Evaluation of nitazoxanide treatment following triclabendazole failure in an outbreak of human fascioliasis in Upper Egypt

Background Fascioliasis is a neglected zoonosis with major public health implications in humans. Although triclabendazole (TCBZ) is the drug of choice, there are records of TCBZ failure worldwide. TCBZ-resistant fascioliasis is treated with alternative approved drugs including nitazoxanide (NTZ), with varying levels of efficacy. Data on NTZ efficacy after TCBZ failure in Egypt is scarce. This study evaluated the efficacy of NTZ in cases of TCBZ failure during an outbreak of fascioliasis in Assiut governorate of Upper Egypt. Methodology/Principal findings This prospective study included 67 patients from the outpatient clinic in Manfalout locality of Assiut governorate with clinical manifestations of acute fascioliasis. These included high eosinophilia (> 6% eosinophils in peripheral blood), positive anti-Fasciola antibodies, and hepatic focal lesions (HFL) or ascites on abdominal ultrasound or computed tomography. All patients initially received TCBZ at recommended doses. Patients were followed up after 1 month to assess response. According to the responses, patients were categorized as non-responders and responders. The non-responders received a trial of NTZ and were re-assessed for response based on clinical manifestations, eosinophil count, and abdominal ultrasound. Patients not responding to NTZ received additional doses of TCBZ. One month after initial TCBZ treatment, 37 patients responded well to TCBZ, while 30 patients failed to respond with persistence of fever, abdominal pain, high eosinophilia, and HFL. Most non-responders were male (56.7%); females predominated among TCBZ responders (62.2%). The mean age of the non-responders was relatively lower, at 20.57 ± 14.47 years (p = 0.004). Following NTZ therapy, HFL disappeared in 9/30 (30%) patients and eosinophil counts normalized in only 2 (6.7%) patients, indicating an overall efficacy of 36.6%. The remaining cases received additional doses of TCBZ with complete clinical, pathological, and radiological resolution. Conclusions/Significance Nitazoxanide was partially effective in TCBZ failure in acute human fascioliasis in Upper Egypt. Further studies with larger samples are highly encouraged and further research is urgently needed to find new therapeutic alternatives to TCBZ.


Introduction
Fascioliasis has emerged as a notable zoonotic disease with considerable impact on veterinary and public health. This prompted the World Health Organization (WHO) to include human fascioliasis among the important neglected tropical diseases (NTDs) [1]. It is a foodborne disease caused by trematodes belonging to the genus Fasciola (F. hepatica and F. gigantica). In the past few decades, the incidence of human fascioliasis has considerably risen in different parts of the world. The rise has been particularly remarkable in South America, Asia, and Africa including Egypt, where the two common species of Fasciola coexist [2]. Recent studies have revealed a large number of cases of fascioliasis (2.4 to 17 million cases) worldwide [3].
Fasciolids are parasites of the hepato-biliary ducts, and the disease is mostly confined to the liver. Therefore, the main pathogenic sequelae are hepatic lesions, fibrosis, and chronic inflammation of the biliary passages. The pre-patent period together with the time to onset of signs/ symptoms of the disease may range from a few days to 2-3 months or longer. There are 2 main clinical stages in fascioliasis. The acute stage coincides with larval migration and mechanical destruction of the liver tissue. This stage extends till worm maturation in the hepatic tissues, and lasts for 2-4 months. The chronic stage coincides with the persistence of adult Fasciola worms in the bile ducts and may last for months or even years [4]. Eosinophilia is the most common clinico-pathological feature against fascioliasis in both stages.
In Egypt, fascioliasis has probably been prevalent for a very long period, since the times of the pharaohs [5,6]. High levels of infestation have been widely described in livestock, [7] resulting in considerable economic losses and expenditure for purchase of anthelmintics, liver condemnation, loss of production due to mortality, lower production of meat, milk, and wool, reduced weight gain, and impaired fertility [8].
The mainstay of treatment in fascioliasis affecting animals and humans is triclabendazole (TCBZ), which targets both the immature stages and mature adult worms [9]. Older drugs, such as tetrachloride, tetrachlorethylene, and bithionol, are currently considered to be less effective, unacceptably toxic, or both [10]. Although TCBZ is the only effective treatment for fascioliasis, it is currently registered for human use in only 4 countries [11].
The widespread use of TCBZ in the livestock industry led to the emergence of resistance in fluke populations affecting ruminants in both, developed and developing countries including Ireland, Spain, Australia, Peru, and Argentina [10]. The zoonotic nature of fascioliasis may raise concerns regarding the transmission of resistant strains to humans, particularly in endemic areas such as Peru, Bolivia, and Egypt [12]. In recent years, a few reports have described the occurrence of TCBZ resistance in humans. The first case was reported in a livestock farmer in the Netherlands, followed by 4, 1, and 7 cases in Chile, Turkey, and Peru, respectively [13][14][15][16]. Unfortunately, despite the prevalence of TCBZ resistance in Egypt, a review of the literature does not reveal any published data.
Reliance on monotherapy poses a risk for the treatment of fascioliasis, particularly in the absence of a vaccine for the prevention of the disease [9]. As cases of TCBZ resistance are continuously being documented from livestock, human cases of TCBZ-resistant fascioliasis are most likely to occur. This is a serious challenge for treatment in humans, with considerable public health implications [8] and emphasizes the urgent need for developing new fasciocidal drugs [17].
Several trials were conducted in the search for effective alternative drugs for fascioliasis. Nitazoxanide (NTZ), which is a broad spectrum antiparasitic agent, has been found to be well tolerated by humans, with adverse effects similar to that of placebo [10]. Across different studies, its efficacy has ranged from 40-100% [18].
The aim of the present study was to investigate the efficacy of nitazoxanide as a treatment for fascioliasis in the face of incomplete response to triclabendazole in Upper Egypt.

