Clinical study on the distribution of acute respiratory pathogens after the unsealing of COVID-19

doi:10.21203/rs.3.rs-4834028/v1

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Research Article

Clinical study on the distribution of acute respiratory pathogens after the unsealing of COVID-19

https://doi.org/10.21203/rs.3.rs-4834028/v1

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Objective

To analyze the distribution characteristics of pathogen infection in patients with acute respiratory tract infection in our hospital, in order to understand the etiology and epidemiological traits of respiratory pathogen infection in Qingyuan area of Guangdong Province after the epidemic, and provide reference for clinical diagnosis and treatment.

Methods

Data of multiple RT-PCR and capillary electrophoresis fragment analysis were collected from hospitalized children aged 0-6 years who were diagnosed and treated for acute respiratory tract infection in Qingyuan People's Hospital from December 2022 to December 2023, and the results were statistically analyzed by SPSS 17.0.

Results

The total positive rate of acute respiratory pathogen infection was 65.49% (2306/3521), and the highest positive rate was HRV (21.81%), followed by HRSV (14.23%, 501/3521) and InfA (8.26%, 291/3521). The positive rate of at least one pathogen in male children was 73.77% (1 640/2223), as in female children was 72.88% (946/1298). The positive rate of respiratory pathogens in preschool children group was 71.47%(987/1381),which was higher than in the early childhood group (694/985,70.46%) and in the babyhood group(625/1155,54.11%), and the difference was statistically significant(c²=98.760，P<0.001).The positive rate of respiratory pathogens was highest in April(80.71%,251/311),then were in December 2023 (78.66%,199/253) and in November(76.92%,210/273), and the difference was statistically significant(c²=423.944，P<0.05).

Conclusion

The main pathogens of acute respiratory tract infection in our hospital are HRV, HRSV and InfA. The positive rate of males is higher than that of females, and the positive rate of preschool children is higher. There is a high incidence of infection during the winter and spring changes.

Acute respiratory tract infection

Epidemiology

Multiplex-PCR

Influenza virus

Respiratory syncytial virus

Acute respiratory infections (ARI) are infectious diseases caused by one or more microorganisms such as viruses, bacteria, mycoplasma and chlamydia that invade the respiratory tract. They include the common cold, otitis media, acute bronchiolitis, pharyngitis and pneumonia. It is one of the leading causes of morbidity and mortality worldwide, especially among children ^[1]. Common pathogens causing ARI include viruses, bacteria and fungi, among which viruses are more common. Respiratory syncytial virus, parainfluenza virus, influenza virus and adenovirus are the main respiratory viruses infected by children ^[2]. There are strict diagnostic criteria for the diagnosis of ARI, but empiric treatment is still common. The clinical symptoms of respiratory infections are often similar, and it is difficult to distinguish viral infections from bacterial infections based on symptoms ^[3]. Therefore, a better understanding of the epidemiological and clinical characteristics that lead to ARI is critical to the successful implementation of prevention, control and treatment strategies.

The COVID-19 pandemic is having a huge impact on global health. To reduce the previously unknown transmission of SARS-CoV-2, preventive measures such as containment, keeping a safe distance, using masks and raising awareness of hand hygiene have been widely adopted ^[4]. These non-pharmaceutical interventions (NPI), also known as public health measures or community mitigation strategies, are thought to be effective against the spread of other respiratory infections as well ^[5]. Within two weeks of the declaration of the COVID-19 epidemic in the United States in March 2020, influenza incidence in the United States suddenly declined ^[6]. Subsequently, global surveillance studies also confirmed a significant reduction in the occurrence of influenza ^[5–8]. Several retrospective studies have reported similar findings for other respiratory pathogens, such as syncytial virus, parainfluenza virus, and metapneumovirus ^[8–11]. However, after the cancellation of NPI, respiratory syncytial virus surged in the summer, while parainfluenza virus and coronavirus are returning to pre-COVID-19 pandemic levels ^[12].

