Epidemiologic investigation
General situation
The school has a total of 33 classes, including grades 1–5.All students are day students. There are two teaching buildings in the school. The first and second grades are located in the A teaching building, and the remaining grades are located in the B teaching building. At the time of the outbreak, the primary school had 1398 students and 100 teachers and other staffs. A school doctor provides students with medical care.
Outbreak finding/outbreak confirmation
On 7 June, we identified 21 students suffering from a respiratory disease characterized by fever and cough in the Class 8 and 9 of Grade 1. Oropharyngeal (throat) swabs were collected from consenting patients presenting with ARD and specimens were tested using two multiplex quantitative polymerase chain reaction (qPCR) assay for 24 common respiratory pathogens (including 9 viruses, 14 bacteria and Pneumocystis pneumonia) at Shunyi CDC to identify the causative agent. Nine of the 12 (75%) specimens were positive for M. pneumonia. We confirmed a M. pneumoniae outbreak in that school according to epidemiological and laboratory data.
Case definition and finding
To investigate the outbreak, the school absenteeism records were reviewed, students, teachers and other staffs in the school were interviewed, beginning June 8, 2018, to find cases of acute respiratory disease among students. The outpatient and inpatient records were reviewed and doctors in the hospitals were interviewed. The information regarding demographics, the process of illness, signs and symptoms, underlying conditions, clinical treatments, and extracurricular custody was collected. Retrospective record review was performed to identify cases diagnosed as early as May 1, 2018.
We defined probable cases as students, teachers and other staffs in the school developed fever (T ≥ 37.5 °C) with cough or sore throat; or a diagnosis of pneumonia during May 1–June 31, 2018. Confirmed cases were probable cases with M. pneumoniae detected in oropharyngeal swabs by qPCR.
Specimen collection and nucleic acid extraction
Oropharyngeal (OP) swabs were obtained from the students identified as probable cases and their close contacts who agreed to testing. OP swabs were placed in 2 ml of universal transport medium (UTM) and transported at 4 °C to the laboratory of the Shunyi CDC. Total nucleic acid (TNA) was extracted from 200 μl of UTM from each swab specimen and eluted into 100 μl using a MagNA Pure compact total nucleic acid isolation kit (Roche Applied Science) in accordance with the manufacturer instructions.
Detection and genotyping
All patient specimens were screened using two multiplex combined qPCR detection kits for Respiratory viruses and bacteria respectively (number product code: CN12–33 and CN13–3, Uninovo Biological Technology Co. Ltd., Jiangsu, China) in order to identify the etiology of the outbreak. The kit for respiratory bacteria were specific for 15 respiratory pathogens, including: acinetobacter baumannii (Ab), chlamydia pneumoniae(C. pneumoniae), escherichia coli (E. coli), haemophilus influenza (Hi), Klebsiella pneumoniae (KP), Legionella pneumophila (Lp), moraxella catarrhalis (MC), mycoplasma pneumoniae (M. pneumoniae), Mycobacterium tuberculosis (Mtb), Staphylococcus aureus (S.aureus), streptococcus pneumoniae (S. pneumoniae), Streptococcus pyogenes (SP), stenotrophomonas maltophilia (SMA), pseudomonas aeruginosa (PAE), and pneumocystis jiroveci pneumonia (PCP). The kit for respiratory viruses were specific for 9 respiratory viruses, including: human parainfluenza virus types 1 to 4 (HPIV), adenovirus (ADV), metapneumovirus (MPV), human coronavirus types OC43/NL63/229E/HKU1 (HCOV), respiratory syncytial virus (RSV), human rhinovirus (HRV), influenza virus types A/B/C (FLU), human enterovirus (HEV), and human bocavirus (HBOV). The multiplex qPCR was performed by previously described methods [20]. A duplex qPCR assay was used for P1 genotyping of M. pneumonia, and primers and probes were as previously described [21].
Detection of macrolide resistance at the gene level
Point mutations associated with resistance in domain V of 23S rRNA were detected. Genomic DNA of all M. pneumoniae was extracted using a QIAamp DNA minikit (Qiagen, Hilden Germany). The extracts were distributed into aliquots and saved at-20 °C. A forward primer extending from position 1758 to position 1775 (GCAGTGAAGAACGAGGGG) and a verse primer extending from position 2684 to position 2664 (GTCCTCGCTTCGGTCCTCTCG) in the sequence of M. pneumoniae 23S rRNA were used to amplify the domain V region of the 23S rRNA gene by PCR methods, as previously described [22]. The amplification products were sequenced by the Invitrogen Life Technologies (Shanghai, China).
Statistical analysis
All data were analyzed with SPSS 25.0 software (IBM, USA). Categorical variables were described with counts and percentages. Differences between medians were tested by the Mann-Whitney U test, and the Chi-square or Fisher’s exact test was used to compare categorical variables. A P value of less than 0.05 was considered statistically significant.