Study design
This prospective study was conducted between August and November 2018.

Ethical approval and consent
The study protocol was approved by the Institutional Review Board of the Faculty of Medicine of Assiut University, Egypt. Written informed consent was obtained from all patients prior to participation in this study.

Study population
A total of 74 patients with diagnosed or suspected fascioliasis were recruited in the study. All these patients were referred to the outpatient clinic in the Tropical Medicine and Gastroenterology Department at the Al-Rajhi Liver University Hospital during an outbreak of fascioliasis in Manfalout locality of Assiut Governorate in Upper Egypt.

Clinical and laboratory investigations
The included patients had symptoms and signs suggestive of fascioliasis such as fever, abdominal pain, jaundice, and hepatomegaly. The complete blood count (CBC), including eosinophil percentage and absolute eosinophil count was individually assessed using the ADVIA 2120i Hematology System (Siemens Healthcare Diagnostics Inc. Tarrytown, NY 10591, USA). Stool examination was also performed for all cases for the qualitative diagnosis of fascioliasis using the native lugol and formalin ethyl acetate sedimentation method [19]. Stool samples were also examined for the presence of other co-existing intestinal parasites that could potentially crossreact or overlap with fascioliasis. Liver function tests were also performed.
Further investigations included; serological analysis was done by F. hepatica IgG Enzymelinked immune sorbent assay (ELISA) kits (DiaColon Tech Houston, USA) for qualitative diagnosis of fascioliasis. The result was read photometrically at 450 nm (TECAN Sunrise Absorbance Reader). (values greater than 10.0 AU/ml were interpreted as seropositive, cut-off value 0.25 according to the manufacturer's instructions) Indirect hemagglutination assay (IHA) using Distomatose Fumouze (Laboratories Fumouze Diagnostic, Levallois Perret, France) was also done to compare antibody titers (a titer � 1/320 was considered to be positive). Abdominal ultrasound (US), and abdominal computerized tomography (CT) were also done. Endoscopic retrograde cholangiopancreatography (ERCP) was performed in cases presenting with obstructive jaundice and a dilated common bile duct (CBD) on abdominal US and/or CT.

