Parasitological and histopathological features of appendectomy specimens in Fars Province, southern Iran: a retrospective study

S.M.: conceived, analyzed and interpreted the data and prepared the original draft paper. M.S.: collected the data from the hospital information system database and pathologic analysis of the appendectomy specimens. P.V.: collected the data from the hospital information system database. F.M., M.J.A.A.: conceived, analyzed, and interpreted the data. R.A., F.G.: analyzed and interpreted data. A.T.: conceived and designed the study, review, and editing of the manuscript.

The patient’s data included in our study were fully anonymized by the medical report section of the hospitals before our accession to them, and information collected from the study participants was kept confidential. Informed written consents was obtained from all subjects and/or their legal guardian(s).

This study was approved by the research ethics committee of Shiraz University of Medical Sciences with the ethical code: IR.SUMS.MED.REC.1400.194.

This retrospective study included 7628 surgically removed appendices from clinically diagnosed patients with AA. All the surgical operations were performed at Namazi and Shahid Faghihi hospitals (Sa’adi hospital), the two main teaching hospitals affiliated with Shiraz University of Medical Sciences, from April 2016 to March 2021 (a 5-year period). All appendectomy specimens were routinely submitted for pathological assessment. They were fixed in 10% formalin and cut into one longitudinal and two transverse samples. The sections were stained by the hematoxylin and eosin technique and examined under a light microscope. All pathology reports and recorded demographic data of patients were retrospectively reviewed from the available data in the hospital information system database for the patient’s age and sex, as well as the presence of parasites and the histopathological findings. This work has been reported in line with the PROCESS criteria 24 . Analytical and descriptive statistics were carried out using Statistical Package for the Social Sciences (SPSS Inc.) version 22 software. Data were described using frequencies (%) and 95% CI, or mean and median, as appropriate. χ 2 -test was used for testing the association between demographic variables and histopathological features among the studied population. A P value of less than 0.05 was considered to be statistically significant.

A total of 7628 appendectomy materials were evaluated in the present study. The results of the sociodemographic characteristics of study participants are shown in Table 1 . Of the total participants, 4528 (59.4%, 95% CI: 58.2–60.5) were males, and 3100 (40.6%, 95% CI: 39.5–41.8) were females. The median [interquartile range (IQR] of age was 21 years old 13 – 31 . Participants were divided into seven major age groups of less than or equal to 10, 11–20, 21–30, 31–40, 41–50, 51–60, and greater than 60. The largest age group was 11–20 age range (33.7%, 95% CI: 32.6–34.7). The smallest number of cases was in the greater than 60 group. The majority of the specimens (1779/7628) were taken from April 2016 to March 2017 (23.3%, 95% CI: 22.4–24.3). Demographic features of the cases ( n =7628) The distribution of various histopathological findings of appendicitis is shown in Table 2 . When our cases were evaluated in terms of histopathological diagnoses, 5813 (76.2%), 65 (0.9%), and 1750 (22.9%) were diagnosed as inflamed appendix, neoplasm of the appendix, and others, respectively (Table 2 ). Distribution of various histopathological findings of appendicitis ( n =7628) The distribution of patients according to histopathological features in appendicitis based on demographic features including sex, age, and year is shown in Table 3 . The inflamed appendix was higher in males (64%), while other histopathological features were higher in females (55.5%). There was a statistically significant difference between histopathological findings and sex ( P <0.001). Inflamed appendix and other histopathological features were most prevalent among participants aged 11–20 years old (32.3 and 39.1%, respectively), and the difference between the age and histopathological features was significant ( P <0.001). In contrast, most cases of neoplasm of the appendix were greater than 30 (70.8%). Distribution of histopathological features in appendicitis based on the demographic features ( n =7628) Overall, E. vermicularis was only the parasite identified in 20 appendectomy specimens (Fig. 1 ). The mean and median ages of patients with E. vermicularis infection was 18.1±12.8 and 13.5 (IQR=14.5) years old, respectively; 65% (95% CI: 40.8–84.6) of them were female and 35% (95% CI: 15.4–59.2) male. However, this difference was not statistically significant between the sexes of patients with E. vermicularis infection ( P =0.18). Out of 20 cases with E. vermicularis infection, 14 cases (70%, 95% CI: 45.7–88.1) were younger than 20 years old (Table 4 ). However, this difference was not statistically significant ( P =0.074). Pathologic analysis of the appendectomy specimens with E. vermicularis indicated follicular hyperplasia in 11 (55%), AA in six (30%), normal-structured appendix in two (10%), and gangrenous appendicitis in one (5%) (Table 4 ). In all cases of parasitic infection, either the parasite or its eggs were identified in the lumen of the appendix. (A) Enterobius vermicularis worms in the appendix. (B) Triangular alae of E. vermicularis. (C) Section of female worm of E. vermicularis containing eggs in uterus, hematoxylin and eosin staining. Demographic data and histopathological findings of the patients with Enterobius vermicularis infection of the appendix