At present, traditional immunological techniques are still the main detection techniques for respiratory pathogens in clinical practice, but its methodology has shortcomings such as low positive detection rate, the possibility of false positives, and the inability to distinguish between existing infections and past infections, thus affecting subsequent treatment. The multiple RT-PCR-capillary electrophoresis fragment analysis technology has the advantages of simultaneous detection of 13 kinds of respiratory pathogen nucleic acids through 1 specimen 1 time, triple quality control exclusion of specimens and false negatives caused by experimental process, and the use of UDG enzyme to prevent false positives caused by sample contamination, therefore, the positive detection rate of acute respiratory infection pathogens is high, 13 pathogens can be detected simultaneously ,including Influenza A virus (InfA, including subtypes 2009H1N1 and H3), Influenza B virus (InfB), Human respiratory syncytial virus (HRSV), Human parainfluenza virus (Human parainfluenza virus) HPIV, Human Metapneumovirus (HMPV), Human adenovirus (HADV), Human rhinovirus (HRV), Boca virus (Bocaparvovirus) Boca, Human coronavirus (HCoV), Mycoplasma pneumoniae (Mp) and Chlamydia (Ch). From the perspective of health economics, multiple PCR detection has strong specificity and high sensitivity, and can detect 13 pathogens at the same time, greatly shortening the time of such results, making the diagnosis more accurate, thus enabling clinicians to use more precise drugs, avoid the abuse of antibiotics, effectively shorten the hospitalization period of patients, improve the turnover rate of hospital beds, and reduce the hospitalization cost of patients. Savings in medical costs ^[13].

This study retrospectively analyzed the nucleic acids of 13 respiratory pathogens in 3 521 hospitalized children with acute respiratory infection treated in Qingyuan People's Hospital after after NPI were lifted, and analyzed the detection results. It is expected to help clinical early diagnosis and early treatment, precision medicine, so as to prevent antibiotic abuse, shorten the hospitalization period of patients, improve the bed turnover rate, and reduce the waste of medical resources.

2.1 Specimen collection

A total of 3 521 hospitalized children with acute respiratory tract infection were included in the study from December 2022 to December 2023 in Qingyuan People's Hospital, of those2 223 males and 1 298 females ranged in age from 0 to 6 years old. The sample type was nasopharyngeal swab or sputum.

2.2 Methods

2.2.1 The principle of method Specific primers were designed for 13 respiratory pathogens. Multiple PCR technology was used to amplify multiple targets in a reaction tube at the same time, and then capillary electrophoresis was used to distinguish specific pathogens according to different lengths of amplified fragments.

2.2.2 Instruments and reagents The amplification instrument was purchased from Hangzhou Langi Scientific Instrument Co., LTD., the first generation sequencer ABI3500DX and supporting reagents such as DNA molecular length standard-500 (size-500), formamide solution (HiDi), separation glue, anode and cathode buffer were purchased from ABI Company. Nucleic acid extraction and purification reagent and multiple detection kit of 13 kinds of respiratory pathogens were purchased from Ningbo Health Gene Technologies Ltd..

2.2.3 Sample collection The sample type was nasopharyngeal swab or sputum. Immediately after collection, the sample should be placed vertically in the ice bag, not tilted or overturned, and immediately submitted for inspection. If it cannot be detected immediately, the throat swab sample should be stored in a -20 ℃ refrigerator for no more than 3 days.

2.2.4 Pathogen DNA / RNA extraction The pathogen nucleic acid was extracted by magnetic bead method according to the instructions. After the extraction process, the nucleic acid in the last well was collected and needed to be amplified.

2.2.5 RT-PCR reaction 5 μL of the extracted nucleic acid was added to the 15 μL reverse recording system (14 μL premix + 1 μLRT-PCR enzyme solution). The corresponding amplification procedure was set in the PCR instrument according to the reagent instructions, and then the amplification was performed for 2 h 28 min, and the amplification product was stored for 4℃.

2.2.6 Capillary electrophoresis fragment analysis 9 μL AB Master Mix (Hi-Di ™ Formamide containing 2.5% SIZE-500) and 1 μL PCR product were added to the 96-well plate, and the corresponding specimen number and analysis method were set in the ABI-3500DX gene analyzer. After the detection is complete, the data is imported into the GENEMAPPER software for analysis, and the final detection result is obtained.

2.3 Statistical method

Statistical analysis was performed using the SPSS17.0 software. The data were processed by using the Excel. Count data were expressed as n (%), and differences were compared using the χ² test, and P <0.05 was considered as statistically significant.

