups.


| INTRODUC TI ON
Community-acquired pneumonia (CAP) is a common infectious disease worldwide, with a mortality rate of 2%-14%. [1][2][3][4][5] Recently, it has been found that the importance of atypical bacterial pathogens and viruses in CAP has been underestimated. 6,7 Previous study has shown that some of CAP were caused by atypical bacterial pathogens and viruses. 8 Jain et al studied 3634 American adults with CAP and found that the most common pathogens were human rhinovirus (in 9% of patients), influenza virus (in 6%), and Streptococcus pneumoniae (in 5%). Chen et al studied 1204 children with pneumonia and found that MP was the most dominant pathogen, followed by influenza B (INFB), parainfluenza virus (PIVs), and respiratory syncytial virus (RSV). 9 Virus, as a cause of CAP, is more common in children than in adults. [10][11][12] However, the study of its importance in adults is relatively insufficient. 13 The assessments of the prevalence of viral infection in adults with CAP based on a large study population in China have only been performed in Beijing, Shanghai, and Jinan. [14][15][16] With the understanding of atypical pathogens, 6,7 evaluations of their role in lower respiratory tract infections have been gradually increasing.
However, reports of viruses and atypical pathogens in adults with CAP remain scarce. [17][18][19] Currently, the greatest challenge in the diagnosis and treatment of CAP in China is the lack of etiological diagnosis in everyday clinical practice. 20,21 The available methods for etiological diagnosis of infection are serology, culture, and PCR. 22

| Patients
This study was reviewed and approved by the ethics committee of Guangdong Provincial Hospital of Chinese Medicine. Because this was a retrospective study of medical record data with no patient contact and no collection of personal data, the study was exempt

| Sample collection
A total of 3 mL of venous blood were drawn from each patient within 24 hours after admission. The specimens were sent to the clinical laboratory immediately and were centrifuged at 11268g for 20 minutes at 4°C. The serum was obtained and stored at −20°C until assayed with the Pneumoslide IgM test.
Then, they were added to every well containing the Pneumoslide IgM slide, respectively. After incubating for 90 minutes at 37°C, the slide was washed twice with PBS and dried. The fluorescent secondary antibody was added and incubated at 37°C for 30 minutes.

| Statistical analysis
Statistical analysis was performed using the SPSS 17.0 software program (SPSS). Quantitative data were presented as the means ± standard deviation (SD). The categorical variables were expressed as frequencies and percentages and were compared using chi-square or Fisher's exact test. Differences for the pathogen detection rates in the various groups were examined using the chi-square test. The positive rates in different age groups were compared using the chisquare test. Comparisons of the mean age between adults with the three most prominent pathogens and controls were performed by analysis of variance (ANOVA). P < .05 was considered to be statistically significant.

| Demographics
From September 2014 to October 2015, 685 hospitalized CAP adult patients and 108 non-CAP control patients were included in the study. The demographic characteristics of the CAP patients were shown in Table 1. There was no significant difference in gender and comorbidities (liver disease, cerebrovascular disease, hypertension, and diabetes mellitus) between the two groups. The age of patients with CAP was higher than that of control patients (68.77 ± 18.56 vs 60.9 ± 20.53) (P < .01). The percentage of kidney disease in CAP patients was significantly higher than that in control patients (18.8% vs 10.2%) (P < .05).

| Breakdown of the detected pathogens
The results were shown in Table 2. Among the 685 patients, the total positive rate was 35.4%. A positive rate of 17.11% was observed for MP, which was the highest among the nine examined pathogens. The next most commonly detected pathogen was INFB (5.56%). The detection rates of LP1, COX, INFA, PIVs, RSV, CP, and ADV were 3.07%, 2.63%, 2.34%, 1.90%, 1.61%, 0.88%, and 0.29%, respectively. The positive rate of LP1 in CAP patients was higher than that of controls (3.07% vs 0%). The positive rates of other pathogens were similar between the two groups (P > .05).

| Mixed infection modes of the pathogens
There were 30 patients in which two or more pathogens were detected, representing 4.37% of the samples, and the modes of mixed infection were complex (Table 3). Among the specimens shown infection with two pathogens, the MP + PIV mixed infection was the most common, with 5 cases. The next mixed infections were MP + INFA, MP + INFB, and MP + LP1, with 3 cases in each. In addition, 5 cases of co-infections with three pathogens were also been observed in Table 3.

| The positive rates of the pathogens isolated from different age groups
Overall, the positive rates for the 9 pathogens were 86.7% in the (

| Clinical characteristics and blood test values of CAP patients
The patients' clinical characteristics were summarized in  In addition, we found that the age of patients with CAP was higher than that of controls. We speculated that the difference in age between patients with CAP and individuals with non-CAP may be attributed to the increasing comorbidities and decreasing immunity with age. It indicated that older patients with atypical pathogens and respiratory virus infections may be more susceptible to CAP than younger patients.

| D ISCUSS I ON
In studies from China and other Asian countries, MP was the most common atypical pathogen detected in patients with CAP. 15,22,26,27 We detected MP in all age groups, and the results showed that patients with MP were older than controls, and there was a greater frequency of MP in the (18-24) age groups. Those finding suggested that MP was an important CAP-related pathogen in the (18-24) age groups in patients in Guangzhou.
Viruses were detected in approximately one-third of patients, which was in agreement with the 13%-56% range reported in previous studies. 5,8,28 In agreement with these studies, our study revealed that the detection rate of viruses was not high in adult patients with CAP, which may be because the total number of an- study. 31,32 Paranhos-Baccala et al reported that according to clinical research, co-infections were more severe than single infections. 33 The proportions of mixed infections reported in adults with CAP range from 5.1% to 10.5% in China. 15,16,26 This is due to diverse pathogens, test methods, and study designs. In our study, 4.37%    Note: Data in symptoms were expressed as n (%). Data in blood test were expressed as means ± SD.
*ANOVA was used to compare the differences for quantitative data, and chi-square or Fisher's exact test was used to compare the differences for categorical variables.
clinically insignificant unless combined with the identification of atypical pathogens or respiratory viruses.

| CON CLUS IONS
In conclusion, our results suggested that MP was the most common causative pathogen in adult patients with CAP and had the highest positive rate in the (18-24) age groups. Compared with the controls, those with pre-existing kidney disease were at a higher risk of developing subsequent CAP. The patients with CAP characterized by cough, expectoration, and fever exhibited obvious systemic and respiratory symptoms. The MP infection in patients with 18-24 years old should be paid more attention to in further study. Furthermore, the study on the etiology and clinical characteristics of adults with CAP in Guangzhou will not only help to determine the etiology of CAP in this area, but also assist in improving the levels of disease diagnosis, treatment, and management in different age groups.

E TH I C A L A PPROVA L A N D CO N S E NT TO PA RTI CI PATE
This study was reviewed and approved by the ethics committee of Guangdong Provincial Hospital of Chinese Medicine. Because this was a retrospective study of medical record data with no patient contact and no collection of personal data, the study was exempt from obtaining informed consent.

CO N S E NT FO R PU B LI C ATI O N
Not applicable.

DATA AVA I L A B I L I T Y S TAT E M E N T
Not applicable.