Efficacy and reinfection with soil-transmitted helminths 18-weeks post-treatment with albendazole-ivermectin, albendazole-mebendazole, albendazole-oxantel pamoate and mebendazole

Background Preventive chemotherapy with albendazole or mebendazole is the current strategy to control soil-transmitted helminth (STH) infections (i.e. Ascaris lumbricoides, hookworm and Trichuris trichiura). STH reinfections, in particular A. lumbricoides and T. trichiura occur rapidly after treatment with the standard drugs. However, their low efficacy against T. trichiura, made an accurate assessment of reinfection patterns impossible. Methods In 2013 a randomised controlled trial was conducted on Pemba Island, Tanzania. School-aged children diagnosed positive for T. trichiura, were randomly allocated to (i) albendazole-ivermectin; (ii) albendazole-mebendazole; (iii) albendazole-oxantel pamoate; or (iv) mebendazole. Here we report the efficacy [cure rates (CR) and egg-reduction rates (ERR)], reinfection rates and new infections determined 18 weeks post-treatment. Results For a total of 405 children complete baseline and follow-up data were available. Similar to the efficacy determined after 3 weeks, 18 weeks after treatment albendazole-oxantel pamoate showed a significantly higher efficacy against T. trichiura (CR: 54.0 %, 95 % CI: 43.7–64.0; ERR: 98.6 %, 95 % CI: 97.8–99.2) compared to the other treatment arms. Children treated with albendazole-oxantel pamoate or albendazole-ivermectin had fewer moderate infections compared to children treated with albendazole. The reinfection rates 18 weeks post-treatment among all treatment arms were 37.2 % for T. trichiura (95 % CI: 28.3–46.8), 34.6 % for A. lumbricoides (95 % CI: 27.3–42.3) and 25.0 % for hookworms (95 % CI: 15.5–36.6). Conclusion The moderate reinfection rates with STHs 18 weeks post-treatment support the concept of regular anthelminthic treatment in highly endemic settings. Combination chemotherapy might achieve decreased morbidity in children since in the albendazole plus oxantel pamoate and albendazole plus ivermectin treatment arms only few moderate T. trichiura infections remained. Further trials should investigate the long term efficacy of albendazole-oxantel pamoate (i.e. 6 and 12 month post-treatment) and after several rounds of treatment in order to develop recommendations for appropriate control approaches for STH infections. Trial registration Current Controlled Trials ISRCTN80245406 Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1406-8) contains supplementary material, which is available to authorized users.


Background
The most common soil-transmitted helminths (STH; Ascaris lumbricoides, hookworms and Trichuris trichiura) infect approximately 1.5 billion people [1] worldwide with the highest prevalences in Asia and Africa. Schoolaged children living in the least developed settings, lacking clean water and sanitation facilities are primarily affected by A. lumbricoides and T. trichiura, while hookworm infections mostly occur in adults [2,3]. Untreated, chronically infected children might suffer from anemia, malnutrition and impairments in cognitive and physical development [2]. The burden of soil-transmitted helminthiasis has been estimated as 5.3 million disability-adjusted life years [1]. Large scale distribution of anthelminthic drugs without prior diagnosis ("preventive chemotherapy") mainly given to school-aged children, is the current strategy to control morbidity [4]. The most common anthelminthic drugs are albendazole and mebendazole [5]. Both drugs have high efficacy against A. lumbricoides, while only albendazole reveals a good performance in the treatment of hookworm infections. For the treatment of T. trichiura both drugs show poor cure rates in single-dose regimen [6].
Preventive chemotherapy does not avert reinfections as demonstrated in earlier studies [7,8]. Six to 12 months after treatment with albendazole or mebendazole, the prevalence of A. lumbricoides reached the pretreatment level [9][10][11], while hookworm reinfection is slow [9]. However, it is difficult to accurately estimate the reinfection rate of T. trichiura since, as mentioned above, the efficacy of the benzimidazoles against T. trichiura is low [6], in particular when children suffer from high infection intensity [12].
In a clinical trial conducted in 2013 on Pemba Island, Tanzania, we examined the efficacy of three drug combinations (i.e. albendazole-ivermectin, albendazolemebendazole, and albendazole-oxantel pamoate) versus mebendazole against T. trichiura and concomitant STH infections [13]. In brief, the combination albendazoleoxantel pamoate revealed a significantly higher cure rate (CR) (68.0 %) and egg-reduction rate (ERR 99.2 %) against T. trichiura than the other treatment regimens. This high efficacy might allow drawing better conclusions on reinfection with T. trichiura and ultimately to develop recommendations for control efforts.
The aim of the present study was to investigate whether the efficacy of albendazole-oxantel pamoate remains superior to the other combinations 18 weeks post-treatment and to monitor reinfection patterns of T. trichuris, A. lumbricoides and hookworms.