This research was financially supported by the office of the Vice-chancellor for research at Shiraz University of Medical Sciences with grant No: 23510.

1. Name of the registry: NA. 2. Unique identifying number or registration ID: NA. 3. Hyperlink to your specific registration (must be publicly accessible and will be checked): NA.

Dr Aref Teimouri, Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. E-mail: [email protected]

In conclusion, the current study indicated that E. vermicularis is one of the most common infectious agents that could be found in the appendix and may increase the risk of appendicitis. Although sanitation and health infrastructures have diminished the risk of human parasitic infections in several countries, the risk of parasite-associated complications is lurking. Therefore, in terms of appendicitis, clinicians and pathologists must be aware of the possibility of the parasitic agents, especially, E. vermicularis to treat and manage the patients sufficiently. Moreover, it seems that an early diagnosis and precise treatment of E. vermicularis infection are conducive to preventing the occurrence of appendicitis, recurrences, and other ectopic infections.

Appendicitis is the most common surgical emergency globally, occurring among people of different ages and sexes 25 . Although the primary causes of appendicitis are questionable, the role of gastrointestinal parasites must not be overlooked 26 . Pinworms are the most common parasitic worms capable of resulting in appendicitis, which first was recovered from the appendix by Fabrius in 1634 27 , 28 . Based on the results of the current study, E. vermicularis was the only reported parasite in the patient’s undergone appendectomy and was reported in 20 of 7628 appendectomy specimens (0.26%) in a 5-year period. The findings of our study are identical to those of a similar study conducted in Qom Province, Iran, with 0.22% appendicitis associated with pinworm infection 29 . The results of a study conducted in Greece showed that E. vermicularis was identified in only in 0.65% of patients with clinical appendicitis 30 . In another study by Lala and Upadhyay on 2923 resected appendices in children aged 3–15 years old in New Zealand over a 10-year period, E. vermicularis was present histologically in 4% of cases 31 . In one study by da Silva et al . 32 , the helminthes were seen in 24 cases out of 1600 appendectomies (1.5%) during a 10-year period, and E. vermicularis was found in 23 of 24 (95.8%) appendectomy cases, while Taenia spp. was detected in only one (4.2%) case. On the other hand, a study conducted in Kerman province, Iran 33 as well as a recent study conducted in Saudi Arabia 34 , reported the incidence of E. vermicularis in appendectomy specimens ~10 folds more than our results of 2.9 and 2.7%, respectively. This variation in different studies could be a results of geographic areas, lifestyle, sanitary and hygiene level, culture, and sociodemographic features, as well as the techniques followed by the pathologists 13 . The prevalence of helminthiasis has been significantly decreased in recent decades globally due to screening programs and public health improvement. However, some helminths, particularly those with direct fecal-oral transmission, such as Enterobius and Hymenolepis , are still common 35 – 37 . The invasion of E. vermicularis and the presence of eggs inside the tissue of the appendix change the histology of appendix tissue and could generate various histopathological manifestations with different appendiceal symptoms 14 . Follicular hyperplasia is the initial pathologic reaction to the presence of E. vermicularis , causing noninflammatory clinical symptoms in the tissue. Numerous studies indicate that the symptoms of appendicitis could merely stem from E. vermicularis infection without any evidence of acute inflammation in histologic observations of the appendix. In other words, E. vermicularis is more commonly associated with noninflamed appendices, and in the current study, noninflamed histological marks were observed in 65% of patients, which is consistent with the results by Lala and Upadhyay in 2004 31 . However, the invasion of pinworms into the tissue could generate inflammatory reactions with hallmarks such as eosinophilic infiltrate and neutrophilic infiltrate, and in our study, 35% of cases indicated acute inflammation with a significant case of gangrenous appendicitis. In the present study, although the majority of patients who had undergone appendicectomy were male, 65% of the appendices containing E. vermicularis belonged to the female sex. This finding is consistent with the previous studies conducted in Iran and other countries globally 31 , 33 , 34 . The results of a recently published systematic review and meta-analysis study by Taghipour et al . 