The school enrolled Tibetan students from 1985. Length of schooling was 4 years. Most of them came from Tibetan countryside. They boarded in the school. The living condition and the hygiene were good overall. The student building was partitioned by a wall on each floor with one side for female students and another side for male students. There were six students in each room with wash stand, toilet and bathroom. The management of building is very strict. The male students and the female students are not allowed to enter rooms of the other side without permission. Each Tibetan student ate same set of food for each meal at the same time in the same dining hall. The school arranged life guardians for Tibetan students. Life guardians were responsible for supervising and guiding students' diet hygiene, dormitory hygiene and personal hygiene, correcting and guiding students' behavior, life style, enforcing rules and regulations after school hours, writing students' activity summary and weekly report to the lead teacher.

We conducted longitudinal study design to investigate epidemiological features, risk factors and effectiveness of intervention for the cluster of appendicitis, which was divided into three stages: 1. We began this study in August of 2005. Retrospectively, we collected epidemiological data and clinical data prior to August of 2005 in order to explore risk factor; 2. After finding risk factor, we conducted intervention to control the cluster of appendicitis in August of 2005; 3. Since the cluster of acute appendicitis was under control in 2011, we conducted surveillance for acute appendicitis from the beginning of 2012 until July 2018 to monitor whether intervention measures could prevent re-occurrence of cluster of appendicitis. Our study was conducted according to epidemiological steps of a cluster investigation and reported compliant with STROBE (24–26).

The epidemiological data included person and time distributions of patients, exposure history and environmental sanitation at this school, which were obtained by visiting the school and interviewing the patients and Tibetan students. Patients were identified by reviewing hospital records. The clinical data included patient history, physical examination, laboratory tests, pathology examination, which were collected on a data collection form. Routine blood tests were performed before surgery for all the patients.

Patients were defined as those who had typical clinical manifestations and who had surgery (appendectomy) and pathological features of acute appendicitis. All pathological slides from appendectomy were re-examined in Department of Pathology, Zhongnan Hospital of Wuhan University. The study was approved by ethic boards at the designated hospital and Wuhan University School of Medicine. Written informed consent for investigating risk factors was obtained from guardians of each respondent.

In August 2005, 72 students were in school for the summer vacation. We interviewed the students and their life guardians to investigate students' lifestyle and health habit and explore possible causes of the cluster, especially causes of higher incidence in female students. Among them, there were 28 patients who had acute appendicitis. Most students and their life guardians thought that the possible causes of higher incidence in female students were as follows: (1) Female students had a habits of staying in bedrooms, in contrast to male students who like outdoor activity; (2) Female students had a habit of eating snacks and sharing them with each other whereas male students did not; (3) The patients liked staying in bedrooms and eating snacks and sharing them more than healthy students; (4) Most patients had a contact history with their preceding patients before onsets of symptoms; (5) Toilets were built in bedrooms and most students do not have a habit of washing hands before eating snack, before meals and after bowel movement.

According to their answers, we designed questionnaire to investigate the following exposure factors between patients and healthy students: gender, eating snack (one bag per day on average), engaging in outdoor activities (after school 5 days a week on average), contact history (contacting their preceding patients in bedroom or living with them in bedroom or escorting patients to the hospital), health habit (washing hands before eating snack, before meals, and after bowel movement).

Based on the investigational results and our suggestion, the school started intervention measures including regular health education for all students since August of 2005, which was conducted by life guardians, especially when new students entered school every year or new patients occurred. The measures were as follows: 1. Treating potential source of infection: life guardians escort patient to the hospital soon to reduce contact opportunities with healthy students once patient appear. 2. Conducting health education to block possible routes of transmission and protect potential susceptible population: (1) washing hands before meal and after bowel movement; (2) flushing and cleaning toilets after using toilet; (3) avoiding contact with patients and cleaning fomites once patients appear; (4) washing hand and clothes if contacting patients. (5) Limiting eating snacks and increasing outdoor activities.

After intervention, we followed up Tibetan students three times a year, including one visit (in July each year) to school and two telephone calls (in March and November each year) in order to evaluate effectiveness of intervention. The following data were collected: incidence rate, date of onset of new patients, risk factors and intervention against risk factors and so forth. During period of surveillance between 2012 and 2018, we followed up in March and November each year by two telephone calls.

Statistically significant differences in incidence rates between the male and female students were assessed using chi-square tests. Univariate and multivariate logistic regressions were conducted to explore potential risk factors of acute appendicitis. Firth's logistic regression, using a penalized likelihood estimation method, was applied as an appropriate method to deal with small sample size. In multivariate logistic regression, all potential factors were included.