Study oversight
The presented data derive from a randomised controlled trial conducted among school-aged children on Pemba Island, Tanzania [13]. Ethical clearance was obtained from the Ministry of Health and Social Welfare in Zanzibar, Tanzania (ZAMREC, reference no. 0001/June/13) and from the ethics committee of Basel, Switzerland (EKBB reference no. 123/13). The trial was registered with www.isrctn.com (identifier: ISRCTN80245406). Prior to the study start, written informed consent was obtained from the parents or legal guardians and verbal assent from the participating children.

Study procedures and patients
The clinical trial was conducted from September 2013 to January 2014. Study setting and trial procedures are presented elsewhere [13]. In brief, school-aged children diagnosed positive for T. trichiura were randomly assigned to one of the following treatment arms: (i) albendazole (400 mg) plus ivermectin (200 μg/kg); (ii) albendazole (400 mg) plus mebendazole (500 mg); (iii) albendazole (400 mg) plus oxantel pamoate (20 mg/kg); and (iv) mebendazole (500 mg). All children were invited 3 and 18 weeks after treatment to provide stool samples on two consecutive days for the diagnosis of STH infections. Duplicate Kato-Katz thick smears were prepared from each stool sample using 41.7 mg templates [14] and quantitatively examined by experienced laboratory technicians for eggs of T. trichiura, hookworms and A. lumbricoides. Slides were read under a light microscope within 60 min after preparation to avoid overclearing of hookworm eggs [15]. Ten percent of the Kato-Katz thick smears were randomly chosen and reexamined to assure high quality of the microscopic diagnosis [16]. In case of discrepancies, the slides were read again and discussed until consensus was reached. At the end of the study, all children remaining infected from the two schools (Mchangamdogo and Shungi) received treatment according to national guidelines [17].

Statistical analysis
Prevalence of infection with T. trichiura, A. lumbricoides and hookworms was calculated for each treatment arm at baseline, 3 and 18 weeks post-treatment for all children with a complete dataset (2 stool samples at each time point). Differences among treatment arms in CRs 3 weeks and extended CRs (extended CR: children positive at baseline and negative at both follow-ups) 18 weeks posttreatment against T. trichiura, A. lumbricoides and hookworms were assessed using logistic regression.
Geometric means (GM) for eggs per gram (EPG) were calculated by adding 1 to each count (to take the logarithm in case of EPG = 0), taking the GM and subtracting 1 (GM = exp ((∑ log (EPG + 1))/n) − 1) [18]. Bootstrap resampling method with 10,000 replicates was used for calculating the 95 % confidence intervals (CI) for the ERRs. We assumed non-overlapping CI as statistical significant difference in ERRs. All arithmetic means (AM) are presented in the Additional file 1: Table S1.
Reinfection rates were defined as children positive at baseline, negative 3 weeks and positive 18 weeks posttreatment. New infections were defined as children negative at baseline and 3 weeks after treatment and positive 18 weeks post-treatment. As all children were by design positive for T. trichiura, new infections according to our definition, were only observed for A. lumbricoides and hookworms. Children negative at baseline and positive 3 weeks later were not included in these calculations (excluded for A. lumbricoides: 4 out of 405 children; and for hookworm: 27 out of 405 children).