Treatment regimen and follow up of patients
All patients received a double dose of triclabendazole (Egaten, Novartis Pharma AG) at a dose of 10 mg/Kg/dose, at 12-hour interval in a joint venture with the WHO. Patients were advised to avoid vegetables that posed a risk for re-infection.
The endpoints for treatment response were evaluated on follow up after 1 month. Evaluation was based on 3 parameters, namely, resolution of clinical symptoms and signs, normalization of eosinophil counts, and improvement of hepatic lesions on US. According to the WHO criteria, persistence of symptoms or signs with either eosinophilia (> 6% eosinophils in peripheral blood) or hepatic focal lesions, was considered to be a probable indicator of treatment failure with TCBZ [1]. Patients were then divided into 2 groups according to treatment response. The first group included the patients who did not respond to TCBZ and were administered NTZ (non-responders), while the second group included patients who successfully responded to TCBZ (responders).
The non-responders received NTZ at a dose of 500 mg orally every 12 hours for 7 days. Patients were clinically assessed for response after 1 month. Resolution of both, eosinophilia in the CBC and/or hepatic focal lesions on US were indicative of response. Patients who failed to respond to NTZ were re-treated with TCBZ at doses similar to the initial dose and were followed up for response.
Patients who received any other anthelminthic drugs within 1 month before TCBZ or NTZ therapy including bithionol, praziquantel, albendazole, dihydroemetine, or emetine hydrochloride, and patients who showed hypersensitivity to nitazoxanide were excluded from this study.

Statistical analysis
Data entry and analysis were performed using the IBM SPSS Statistics for Windows, Version 20.0. (Armonk, NY: IBM Corp) software package. Data were presented as numbers, percentages, means, and standard deviations. The Chi-square and Fisher's exact tests were used to compare qualitative variables. The Mann-Whitney test and the Wilcoxon signed rank test were used to compare variables between independent and dependent groups, respectively. In case of non-parametric data, the Wilcoxon signed rank test was used to compare the quantitative variables before and after treatment. A P-value < 0.05 was considered statistically significant.

Results
In this prospective study, 74 patients were initially recruited. Among them, 67 patients with symptoms and signs suggestive of acute fascioliasis were included for the NTZ trial; 7 patients were excluded as they presented with obstructive jaundice and a dilated CBD on ultrasound (suggesting chronic fascioliasis). These 7 patients underwent endoscopic sphincterotomy and extraction of the adult worm by ERCP followed by TCBZ therapy. The included patients received initial treatment with a double-dose of TCBZ.
The pretreatment demographic, clinical, and laboratory data of the studied patients are shown in Tables 1-3 The studied patients were followed up after 1 month to evaluate the response to first line TCBZ. A total of 37 cases (55.2%) showed good response to TCBZ (the responder group) as evidenced by disappearance of signs and symptoms, normalization of peripheral eosinophil showed persistence of infection, as evidenced by persistence of clinical manifestations, high eosinophilia, and HFL. This group received nitazoxanide and were followed up after 1 month (Fig 2). The demographic, clinical, and sonographic characteristics of both groups, as summarized in Table 4, showed that most patients in non-responder group were male (56.7%), while females were predominant in the responder group (62.2%). Also, the mean age in the responder group (30.89 ± 14.57 years) was significantly higher than that of the non-responder group (20.57 ± 14.47 years) (p = 0.004). However, the clinical presentation and sonographic evidence of HFL were not significantly different between the groups.
As shown in Table 5, the hematological, biochemical, serological and parasitological parameters of patients at baseline were not considerably different between patients in both groups, except for total leucocyte count, and levels of ALT and AST, that were significantly higher in the non-responder group (p = 0.008, p = 0.026, and 0.047, respectively).
Furthermore, the assessment of response to first line TCBZ showed complete resolution of the clinical manifestations in all patients in the responder group; patients in the non-responder group had persistent fever and abdominal pain. The pre-treatment eosinophil counts were not significantly different between the groups (p = 0.081). After treatment, limited improvement in eosinophil counts was observed in the non-responder group, with a reduction from 26.72% ± 13.21 to 20.00% ± 11.28. In the responder group, the counts reduced from 30.47% ± 15.18 to 3.6% ±1.7, showing statistically significant difference between the groups (p = 0.000). After NTZ treatment in the non-responder group, HFL disappeared in 9/30 patients (30%) as opposed to all patients in the TCBZ responder group; this difference was statistically significant (p = 0.015) ( Table 6). In addition, eosinophil counts normalized in only 2 (6.7%) patients after NTZ therapy. Patients who did not show improvement after NTZ therapy received an additional dose of TCBZ, similar to the initial dose, with complete clinical, laboratory, and radiological resolution. Therefore, based on the improvement of eosinophil counts and HFLs in patients with TCBZ failure, nitazoxanide was effective in 11/30 patients (36.6%).