3.1 Demographic Characteristics

A total of 3521 hospitalized children diagnosed and treated with acute respiratory infections at Qingyuan People's Hospital from December 2022 to December 2023 were included. Among them, there were 2223 males and 1298 females; 1155 in the infant group (0–1 years old), 985 in the toddler group (1–3 years old), and 1381 cases in the preschool group (3–6 years old) (Table 1).

Table 1

Demographic data of the hospitalized children admitted with ARIs
Characteristics	n	Constituent ratio
Age group (years)
0 ~ 1	962	35.79%
1 ~ 3	785	29.20%
3 ~ 6	941	35.01%
Gender
male	1708	63.54%
female	980	36.46%

3.2 Overall Prevalence of Respiratory Pathogen Infection

The results of 3 521 cases of acute respiratory infection were statistically analyzed. 2 306 cases were found to be positive for at least one pathogen, with a total positive rate of 65.49%. The highest positive detection rate was HRV, with a positive rate of 21.79% (768 / 3 521), followed by HRSV with 14.21% (501 / 3 521) and InfA (8.26%, 291 / 3 521) (Table 2).

Table 2

Positive rates of 13 respiratory pathogens from December 2022 to December 2023
Respiratory tract pathogen	Positive samples	Positive rate
HRV	768	21.79%
HRSV	501	14.21%
InfA	291	8.26%
HPIV	276	7.83%
Mp	262	7.43%
HMPV	221	6.27%
09H1	202	5.73%
HADV	175	4.96%
H3	74	2.10%
HCOV	68	1.93%
Boca	68	1.93%
InfB	47	1.33%
Ch	24	0.68%

3.3 Gender -related Prevalence of Respiratory Pathogens

Among the 3 521 samples, 1471 of the 2 223 male children were positive for at least one respiratory pathogen (including single infection and mixed infection), with a positive rate of 66.17%; 853 of 1 298 female children were positive for at least one respiratory pathogen(including single infection and mixed infection), with a positive rate of 64.33%. There was no significant difference in the detection rates of 13 respiratory pathogens in different genders (P > 0.05) (Table 3).

Table 3

Positive rates of 13 respiratory pathogens in different genders from December 2022 to December 2023 [n(%)]
Respiratory tract pathogen	male(n = 2 223)	female(n = 1298)	χ²值	P值
InfA	195(8.77)	96(7.4)	2.046	0.153
09H1	135(6.07)	67(5.16)	1.258	0.262
H3	47(2.11)	26(2)	0.050	0.823
InfB	35(1.57)	12(0.92)	2.629	0.105
HPIV	161(7.24)	115(8.86)	2.967	0.085
HRSV	328(14.75)	173(13.33)	1.367	0.242
HADV	119(5.35)	56(4.31)	1.872	0.171
Mp	155(6.97)	105(8.09)	1.495	0.222
Ch	12(0.54)	12(0.92)	1.791	0.181
HRV	500(22.49)	268(20.65)	1.636	0.201
HMPV	129(5.8)	92(7.09)	2.300	0.129
HCOV	43(1.93)	25(1.93)	0.000	0.986
Boca	42(1.89)	26(2)	0.056	0.813

3.4 Age-related Prevalence of Respiratory Pathogens

The 3 521 samples were divided into three age groups to analyze the infection of patients of different ages.Among them, the positive rate of respiratory pathogens in preschool group was the highest (987/1 381,71.47%), followed by the toddler group (694/985, 70.46%), and the infant group was the lowest (625/1155).The detection rate of HRSV was highest in the infant group, with a detection rate of 20.43% (236/1155).The detection rate of HRV was the highest in the infant group and preschool group, with the detection rate of 28.12% (277/985) and 21.80% (301/1381), respectively.Except for HPIV and HCOV, there were statistically significant differences in other pathogens among different age groups (P < 0.05) (Table 4).

