Abstract
Introduction:
The cycle of Echinococcus granulosus and its developmental potential has always been a subject of debate. While the origin of protoscoleces was well defined, the origin of daughter cysts (DC) was still a contentious topic, and several hypotheses have been proposed. The aim of this work was to study the fertility of DC and its predictive factors in order to determine its origin.
Patients and Methods:
This was a prospective pilot study including hepatic echinococcal cysts containing DC operated in the Department of General and Visceral Surgery of Habib Bourguiba University Hospital, Sfax, during a 22-month period. A parasitological study of the DC including the determination of their fertility was done.
Results:
A total of 248 DC collected from 27 multi-vesicular cysts from 21 patients were included in our study. The fertility rate of the DC was 64.5%. Free hooks were present in 130 DC (52.4%) and in only 11(12%) of infertile DC. In a multi-variable analysis, only World Health Organization–Informal Working Groups on Echinococcosis classification type CE2, bilious cyst fluid, number of DC per cyst, cyst size, and DC diameter were factors associated with DC fertility (p < 0.05). Out of 88 infertile DC, 77 (87.5%) were devoid of any hooks.
Conclusion:
Our findings suggest that most of the analyzed DC were fertile, with fertility associated with factors such as cyst size, DC size, CE2 type, and number of DC/cysts. The absence of hooks in infertile DC questions prevailing theories of their origin.
The life cycle of Echinococcus granulosus and its developmental potential has been a subject of debate for authors since the 1800s. Indeed, the question of the mode of formation of daughter cysts (DC) in cystic echinococcosis cysts (CEC) has been taken up many times since Himly in 1809 1 and Eschricht in 1856. 2 Although the origin of protoscoleces was well defined by only one theory, that of the budding of the germinal layer, 3 the origin of DC was still a matter of debate and several hypotheses were proposed. The origin and development of DC have been a subject of scientific inquiry since the 19th century. In the early 20th century, in vivo studies 4 gave evidence on the factors affecting cyst vesiculation, proposing that the vesicular formation of protoscoleces is the most common source of endogenous DC. In the latter studies, 5 the concept of DC originating from brood capsules through budding from the germinal layer was introduced. Since then, no studies have investigated the process behind the formation of protoscoleces in DC in human beings.
These DC could play a pivotal role in the disease’s progression and its clinical manifestations. Understanding the biology, formation, and fertility of DC is essential for effective diagnosis, management, and treatment of hydatid disease. With growing evidence suggesting a watch-and-wait approach to transitional cysts, 6 a proper understanding of the activity of CEC is needed. The aim of this work was to study the fertility of DC of hepatic CEC in human beings and its predictive factors.
Patients and Methods
We conducted a prospective descriptive analytical pilot study of hepatic CEC containing DC, operated in the Department of General and Visceral Surgery in Habib Bourguiba University Hospital of Sfax, Tunisia, during a period of 22 months from March 1, 2018, until December 31, 2019. The contents of the operated hepatic CEC were taken and then forwarded to the Laboratory of Parasitology for study. We included multi-vesicular CEC type CE2 and CE3b of the World Health Organization–Informal Working Groups on Echinococcosis (WHO-IWGE) classification. 7 We did not include patients with severe acute cholangitis or shock, and we excluded destroyed DC and DC not stored at +4°C. The number of DC required for our study was determined from data from a presurvey performed on 20 DC from 5 different CECs. To calculate the sample size, we used the Cochran’s sample size formula. 8
We found that with a fertility rate of 63%, the number of DC needed was estimated to be 248 with a margin of error of 6%. All experimental protocols were approved by the Committee for the Protection of Persons Suitable for Medical or Scientific Experimentation of Medicinal Products for Human Medicine, “CPP” under the reference “CPP Sud No. 0021/2017.” All patients included in the study signed an informed consent.