Discussion
Owing to its activity against juvenile and adult forms of the parasite, TCBZ is the drug of choice in the treatment of F. hepatica and F. gigantica infections in humans [1]. Mass control programs for human fascioliasis in Egypt, Vietnam, Bolivia, and Peru have used TCBZ, which was donated through an agreement between the WHO and the manufacturer [20]. Several previous studies have documented the clinical efficacy of TCBZ with various treatment regimens in different regions including Egypt [21][22][23]. The results of these drug trials are indicative of a dose-response relationship. The WHO currently recommends the administration of a single dose of TCBZ at a dose of 10 mg/kg for the treatment of human fascioliasis, and a double dose of 10 mg/kg, 12 hours apart, in severe cases [1]. In a randomized openlabeled study conducted in Egypt, which compared 1-and 2-dose regimens of TCBZ at 10 mg/kg, the 2-dose regimen showed more favorable results [21].
Indeed, TCBZ is the only first-line medication with reports of high efficacy in humans. Therefore, the effective management of resistance to this drug is of utmost importance [9]. Clinical trials on alternatives to TCBZ are limited. This is probably the first study to evaluate the efficacy of NTZ in the management of cases of acute fascioliasis with TCBZ failure in Egypt.
In the current study, all cases of acute fascioliasis were defined based on clinical manifestations, high eosinophilia, and radiological signs with positive anti-Fasciola antibodies. However, stool examination was positive in only 7 cases (10.4%) with a low egg burden. In the present study we could not rely only on coprological examination for the diagnosis and follow up of cases. This is attributed to many factors, including prepatent or acute infections (where the patients were symptomatic prior to the appearance of eggs in the stool) [24], the inability of adult Fasciola worms to produce eggs (due to its lack of adaptation to the human host), encapsulation of eggs in granulomas or abscesses in the liver, and low egg shedding related to low infection burdens [25]. Coprological examination may also overestimate the response to treatment since the age of the fluke or its anatomic location, which may be associated with increased susceptibility to treatment, may impact the results [26].
In a study previously conducted in Egypt including 23 cases, Fasciola eggs were detected in only 2 cases (8.6%) as the patients were diagnosed in the hepatic phase [27]. An immature worm feeds on liver tissue without producing eggs; the only evidences of infection are eosinophilia and HFL, which are observed in early stages of the infection [28].
The detection of anti-Fasciola antibodies by the ELISA test is a reliable and sensitive test for diagnosis of fascioliasis compared with stool examination. The main advantage is that results are positive as early as 2 weeks post infection. However, since serum antibodies may persist for 4-5 months after successful treatment, it is not a reliable test in the evaluation of response during follow up [24]. Eosinophilia as a host defense mechanism is a common feature of fascioliasis and is encountered in 14%-82% of patients, and may rise and fall during the chronic stage [29].
As described by Marcos et al., the primary outcome measures for clinical cure after treatment are defined by resolution of the clinical picture and eosinophilia during follow up [30]. Therefore, in the current study, post-treatment follow-up was based mainly on the persistence of clinical manifestations with either high eosinophilia with or without radiological signs.
In the present study, as evidenced by the disappearance of signs and symptoms, normalization of peripheral eosinophil counts, and resolution of HFL, 37 patients (55.2%) showed good response to TCBZ. The remaining 30 cases (44.8%) were suspected to have TCBZ failure and were treated with NTZ. The mean age of the non-responder group was lower than that of the TCBZ responders; this may have had an impact on the treatment response. A double blinded placebo-controlled trial in Peru, which employed NTZ for the treatment of chronic fascioliasis, has shown a low cure rate in children (40%) and a slightly higher efficacy in adults (60%) [9].
In our cohort, most non-responders were male (56.7%); females were predominant among the responders (62.2%). The gender of the studied patients did not significantly differ between groups. However, previous studies have indicated differences in sensitivity to flukicides depending on the sex of the host animals infected with F. hepatica [31].