Interrupted time series analysis was applied to explore change in incidence rate before and after health education. An interaction between year and group (0-before health education, 1-after health education) was created for hypothesis test. Durbin-Watson statistics and residual autocorrelations were assessed and a generalized ordinary least square regression with Prais-Winsten estimation was applied to explore whether or not the interaction was statistically significant. A P-value <0.05 was considered statistically significant. SPSS 26.0 was used for statistical analysis.

According to study design, three stages of longitudinal study was showed in Figure 1. Epidemiological steps of the cluster investigation were listed in Table 1. Step 1 to step 8 in Table 1 match stage 1 in Figure 1. Step 9 and step 10 match stage 2 and stage 3, respectively.

Before 2000, there were clusters of tuberculosis, varicella, mumps and so forth. From 1998, the incidence rate of acute appendicitis increased in Tibetan students. After 2000, the patients were admitted to the designated hospital for treatment. From January of 2000 to December of 2010, 973 (474 male and 499 female) Tibetan students were enrolled at this high school. Among them, there were 120 students suffered from acute appendicitis (102 females and 18 males). Patients' age ranged from 12 to 17 years. The mean age (SD) was 14.3 (1.1) years. Person and time distribution of the patients were showed in Table 2. There was a female preponderance (female 20.4%, 102 of 499; male 3.8%, 18 of 474; chi-square = 62.280, P ≤ 0.001). The incidence peak appeared in 2005. In 2003, epidemic of severe acute respiratory syndrome (SARS) occurred in China. Because refusal to visitors, disinfection and personal protection were carried out at this school for prevention of epidemic of SARS, there was only one patient with acute appendicitis. The 4-year cumulative incidence rates in female students enrolled in 2001, 2002, 2003, 2004, 2005, 2006 were 26.8% (11 of 41), 27.1% (13 of 48), 44.7% (21 of 47), 42.4% (14 of 33), 23.1% (9 of 39), and 19.3% (11 of 57), respectively before their graduation. There were no obvious seasonal variations of patient occurrence among the Tibetan students.

A history of mutual contact was found in these patients and the onset time of them is close. Most of the patients were classmates (Table 3) or roommates (Table 4). Because there were large number of patients occurring from 2000 to 2010, we only chose the patients occurring in 2004 to be presented in Table 3. With the exception of case 17, all patients occurred in cluster. In Table 4, contact history recall is reliable as 17 were roommates among 19 patients who had contact history.

The patients' clinical signs and symptoms had features of typical acute appendicitis, whereas axilla temperature, white blood cell counts and neutrophil level were normal among the most patients. Pathologically, the resected appendices exhibited focal or diffuse hemorrhages in mucosa and/or submucosa, and infiltration by eosinophil and by lymphocytes. These features were identical to the features of the clustered patients in Wuhan (23). Among these patients, 1.7% (2 of 120) had acute gangrenous appendicitis, 6.7% (8 of 120) had acute suppurative appendicitis and the rest had acute simple appendicitis.

The investigational result of risk factors for 72 students was shown in Table 4. In univariate analyses, contact history was the only risk factor of acute appendicitis (OR, 5.34; 95% CI: 1.91–14.91; P = 0.001). Gender, eating snack, outdoor activity, and health habit were not associated significantly with risk of acute appendicitis. In multivariate analyses, contact history was also the only risk factor of acute appendicitis (OR, 4.89; 95% CI: 1.67–14.35; P = 0.004).

The reason why eating snack, outdoor activity and health habit were not associated with acute appendicitis is likely due to the fact that the vast majority of patients and healthy students had these factors. Therefore, statistical differences in these factors between patients and healthy students cannot be found. However, once a new student came along, these factors may increase the risk of acute appendicitis. So we also took targeted measures to these factors.

Comparison of results before and after intervention was shown in Tables 2, 5 and Figure 2.

Using generalized ordinary least square regression with Prais-Winsten estimation, the interaction between group and time were significant among total students and female students and not significant among male students.

Before intervention, the incidence rate increased year by year from 2000 and peaked in 2005, with the exception in 2003 when there was a reduction of the incidence rate by the implementation of no visitor policy, disinfection and personal protection due to the SARS epidemic. After intervention from 2005, the incidence rate showed a decreasing trend year by year. No new patients occurred in 2011. Under surveillance from January of 2012 to July of 2018, only four sporadic patients occurred at this school.

In summary, we investigated the cluster of acute appendicitis among Tibetan students at a high school in eastern China. The cluster lasted for 11 years. The incidence rate of acute appendicitis was much higher among female students. Female students' lifestyle and the school's management measures for Tibetan students resulted in higher incidence rate among female students. Contact history with patients was risk factor of appendicitis. The patients occurred in cluster. The possible transmission route may be fecal-oral infection. The cluster of acute appendicitis can be controlled and prevented by interventions targeting risk factors and the route of transmission without knowing the pathogen. The study may also be used for prevention of sporadic patients and be generalized in other populations. Future work will focus on finding new clusters to generalize the study and identify precipitating infectious agents.