The primary endpoint was daughter vesicle fertility. Fertility was defined by the presence of protoscoleces in the daughter vesicle by analogy with the fertility of the CE cyst. 9 The protoscoleces could be invaginated (containing a double crown of hooks located within their membrane) or evaginated (containing a double crown of hooks apparent in their anterior part).9,10 The secondary endpoint was the presence or absence of hooks. For the study of predictive factors of DC fertility based on clinical parameters, we considered the proportion of fertile DC per cyst as the unit of measurement for fertility. This proportion, expressed as a percentage, was calculated by dividing the number of fertile DC by the total number of DC collected. This approach was chosen to avoid biases, particularly redundancies that could arise if each DC were analyzed individually. For parameters related to the DC itself, the unit of study was the DC, with predictive factors calculated based on both cyst-specific and DC-related parameters.
A pre-established form was filled in with the data of the operated patient, the cyst, and the DC for which a macroscopic and a microscopic study was performed. The study protocol was standardized: A standard workup and systematic preoperative imaging in the form of abdominal ultrasound and computed tomography (CT) scan was performed. Abdominal ultrasound is the basis of CEC diagnosis in abdominal locations, especially in the liver. 11 However, contrast-enhanced CT allows for a more thorough assessment of the cyst with details of adjacent vessels that might be of concern during the surgical procedure. 12 No patients received albendazole preoperatively. In the operating room, all patients had an intra-operative ultrasound followed by a systematic measurement of the intra-cystic pressure. Then, we performed for each cyst a puncture and aspiration of the cyst fluid (CF) with a dosed syringe, a cystotomy, and a collection of the DC with a sterile spoon. The cyst contents (CF + DC) were sent to the Parasitology Laboratory on the same day of the operation or at the latest the next day after conservation at +4°C. DC were processed in the laboratory with a standardized technique: we randomly collected 10 intact DC in a petri dish if the cyst contained a number ≥10 DC or all DC if the number of DC/cyst was <10. The DC randomly collected was of different size and appearance to avoid selection bias. In order to prevent destruction of protoscoleces, the scolicide, a hypertonic 30% physiologic saline, is used to sterilize the residual cavity only after the cyst material has been collected.
For the macroscopic study of the cyst contents, a visual description of the CF and the DC was performed. We described the CF (clear, bilious, purulent, or gelatinous) and studied the consistency of the DC (tense or flaccid) and their transparency (opaque or transparent).
Then, the cysts were washed with a sterile 0.9% sodium chloride solution, and the volume of each DC was measured using a test tube or a dosed syringe. Then, the vesicle fluid from each DC was withdrawn by a sterile syringe and allowed to sediment for 30 min.
For the microscopic study, we performed a fertility study of the cyst and each vesicle fluid. A drop (100 mcL) of the sedimentation pellet of the liquid was placed between the slide and coverslip and examined directly under the × 10 lens light microscope. This examination allowed us to evaluate whether protoscolex, evaginated, or invaginated protoscolex are existing or not and whether hooks also are existing or not. The presence of at least one protoscolex allowed the DC to be considered fertile (Fig. 1).
Direct observation at ×10 magnification showing protoscolex (red arrows) in a fertile daughter cyst.
For the statistical study, data entry and analysis were done using SPSS 25.0 software. The quantitative parameters were described using the means, standard deviation, and limits for the parameters whose distribution was Gaussian; in the opposite case, the median and extremes or semi-interquartile range (SIQR). The normality of the distribution of quantitative parameters was studied using the Kolmogorov-Smirnov test. The qualitative parameters were described using the calculation of the observed numbers and relative frequencies (percentages). We used the “Chi-square” test for the comparison of two or more frequencies and the “t-test” for the comparison of two averages when the conditions of application were verified and the Mann-Whitney test in the opposite case.
To determine the thresholds of the quantitative parameters, we used the receiver operating characteristic (ROC) curve. Multi-variable analyses were performed using a logistic regression model adjusted for covariates significant at p ≤ 0.1 on a uni-variable analysis. The significance level was set with an α risk at 5%.