Table 4

Positive rates of 13 respiratory pathogens in different age groups from December 2022 to December 2023 [n(%)]
Respiratory tract pathogen	Infant (0–1 years old)	toddler (1–3 years old)	preschool (3–6 years old)	χ²值	P值
InfA	55(4.76)	64(6.5)	172(12.45)ab	54.728	0.000
09H1	42(3.64)	41(4.16)	119(8.62)ab	35.121	0.000
H3	11(0.95)	18(1.83)	44(3.19)ab	15.864	0.000
InfB	8(0.69)	8(0.81)	31(2.24)ab	14.341	0.001
HPIV	103(8.92)	80(8.12)	93(6.73)a	4.303	0.116
HRSV	236(20.43)	149(15.13)a	116(8.4)ab	75.527	0.000
HADV	12(1.04)	42(4.26)a	121(8.76)ab	80.867	0.000
Mp	26(2.25)	58(5.89)a	176(12.74)ab	105.740	0.000
Ch	18(1.56)	4(0.41)a	2(0.14)a	20.100	0.000
HRV	190(16.45)	277(28.12)a	301(21.8)ab	42.465	0.000
HMPV	49(4.24)	67(6.8)a	105(7.6)a	12.718	0.002
HCOV	15(1.3)	21(2.13)	32(2.32)	3.736	0.154
Boca	8(0.69)	40(4.06)a	20(1.45)b	34.645	0.000
Total positives	625 (54.11)	694 (70.46)	987 (71.47)	98.760	0.000
Note:^aP<0.05,compared with the infant group;^bP<0.05,compared with the toddler group.

3.5 Monthly Distribution

According to the statistical analysis by month, the total detection rate of pathogens was the highest in April 2023, 80.71% (251/311),followed by December of the same year, 78.66% (199 / 253) and November,76.92% (210 / 273), and the difference in detection rate in different months was statistically significant (χ² = 423.944, P < 0.05).The detection rate of InfA was the highest in April, 41.48% (129 / 311), followed by March ,24.52% (64 / 261),the detection rate of different months was statistically significant (χ² = 707.091, P < 0.05);The detection rate of HRSV was the highest in May, 38.30% (149 / 389), followed by April, 26.69% (83 / 311),the detection rate of different months was statistically significant (χ² = 360.229, P < 0.05);The detection rate of HRV was the highest in March, 35.63% (93 / 261), followed by November 34.43% (94 / 273), the detection rate of different months was statistically significant ((2 = 160.410, P < 0.05) (Fig. 1).

3.6 Analysis of the Major Pathogens of Community-acquired Pneumonia

In this study, we found that of 3521 clinical diagnosis of ARI children aged 0 to 6 years, 1880 children were diagnosed with community-acquired pneumonia, accounting for 53.39%. Among them, 383 cases (20.37%) were positive for HRSV, 239 cases (12.71%) were positive for Mp, and 87 cases (4.63%) were positive for HADV. The detection rate of the three respiratory pathogens was statistically different (χ² = 1418.00, P < 0.05).

Acute respiratory infections (ARIs) can cause many deaths around the world every year, especially in children. Because children are in the developmental stage, the functions of the respiratory system and immune system are not fully developed, and they are vulnerable to infection by a variety of pathogens, causing acute respiratory diseases, which is a common threat to public safety. Therefore, accurate and rapid identification of pathogens that cause acute respiratory infections is essential for accurate antiviral therapy and prevention of overuse of antibiotics.

A total of 3521 children with acute respiratory tract infection were included in this study, the pathogen positive rate was 65.49%, and the main detection rates were HRV, HRSV and InfA. Min Gao ^[14] et al. found that 14.45% were positive for at least one pathogen.,among them HRSV was the most frequent pathogen, followed by HPIV, HADV,and MP, which was different from this study. The main types of prevalent pathogens are different, which may be related to various reasons such as the types of specimens included, the geographical location of different regions, climate, social factors, and so on.

The children included in this study were more male than female, with a male to female ratio of 1.71:1. It has been reported ^{[14, 15]} that boys are more likely to suffer from respiratory diseases than girls. In this study, there was no statistically significant difference between gender and respiratory pathogen infection, indicating that boys may not be related to susceptibility to respiratory virus infection. However, it has also been reported that boys are more susceptible to HRSV than girls. The relationship between gender and viral infection is complex and could be further studied in the future.

The positive rate of children in different ages is different. In this study, the total positive rate of pathogens in the infant group is significantly lower than that in other groups, while the positive rate in the preschool group is the highest, which is consistent with the report in Chengdu ^[16]. The reason may be that after the epidemic is released, some prevention awareness during the epidemic still persists, and the opportunity for low-month-old babies to go out is far less than the opportunity for preschool children aged 3 to 6. The results of this study showed that the positive detection rate of MP, InfA, HADV and HMPV was the highest in preschool children,while the positive rate of HRSV in the infant group was as high as 20.43%, and the positive rate gradually decreased with the increase of age. Previous studies have also found a similar trend in HRSV infection ^{[14, 17, 18, 19]}, which may be related to the continuous improvement of body immunity.