Efficacy of drug combinations against T. trichiura 3 and 18 weeks post-treatment
In total 405 children infected with T. trichiuria were allocated to four different treatment regimens and provided all six stool samples (i.e. duplicate stool samples at baseline, 3 and 18 weeks follow-up; Fig. 1). The efficacies 3 weeks post-treatment observed with the different treatment regimens are presented elsewhere [13] and summarised in Table 1. The CRs documented 18 weeks post-treatment were 54.0 % (43.7-64.0) for albendazole-oxantel pamoate, 20.0 % (12.7-29.2) for albendazole-ivermectin, 13.9 % (7.8-22.2) for albendazole-mebendazole and 10.6 % for mebendazole (5.4-18.1). At the second follow-up, the efficacy of the other treatment arms remained significantly lower compared to albendazole-oxantel pamoate in terms of CR (P-values in Table 1) and ERR (98.6, CI: 97.8-99.2) compared to the other treatments at the second follow up. Considering the arithmetic ERRs, albendazole combined with ivermectin and albendazole-oxantel pamoate were significantly higher compared to the other treatments 18 weeks post-treatment (Additional file 1: Table S1).  Table 1 Cure rates (CR), extended CRs, egg-reduction rates (ERR), extended ERRs and reinfection data for the four different treatments against T. trichiura infections. Results of baseline and 3 weeks follow-up have been reported elsewhere [13] Trichuris trichiura Baseline infection intensities among treatment arms were equally balanced; 70.9 % of all children harboured light, 28.6 % moderate and 0.5 % heavy infections, stratified according to The World Health Organisation (WHO) cut-offs [18]. The number of children with light, moderate and heavy infection intensities at baseline, 3 and 18 weeks post-treatment for each treatment arm are summarised in Fig. 2. Albendazole plus ivermectin and albendazole plus oxantel pamoate caused a higher reduction of moderate T. trichiura infections 3 and 18 weeks post-treatment compared to albendazole-mebendazole and mebendazole. At the 18 weeks follow-up, the number of moderate infections remained higher for albendazolemebendazole (n = 16) and mebendazole (n = 17) unlike albendazole-ivermectin (n = 2) and albendazole-oxantel pamoate (n = 3; see Fig. 2).

Efficacy against A. lumbricoides and reinfection dynamics
At baseline, 169 (41.7 %) out of 405 children were infected with A. lumbricoides. All treatment arms achieved high CRs at the first follow up (  Fig. 3). In total 58 out of 232 (25.0 %; 19.7-31.1) children had acquired a new light (n = 50) or moderate (n = 8) A. lumbricoides infection.