Results
During the study period, 66 hepatic CECs were operated on. Out of these, 27 cysts (41%) from 21 patients were found to contain DC and were classified as CE2 and CE3b. These 27 cysts, containing a total of 248 DC, were subsequently investigated in the Parasitology Laboratory. Fifteen cysts (56%) were CE2 and contained 161 DC (65%). The median cyst size was 100 mm (SIQR: 35 mm). The median cyst wall thickness was 2 mm (SIQR: 0.7 mm). The number of DC per cyst was variable. Fifteen cysts (57%) had a number of DC ≥10. Table 1 shows the baseline characteristics of the patients, the cysts, and the DC.
Characteristics of the Patients, the Hepatic CE Cyst, and the DC
CE = cystic echinococcosis; SIQR = semi-interquartile range; WHO-IWGE = World Health Organization–Informal Working Groups on Echinococcosis; CF = cyst fluid; DC = daughter cyst.
CF characteristics
On macroscopic study, the CF was predominantly clear in 9 cysts (33%) and bilious in 10 cysts (37%). The 27 CFs were analyzed, and 22 cysts were found to have fertile CF (81%). In the five remaining CFs, no hooks or protoscoleces were found. Bivariate analysis revealed that CE2 type was associated with the fertility of CF. Notably, 93.4% of CE2 cysts (14/15) were fertile (p = 0.043), whereas only 58.4% of CE3b cysts (7/12) were fertile. Additionally, gelatinous CF was associated with non-fertility of CF, with 87% of non-gelatinous CF samples (20/23) being fertile compared with only 25% of gelatinous samples (1/4), with a p value of 0.025. However, other patient and cyst characteristics were not associated with CF fertility. Notably, cyst size showed no substantial difference between fertile and non-fertile CF (p = 0.786).
DC Characteristics
As for DC, the median size of the collected DC was 16 mm (SIQR: 6 mm). The median volume of the DC was 2 mL (Semi Interquartile Range: 2.3 mL). The majority of DC were tense and opaque, whereas the majority of small DC were translucent. Only one DC (0.4%) contained “granddaughter” vesicles (DC within another DC) (Fig. 2).
“Granddaughter” cyst (DC within a DC). DC = daughter cyst.
Microscopic study showed a fertility rate of DC of about 64.5%. The majority of protoscolex were invaginated (70.2%) (Table 1).
Free hooks were present in 130 DC (52.4%). However, 11 DC (4.4%) with a median size of 10.5 mm had hooks without protoscolex including 5 from CE3b cysts and 6 from CE2 cysts. Seventy-seven DC had a median size of 9.8 mm, were empty (no protoscolex or hooks), and were predominantly translucent (Table 2). Out of 88 infertile DC, 77 (87.5%) did not have any hooks.
Non-fertile Daughter Vesicles: Comparative Table of Those Containing Hooks and Those Not Containing Hooks
WHO-IWGE = World Health Organization-Informal Working Groups on Echinococcosis; CE = cystic echinococcosis; DC = daughter cyst.
Bivariate analysis showed that no clinical or biological characteristics were associated with the proportion of fertile cysts for each patient (Table 3).
Predictive Factors of the Fertility of Daughter Cysts in Bivariate Study (Clinical and Biological Parameters)
DC = daughter cyst; SIQR = semi-interquartile range; CE = cystic echinococcosis.
As for cyst characteristics in relation to DC, we found that WHO-IWGE classification type CE2, bilious CF, cyst size, cyst wall thickness, opaque wall, DC diameter, DC volume, and number of DC/cyst were positively correlated with DC fertility. Purulent CF was associated with infertile DC (Table 4 and Table 5). The ROC curve (Fig. 3) helped us determine the optimal sensitivity thresholds for fertility of the DC to be a cyst size >47 mm and DC size >6.5 mm.
ROC curve.
Predictive Factors of the Fertility of DC in Bivariate Study (Cyst Parameters)
DC = daughter cyst; WHO-IWGE = World Health Organization–Informal Working Groups on Echinococcosis; CE = cystic echinococcosis; CF = cyst fluid. Parameters highlighted in bold indicate predictive factors significantly associated with DC fertility.
Predictive Factors of Daughter Cysts Fertility in Bivariate Study
SIQR = semi-interquartile range; DC = daughter cyst. Parameters highlighted in bold indicate predictive factors significantly associated with DC fertility.