Notably, in the current study, patients who did not initially respond to TCBZ in the acute stage, responded to the subsequent trial of TCBZ administered 2 months after the initial dose, in the chronic stage. This relationship between response to TCBZ and the stage of the disease has also been previously mentioned by Marcos et al. [30], who reported the amelioration of eosinophil counts after a single dose of TCBZ in 10 patients with acute Fasciola infection. However, parasitological cure (the absence of eggs in the stools) was not reported during follow up.
The difference in TCBZ susceptibility between juvenile and adult parasites has been previously described in an in vitro study with Fasciola hepatica infection [17]. However, this has not been thoroughly described in case series including patients with acute fascioliasis [32,33].
According to our results, 30 patients showed clinical evidence of the presence of TCBZresistant F. hepatica infection, which is considered a large number. They received a trial with NTZ at a dose of 500 mg twice daily for 7 days, that showed an overall efficacy of 36.6% (11/30 patients), based on the improvement of eosinophil counts and HFLs.
NTZ has been widely used in the management of different parasitic infections with reportedly high efficacy and tolerability. The efficacy of NTZ against Fasciola has been studied in rabbits experimentally infected with F. gigantica. NTZ was found to be partially effective (47%) against the juvenile stages of the parasite, but completely effective (100%) against the adult stage [34].
A few clinical trials have been conducted on the efficacy of NTZ in the treatment of human fascioliasis with considerably variable efficacy. In Egypt, an open-label clinical study including 125 Egyptian patients with chronic fascioliasis demonstrated 97% clearance of F. hepatica eggs in the stool on day 30 after treatment with NTZ; the serological and eosinophilic patterns had also improved [35]. A second report from Egypt showed a slightly lower cure rate with NTZ (82.4%) [36]. Similar results were observed in a study conducted on schoolchildren in Mexico that documented the efficacy rates of NTZ against chronic fascioliasis to be 94.0% and 100% after the first and second treatment courses, respectively [37].
A much lower efficacy rate was observed in a double-blinded placebo-controlled study in northern Peru, where 50 adults and 50 children infected with F. hepatica received a 7-day course of NTZ. Compared to the placebo group, 60% adults and 40% children were cured [10]. These results suggest that NTZ may be a reasonable option at least in the chronic stage of fascioliasis, and is a good alternative to TCBZ.
Conversely, some studies have revealed a lack of efficacy of NTZ in 24 cases of liver fluke infection in Cuba [38] and in a patient with apparent TCBZ failure in the Netherlands [13]. Cabada and colleagues have reported that a cohort of 7 patients, infected by ingesting watercress in the Cusco region of Peru, had failed to respond to multiple courses of TCBZ in combination with NTZ [16]. The wide variances in fasciolid susceptibility to NTZ may be attributed to differences in geographical strains of Fasciola in various regions [16]. This indicates the urgent need for further controlled clinical trials to evaluate the efficacy of NTZ in the control of fascioliasis.
Although TCBZ resistant fascioliasis has been widely described in livestock, the understanding of the mechanism of resistance to TCBZ remains incomplete, with a knowledge gap in terms of its capacity to spread and strategies for control [39]. It has been suggested that resistant fasciolid strains may have alterations in drug uptake, efflux, and detoxification, including the conversion of TCBZ sulfoxide into the less active forms. However, this has not been verified in large studies using other parasite strains. Poor response to TCBZ may also be attributed to its poor water-solubility and limiting drug concentration in the organs [40][41][42].
In contrast to veterinary medicine where other treatment options for Fasciola exist, there is no documented strategy for the management of TCBZ treatment failure in humans. To minimize the development of drug resistance, the use of synergistic drug combinations has been suggested [43]. However, this approach carries the risk of building up resistance to multiple drugs [44].
Although the small sample size has limited the scope of this study, to the best of our knowledge, this is the first report of TCBZ failure in humans with acute fascioliasis in Egypt. Further multicenter randomized studies including larger sample sizes are required to evaluate predictors of TCBZ failure. This will help to determine the optimum timing for repeating TCBZ after failure of the initial dose. Also, further research is urgently needed to find new therapeutic alternatives to TCBZ for controlling fascioliasis.
In this first report of TCBZ failure in acute human fascioliasis in Upper Egypt, NTZ proved to be partially effective.