13 on 59 studies involving 103 195 appendix tissue samples showed that the prevalence of E. vermicularis in females was significantly higher than males (odds ratio: 0.47; 95% CI: 0.38–0.59). Moreover, it is noteworthy that a female predominance of pinworm infection is tangible in those between the ages of 5 and 14 years, and sex-based differences and behavioral patterns might be associated with higher infection rates in females 13 . In the current study, the mean and median ages of patients with E. vermicularis infection were 18.1±12.8 and 13.5 (IQR=14.5) years, respectively, and of 20 cases of E. vermicularis infection, 14 cases (70%, 95% CI: 45.7–88.1) were less than 20 years old. These findings are consistent with a study by Hamdona et al . 38 in Palestine, which showed the highest incidence of patients with E. vermicularis in appendices occurred in the less than 18-year-old age group. Another study carried out on 2267 appendices in Copenhagen revealed that the highest incidence of pinworms in appendectomy specimens occurred in the age group of 6–15 years 39 . A survey by Gialamas and colleagues revealed that the ages of the patients with histologically proven E. vermicularis in appendices ranged from 15 to 33 years, with a mean age of 25 years 18 . Moreover, studies revealed that patients with E. vermicularis were younger than patients with Taenia spp. 40 , 41 . Age is considered a risk factor for AA, and its association with Enterobius infection in both sexes has been documented 13 , 33 . In the present study, the inflamed appendix was most prevalent among participants aged 11–30 years (58.3%), and the difference between the age and histopathological features was statistically significant ( P <0.001). In contrast, most cases (70.8%) of neoplasms of the appendix were greater than 30. Appendiceal neoplasms are regarded as rare gastrointestinal cancers manifesting the symptoms of appendicitis, with the majority of cases categorized as epithelial appendiceal and neuroendocrine tumors 42 . Although this type of neoplasm may occur at any age, it is more common at ages greater than 50, regardless of sex, and this is congruous with the results of our study 43 . It should be noted that the appendectomy must not merely be considered a therapeutic intervention for the patients suffering appendicitis associated with E. vermicularis , as the adult parasites might be present in the lumen of the colon, usually in the cecum. Therefore, clinicians should be vigilant in treating patients after surgery as well as their households with adequate anthelminthic medications such as benzimidazole derivatives or pyrantel pamoate, followed by a repeat dose in 2 weeks to halt the occurrence of reinfection or autoinfection 44 . In this regard, mebendazole and albendazole are considered promising anthelmintic drugs for the precise treatment of enterobiasis, as they both have adulticidal and ovicidal effects 45 , 46 . The existing results should be regarded in the context of their strengths and limitations. In this study, the patient’s data were collected retrospectively during a certain 5-year period. Due to the significant role of our results in the prevention of negative appendectomy and definitive treatment of infections, prospective, longer-duration, and multicenter studies on this topic are thus affirmed. The main strength of this study is its novelty. In addition, the large sample size and coverage of two main hospitals in Shiraz County are the other strengths of this study.

Not commissioned, externally peer reviewed.

The authors declared no potential conflicts of interests.