In the multi-variable analysis, we found that the WHO-IWGE classification type CE2, bilious CF, number of DC per cyst, cyst size, and DC diameter were associated with DC fertility as shown in Table 6.
Predictive Factors of Daughter Vesicle Fertility in Multi-variable Study
DC = daughter cyst; WHO-IWGE = World Health Organization–Informal Working Groups on Echinococcosis; CE = cystic echinococcosis.
Discussion
In human beings, studying the infectious potential of CEC is crucial to prevent the spread of protoscoleces and secondary hydatid formation, which poses a substantial operative risk if protoscoleces accidentally spill into the abdominal cavity. While the parasitological contents of CF have been explored in a few human studies,13–15 no studies have addressed the fertility and viability of protoscoleces within daughter vesicles. Understanding the formation of daughter vesicles could prove to be important for the management of multi-vesicular cysts. A practical example of the clinical relevance of our study is in the case of accidental spillage of DC during cyst opening. Indeed, the consequences of spilling CF might be inferred from the previous studies about CF. However, in the case of spillage of DC into the cavity, the outcomes are less predictable because the contents of DC remain unknown.
The study on CF revealed no substantial correlation between cyst size and CF fertility. However, cyst size may still influence other aspects of fertility. The study on DC showed that DC fertility was associated with cyst size, the number of DC per cyst, DC size, and WHO-IWGE classification type CE2. This indicates that larger cysts tend to harbor a greater number of DC, which are also more fertile. In contrast, gelatinous cysts were linked to fewer fertile DC, supporting the observation that CE2 cysts were more fertile than CE3b cysts. Overall, these findings suggest that more active cysts are likely to contain a greater number of fertile DC.
DC fertility was also associated with bilious appearance of the CF, which could allow us to conclude that fertility would also depend on aggression on the cyst. In addition, small DC were non-fertile or contained only hooks or both.
Could this information help us to establish hypotheses concerning the origin of the DC?
The origin of DC has always been a subject of debate since the 19th century. The first questions concerning the origin of DC have been asked since 1809 with Himly 1 then Eshricht in 1856 2 then D-J Thomas in 1884. 16 It is only in 1902 that the first in vivo results considering the general factors affecting cyst vesiculation were published by Dévé. 4 He mentioned that “the vesicular evolution of protoscoleces constitutes the most common and most important source of endogenous daughter cysts.” The experiments on this subject were continued by the Australian author Dew 5 in 1917 and then in 1925, who, for the first time, evoked the notion of DC, from the brood capsules (budding from the germinal layer). During the same year, Coutelen 17 completed Dévé’s work and confirmed, in vitro, the vesicular development of echinococcal protoscoleces observed by his master Dévé in vivo in 1902. He proposed in his thesis, for the first time, the formation of evaginated protoscoleces, which had a vesiculation at the level of the tail and invaginated protoscoleces attached to the brood capsules and which had a global vesiculation.
But, in spite of the good results of these authors, the scoliculture did not give rise to any research until the 1960s when a new series of works was published by other authors: Benex, Webster, and others.18–23 Indeed, Benex24,25 published his two articles concerning the in vitro formation using much more complex media of Echinococcus granulosus larvae and admitted two origins for the formation of daughter vesicles: “the germinal layer” and “the protoscolex.” However, he considered that the germinal layer is indispensable for the formation of DC and secondary cysts, whereas the protoscoleces after total and apical vesiculation evolved toward abortive vesicular forms after the failure of proglottid formation (apart from the definitive host: the dog).
Thus, two theories for obtaining DC were then accepted:
First, the theory of obtaining DC from protoscoleces19,25,26: by a total vesiculation of an invaginated protoscolex or a caudal or apical vesiculation of an evaginated protoscolex. The experiments carried out to explain this theory were done in vitro by Coutelen, Webster, and Benex. They were based on the principle of the destruction of the germinal layer by trypsin to cancel the effect of the latter.
16
Second, the theory of obtaining DC from the germinal layer
24
by stretching in finger-like projections of the membrane giving small DC by budding.