Discussion
In preventive chemotherapy control programs albendazole and mebendazole are the treatment of choice against infections with all three STHs, yet both reveal a poor efficacy against T. trichiura [6]. In the recent past, oxantel pamoate has emerged as frontrunner for the treatment of infections with T. trichiura [13,19,20]. In more detail, in our recent studies albendazole-oxantel pamoate revealed good CRs and high ERRs, while mebendazole achieved CRs of 11.8 % [19] and 8.4 % [13] against T. trichiura infection. This low efficacy of mebendazole in the Pemba setting might be due to the occurrence of drug resistance, although molecular studies were not carried out to demonstrate it. In this scenario that is common also to other STH endemic areas, the need of new drug combinations in order to expand the armamentarium of treatments available for preventive chemotherapy strategy is of utmost importance [21].
In addition, albendazole-oxantel pamoate achieved a high reduction of moderate T. trichiura infections (persisting at 18 weeks post-treatment) in contrast to mebendazole. Note that, the goal of preventive chemotherapy is to reduce the morbidity from STH in pre-and school-aged children, by decreasing moderate and heavy infection intensities to a level below 1 % [22]. The combination albendazole-oxantel pamoate might contribute to achieve this goal.
Infections after treatment re-appear fast, particularly for A. lumbricoides and hence have a huge impact on the success of preventive chemotherapy [9]. This study presents detailed insights about the impact of drug combinations including the effective oxantel pamoate combination on the reinfection dynamics of the three STHs in a highly endemic area on Pemba Island, Tanzania. Earlier studies on reinfection struggled with the low CR Table 3 Cure rates (CR), extended CRs, egg-reduction rates (ERR), extended ERRs and reinfection data for the four different treatments against hookworms infections. Results of baseline and 3 weeks follow-up have been reported elsewhere [13] Hookworms Weeks posttreatment of the standard drugs against T. trichiura which complicated drawing sound conclusions against this helminth species [9,23]. Previous studies on reinfection mainly described the reacquired level of infection after treatment, in comparison to the pre-treatment level and presented the prevalence risk ratio [9,24,25]. With the focus only on prevalence before and after treatment, new infections are falsely considered as reinfection. Furthermore, prevalence risk ratios are strongly influenced by the achieved CRs of the treatments. Hence, in this study we distinguish and present both; reinfections rates (positive children at baseline, negative at the 3 week examination point and positive 18 weeks after treatment) and new infections (children with an infection exclusively 18 weeks post-treatment). Note that, children negative at baseline and positive 3 weeks later, which was observed for A. lumbricoides and hookworms, were not considered as new infections. We assume, that these children either harboured a non-patent infection or were wrongly diagnosed as negative at baseline [26]. We are confident that our differentiation between reinfections and new infections holds true given that the majority of A. lumbricoides and hookworm infections were of moderate intensities at baseline. However, obviously the low sensitivity of the Kato-Katz technique mainly for light infection intensities [26][27][28] reflects a general limitation of our study. Please note that in the present study four slides were examined by Kato-Katz. However, even when examining multiple thick smears the Kato-Katz method only reaches moderate sensitivity [29]. Therefore our results (i.e. re-infection, new infection) have to be interpreted with caution.
Earlier studies on reinfection with T. trichiura reported prevalence to pre-treatment level between 6 and 12 months [9-11, 24, 30]. In our study, the overall reinfection rate 18 weeks post-treatment for T. trichiura was 37.2 %, which is comparable to the 3 month post treatment prevalence risk ratio of 36.0 % reported by Sinniah [31] and 39.7 % by Al-Mekhlafi et al. [24]. A similar reinfection rate was observed for A. lumbricoides (34.6 %), which is in agreement with the 3 month posttreatment data of Jia et al. [9]. On the other hand, Yap et al. [30] documented a higher reinfection rate 4 months after treatment. Additionally, 58 (25 %) of the children negative at baseline acquired a new A. lumbricoides infection 18 weeks post-treatment. Interestingly, 24 children  6.0 %) who were negative 3 weeks post-treatment harboured already a moderate infection 18 weeks posttreatment, indicating the fast reinfection potential of A. lumbricoides [22].
The three drug combinations cured nearly half of the hookworm-infected children 3 weeks post-treatment, while as expected mebendazole achieved only low CRs [32][33][34][35]. Surprisingly, the extended CRs and ERRs among all treatment arms (except the ERR from albendazole-ivermectin) increased at the second compared to the first follow-up. For example, in the mebendazole group, 14 children identified as hookworm-positive 3 weeks post-treatment were negative at the second follow up. This finding is most likely due to a diagnostic issue, i.e. the low sensitivity of Kato-Katz for low egg-counts [36] and a delayed reading of the microscope slides [15,37], which could have led to fluctuations of Kato-Katz results. Overall, the reinfection rate was slower and less new infections with hookworms were observed compared to A. lumbricoides and T. trichiura, which is in agreement with other studies [9].

Conclusions
In conclusion, our study has reconfirmed the excellent efficacy of an albendazole-oxantel pamoate combination against T. trichiura infections. This combination could become a key element in controlling STH infections, especially in highly endemic settings. Further trials, should evaluate reinfection rates with oxantel pamoate six and 12 month after treatment and ideally after several rounds of treatment.
The moderate reinfection rate observed for T. trichiura and A. lumbricoides is worrying. This finding supports the necessity of an integrated control approaches including regular treatment, improved sanitation and health education [38][39][40][41], in order to reduce the burden of STH infections.

Additional file
Additional file 1: Table S1. Arithmetic mean egg per gram (EPG), egg-reduction rates (ERR) and extended ERRs for the four different treatments against T. trichiura, A. lumbricoides and hookworms infections. (DOCX 17 kb)