Epidemiology and Molecular Identification of Mycoplasma pneumoniae associated respiratory tract, Zhejiang province, China, 2008-2017

Mycoplasma pneumoniae is a common cause of respiratory infections in humans. The aim of this study was to investigate the infection of Mycoplasma pneumoniae (MP) in patients with acute respiratory tract infections in Zhejiang Province from 2008 to 2017, and to provide evidence for the early diagnosis and prevention of MP pneumonia. MP-DNA was detected in nasopharyngeal swabs of patients with acute respiratory tract infection by real-time fluorescent PCR (Taqman probe). Statistical analysis and epidemiological investigation were carried out on the test results.

(211 cases), 20.9% (350 cases), 5.5% (70 cases), 11  Mycoplasma pneumonia (MP) is one of the most important pathogens of respiratory tract infection, it is mainly transmitted by respiratory droplets [1,2] . It belongs to the class of flexible membranes. Mycoplasma is the smallest pathogenic microorganism between bacteria and viruses that can live independently. It has no cell wall and is naturally resistant to antibiotics acting on the cell wall [3] . MP can cause acute, chronic respiratory infections, bronchitis, asthma and other respiratory diseases [4,5] . It can also cause encephalitis, nephritis, myocarditis and other extrapulmonary complications through blood dissemination or immune mechanism, especially in children's health [6,7] . In order to grasp

Specimen sources
Clinical data were collected from the information system of Zhejiang Provincial People's Hospital Inspection Center. This study was approved through the local ethics committee of Zhejiang Provincial People's Hospital. 10296 nasopharyngeal secretion specimens were collected from patients in Zhejiang Provincial People's Hospital who were clinically diagnosed as respiratory tract infections between 2008 and 2017. Among them, 4387 specimens were collected from female patients and 5909 specimens were collected from male patients. The throat swab specimens were placed in a sterile closed tube and stored at -20 °C for testing. The 10296 patients were divided into six age groups: < 18 years old, 18-29 years old, 30-39 years old, 40-49 years old, 50-59 years old and ≥ 60 years old.

The testing of MP
The samples were washed thoroughly by adding 1 ml sterile normal saline to the swab samples and shaking for 30 seconds. The samples were transferred to 1.5 mL centrifuge tube by sterile suction tube. The samples were centrifuged for 10 minutes with 14000 rpm of Eppendorf freezing centrifuge. The supernatant was removed and the precipitate was shaken and mixed with 50 μL DNA extractsolution, and then bathed at 100 ℃ for 10 minutes. The supernatant was centrifuged for 5 minutes with 12000 rpm and the supernatant wasused as a template for PCR detection. Mycoplasma pneumoniae nucleic acid quantitative detection kit (PCR fluorescent probe method) was purchased from Da'an Co., Ltd.(National Instrument Accreditation 20153402024), and operated strictly according to the instructions. Lightcycle 480 was used for amplification reaction. The reaction procedure was preheated at 93 ℃ for 2 minutes, reacted at 93℃ for 5 seconds, and reacted at 57℃ for 45 seconds, with 40 cycles. The sensitivity of the kit was 1.0×10 4 copies and the linear range was 1.0×10 4~ 1.0×10 8 copies. If the amplification curve is Stype and the CT value < 40, the sample is positive for Mycoplasma pneumoniae (+); if the CT is blank or the growth curve is not S-type, it is negative for Mycoplasma pneumoniae (< 500 copies).

Statistical analysis
Categorical variables were described with counts and percentages. The χ2 test was used in comparisons of categorical variables. Statistical analyses were performed with SPSS 17.0 software. P value < 0.05 was considered significant.

MP mainly transmitted by droplets with a latency of 2-3 weeks. After infection, it can
adhere to the surface of respiratory tract cells, then extend microtubules into cells to absorb nutrients and damage cell membranes, and then release nuclease, hydrogen peroxide and other metabolic products to cause cell lysis and swelling and necrosis of epithelial cells [8][9][10] . Mycoplasma pneumoniae infection can cause a variety of respiratory diseases, and also can cause a variety of extrapulmonary complications after blood dissemination [6,11] . The pathological changes of mycoplasma pneumonia were mainly interstitial pneumonia, and the incidence of mycoplasma pneumonia was the highest among adolescents. The clinical manifestations and chest X-ray examination of mycoplasma pneumonia are not characteristic [11][12][13] . Diagnosis cannot be made solely by clinical manifestations and chest X-ray examination. It is easy to cause clinical missed diagnosis. To make a definite diagnosis, pathogen detection is needed.
MP lacks cell wall and is highly pleomorphic, and can filter bacteria [3,14] . Therefore, penicillin, cephalosporin and other antibiotics acting on bacterial cell walls have poor therapeutic effect. MP is generally sensitive to macrolides and tetracyclines, so rapid detection of MP is of great value for early clinical diagnosis and antibiotic treatment.
At present, the detection methods for MP mainly include MP separation culture method, immune serum method and PCR method [15,16] . Although isolation and culture of MP is decisive for diagnosis and differential diagnosis, its operation is complicated, timeconsuming and low in detection rate, and often cannot meet clinical needs [17] . Serological methods are often used in clinical practice. However, there are fewer anti-MP-IgM antibodies produced in the early stage of infection, and it usually takes about one week to be detected by serology, which delays the optimal treatment timing [18] . And for patients with immune system development or immune function defects, the detection rate of anti-MP-IgM antibodies is low. Therefore, detection of MP-DNA by real-time fluorescent PCR has become more and more popular.
The positive detection rate of MP varies greatly among different countries and regions, different populations and ages, different years and seasons [1,19,20] . In order to grasp the  8%, 20.9%, 20.9%, 5.5%, 11.7%, 15.2%, 7.8%, 5.9%, 7.8% and 6.0%, respectively. Of 1251 MP-DNA positive patients, 1243 (99.4%) were younger than 18 years old. It indicates that MP infection is mainly concentrated in the age group below 18, suggesting that clinical attention should be paid to the early diagnosis and prevention of MP infection in adolescents.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author Yumei Ge on reasonable request.

Competing interests
The authors declare that they have no conflict of interest. P value: statistical analyses were performed within MP cases.  Table 3. Analyses on MP cases by age groups