From our results, we found that on one hand, the fertility of the DC was statistically associated with the number of DC per cyst. Thus, the greater the number of DC in the cyst, the more protoscoleces the DC contained. Moreover, DC <6.5 mm with translucent walls (young DC) were empty (non-fertile). Furthermore, as a unique finding from our results, we found a DC that containing multiple “granddaughter cysts.” This disproves the malformation theory of the DC 16 that they arise from a degenerative process of germinal layer. Hence, the results of our study could confirm the theory of Benex. 24 (The germinal layer is essential for the formation of DC.) We could infer that the DC could have as origin the germinal layer.
On the other hand, the small translucent DC contained isolated hooks reminiscent of the total vesiculation of protoscoleces by leaving the hooks inside the DC, which could confirm the theory evoked by Coutelen 17 and Dévé. 16 Similarly, the majority of evaginated protoscolex were non-viable, which could explain that the caudal vesiculation is only a degenerative phenomenon. Thus, Benex’s deductions 25 (protoscolex vesiculation is a degenerative phenomenon and never produces DC and secondary cysts) concerned only caudal vesiculation. Moreover, in the literature, injection of protoscolex into the peritoneum of mice produces secondary cysts in the studies of Sarciron 27 and Elissondo. 28 This shows that the DC could also have as origin the invaginated protoscoleces. Our study shows that hooks are present in only 12% of infertile DC, whereas 88% do not contain hooks. This finding undermines the theory that these DC form through total vesiculation of protoscoleces. These findings may suggest that the majority of DC could originate from the germinal layer. At the end of our study and after a review of the literature, our results are in favor of both theories for the birth of DC. We hypothesize that when the germinal layer is intact, the majority of the DC could come from it, but, when the germinal layer is destroyed, the DC could rather come from the vesiculation of the protoscoleces.
This deduction triggers several questions: At what point does the birth of DC begin because one can have large (>10 cm) univesicular cysts and small (<5 cm) multi-vesicular cysts? Are there determinants of both pathways (the germinal layer pathway or the protoscolex pathway)?
Further studies including an anatomopathological study of the germinal layer and its developmental process are needed to confirm these hypotheses and answer these questions.
The current study aimed to elucidate the factors influencing the fertility of DC in CEC. While our findings provide substantial insights, some limitations warrant discussion, particularly concerning the sampling methodology. In this study, we established a sampling tactic, where we fixed the number of 10 DC per cyst, if the cyst contained more than 10 DC. During the sampling process, we attempted to collect DC with varied appearance and morphology to avoid selection bias.
Sampling all or a large number of DC would have allowed for a more thorough examination of the influence of DC characteristics on fertility and protoscolex viability within the cyst. However, this would engender redundancies in terms of cyst characteristics. In contrast, sampling one DC per cyst might mitigate this but does not allow the determination of predictive factors of DC within the cyst such as characteristics of DC. Therefore, choosing a fixed number of DC per cyst and aiming for morphological diversity will allow determination of predictive factors of cyst characteristics as well as DC characteristics.
Despite these potential biases, our multi-variable analysis revealed substantial associations between DC fertility and factors such as transaminase levels, total bilirubin levels, WHO-IWGE classification type, cyst size, number of DC per cyst, and DC size. These findings suggest that while sampling limitations exist, the identified predictive factors are robust and clinically relevant.
Conclusion
Our findings indicated that 64% of the analyzed DC were fertile. Fertility of DC was associated with WHO-IWGE classification type CE2, bilious CF, number of DC per cyst, cyst size, and DC diameter. The absence of hooks in small, young, and infertile DC makes the vesiculation theory of their origin less likely. Moreover, 88% of infertile DC lack hooks, undermining the theory that vesiculation of protoscoleces contributes to the formation of these DC.
Footnotes
Authors’ Contributions
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Disclaimer
This article has not been published in nor is being submitted to any other journal. To our knowledge, all the claims, statements, and conclusions are true and are our jointly held opinions.
Author Disclosure Statement
We have no conflicts of interest to disclose.
Funding Information
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.