The positive rate of respiratory pathogens was different in different months. In this study, the positive rate of respiratory pathogens showed a general upward trend with the distribution of each month. Among them, the virus detection rate in January is significantly lower than other months, one possible reason is that the Chinese Lunar New Year occurs in January, many children go home for the New Year as their families, and the population gathering in public places is significantly reduced. This is consistent with previous studies ^[9]. The positive rate reached the highest in April, possibly due to the influenza A virus epidemic in Qingyuan at that time, resulting in 80.71% positive rate of acute respiratory pathogens. The second is December, with a positive rate of 78.66%, which may be greatly related to the worsening of cold in December, the reduction of outdoor activities of many children, and the reduction of the frequency of ventilation by opening windows. That is consistent with findings reported in Spain, the USA ^{[20, 21]}, and Shanghai, China ^[22].Therefore, seasonal prevention is of great significance. In autumn and winter, when it is impossible to avoid school gatherings, hand hygiene awareness during the epidemic period, ventilation, wearing masks and other preventive measures can greatly reduce the risk of acute respiratory infection.

Respiratory viruses are the main pathogens causing community-acquired pneumonia in infants and preschool children ^[23–25]. In this study, 53.39% of the diseases caused by acute respiratory infection were diagnosed as community-acquired pneumonia. Among them, the positive rate of children infected with HRSV was 20.37%, which was higher than that of children infected with MP and HADV. Because the mucosal barrier defense system of children aged 0 to 6 years is still underdeveloped, they are more susceptible to respiratory syncytial virus infection once they come into contact with secretions containing the virus. In view of the high incidence of HRSV in young children, it is recommended that infants and young children should be the focus of prevention work, such as children with high risk factors such as immune deficiency can be vaccinated as early as possible.

In summary, different pathogens have different morbidity characteristics in different regions, different ages and different months. By analyzing the detection and epidemic characteristics of pathogens in children with acute respiratory infection, it is conducive to early clinical diagnosis and treatment, precision medicine and prevention of antibiotic abuse. At the same time, it also helps to guide the disease prevention and control in the region and reduce the waste of medical resources.

Ethics approval and consent to participate

This was a retrospective, observational study. This study has been approved by the Ethics Committee of The Affliated Qingyuan Hospital of Guangzhou Medical University (Clinical Trial Number : MR-44-23-045882; Ethics approval number: IRB-2023-163).

Consent for publication

Not applicable.

Availability of data and material

The data and materials supporting the conclusions of the study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This study was supported by fund from the Guangdong Zhong Nanshan Medical Foundation research Fund(ZNSXS-20240010)

Authors' contributions

YWG,CB ,TG designed the research;CB,YJL,CC,QHX, ZMF performed the research and acquired data;CB,TG, LYM analyzed the data;CB,TG and YWG wrote the paper; XLQ and YWG revised the manuscript; YWG approved the final version to be published. All authors read and approved the final manuscript.

Acknowledgements

We gratefully thank to all patients who participated in this study, and thank our colleagues at the Affliated Qingyuan Hospital(Qingyuan People's Hospital), Guangzhou Medical University, for their support during the study.

Authors' information

¹The Affliated Qingyuan Hospital(Qingyuan People's Hospital), Guangzhou Medical University,Qingyuan, Guangdong, China, 511518;² Kingmed School of Laboratory Medicine, Guangzhou Medical University,Guangzhou,Guangdong, China, 510000;

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No competing interests reported.

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Clinical study on the distribution of acute respiratory pathogens after the unsealing of COVID-19

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Abstract

Figures

1. Introduction

2. Materials and Methods

3. Results

3.1 Demographic Characteristics

3.2 Overall Prevalence of Respiratory Pathogen Infection

3.3 Gender -related Prevalence of Respiratory Pathogens

3.4 Age-related Prevalence of Respiratory Pathogens

3.5 Monthly Distribution

3.6 Analysis of the Major Pathogens of Community-acquired Pneumonia

4. Discussion

Declarations

References

Additional Declarations

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