A self-detection tool of tapeworm carriers and microscopy

Expulsion of proglottids is a specific symptom in taeniasis-infected persons. Carriers may report the presence of proglottids in faeces, in a toilet towel or, not uncommonly, felt in the under-garments (Ito et al. Reference Ito, Li, Chen, Long, Yanagida, Nakao, Sako, Okamoto, Wu, Raoul, Giraudoux and Craig2013). With T. saginata and T. asiatica, tapeworm proglottids are frequently expelled, and expulsion is spontaneous (independent of defaecation), whereas in T. solium taeniasis carriers expulsion of proglottids is passive (together with faeces) and occasional. T. saginata/T. asiatica carriers are often aware of the presence of a tapeworm, but this is not necessarily the case for T. solium. Therefore, questioning about the history of expulsion of proglottids can be used as an auxiliary method for the diagnosis of taeniasis infection. The feasibility of self-detection of tapeworm carriers has been previously confirmed not only for T. saginata and T. asiatica, but also for T. solium (Sarti et al. Reference Sarti, Schantz, Plancarte, Wilson, Gutierrez, Lopez, Robert and Flisser1992; Schantz et al. Reference Schantz, Cruz, Sarti and Pawlowski1993; Flisser et al. Reference Flisser, Vazquez-Mendoza, Martinez-Ocana, Gomez-Colin, Leyva and Medina-Santillan2005; Li et al. 2012). The reliability of the clinical history of proglottid expulsion was largely different based on previous reports, and ranged from less than 50% in Honduras to over 80% in Sichuan, China (De Kaminsky, Reference De Kaminsky1991; Li et al. Reference Li, Ito, Chen, Long, Okamoto, Raoul, Giraudoux, Yanagida, Nakao, Xiao and Craig2012). The differences might be attributable to the predominant Taenia species, and habits/customs of local people. To improve reliability, it is advisable to carry out a public health education intervention in advance (Flisser et al. Reference Flisser, Vazquez-Mendoza, Martinez-Ocana, Gomez-Colin, Leyva and Medina-Santillan2005).

Stool microscopy is one of the conventional techniques for diagnosis of taeniasis. It is generally agreed that microscopy lacks sensitivity (Pawlowski and Schulz, Reference Pawlowski and Schulz1972), but repeated microscopic examinations are able to improve the diagnostic value (Hall et al. Reference Hall, Latham, Crompton and Stephenson1981). Concentration techniques such as ether sedimentation and formalin–ether concentration can be applied, which may detect about 62–68% of T. saginata and 38% of T. solium cases (Hall et al. Reference Hall, Latham, Crompton and Stephenson1981; Deplazes et al. Reference Deplazes, Eckert, Pawlowski, Machowska and Gottstein1991; Allan et al. Reference Allan, Velasquez-Tohom, Torres-Alvarez, Yurrita and Garcia-Noval1996). In addition, ‘Scotch’ tape peri-anal swabbing is also used for detection of T. saginata and T. solium eggs (Pawlowski and Schulz, Reference Pawlowski and Schulz1972; Schantz and Sarti-Guttierez, Reference Schantz and Sarti-Guttierez1989; De Kaminsky, Reference De Kaminsky1991), which is more sensitive than a single coprological examination with T. saginata (Pawlowski and Schulz, Reference Pawlowski and Schulz1972). Anthelmintic treatment can detect many more tapeworm carriers than either coprological techniques or questioning (Hall et al. Reference Hall, Latham, Crompton and Stephenson1981; De Kaminsky, Reference De Kaminsky1991; Allan et al. Reference Allan, Velasquez-Tohom, Torres-Alvarez, Yurrita and Garcia-Noval1996).

Coproantigen detection

Research in the 1980s/90s on experimental taeniases and hymenolepiasis in laboratory animals and dogs indicated that tapeworm-derived antigens could be detected by ELISA in host faeces (i.e. coproantigens) (Allan and Craig, Reference Allan and Craig1989; Allan et al. Reference Allan, Avila, Garcia Noval, Flisser and Craig1990; Deplazes et al. Reference Deplazes, Gottstein, Stingelin and Eckert1990). Polyclonal antibodies from hyperimmunized rabbits against somatic or excretory/secretory (ES) tapeworm antigens were efficacious in robust detection of coproantigens before and during patency in those experimental infections, and showed that coproantigen levels in humans and animals rapidly declined (usually within 5–15 days) following successful anthelmintic treatment (Allan and Craig, Reference Allan and Craig2006; Bustos et al. Reference Bustos, Rodriguez, Jiminez, Moyano, Castillo, Ayvar, Allan, Craig, Gonzalez, Gilman, Tsang and Garcia2012).

Application of the coproantigen ELISA approach for detection of human taeniases quickly followed, and more than doubled the sensitivity compared to microscopy for the diagnosis of T. solium. Very high genus level (i.e. Taenia spp.) specificity was also apparent with specificities >98% when assessed against other non-Taenia spp. gut helminth infections including nematodiases and hymenolepiasis (Deplazes et al. Reference Deplazes, Eckert, Pawlowski, Machowska and Gottstein1991; Allan et al. Reference Allan, Craig, Garcia Noval, Mencos, Liu, Wang, Wen, Zhou, Stringer, Rogan and Zeyhle1992). Subsequent community application of Taenia copro ELISAs generally indicated, when compared to treatment/purge follow-up, very good detection ability for T. solium and T. saginata taeniases in Latin America (e.g. Garcia-Noval et al. Reference Garcia-Noval, Allan, Fletes, Moreno, de Mata, Torres-Alvarez, Soto de Alfaro, Yurrita, Higueros-Morales, Mencos and Craig1996; Rodriguez-Canul et al. Reference Rodriguez-Canul, Fraser, Allan, Dominguez-Alpizar, Argaez-Rodriguez and Craig1999; Lescano et al. Reference Lescano, Garcia, Gilman, Gavidia, Tsang, Rodriguez, Moulton, Villaran, Montano and Gonzalez2009) and for all 3 human Taenia species, including T. asiatica, in SE Asia (Li et al. Reference Li, Craig, Ito, Chen, Qiu, Qiu, Sato, Wandra, Bradshow, Li, Yang and Wang2006; Somers et al. Reference Somers, Dorny, Nguyen, Dang, Goddeeris, Craig and Vercruysse2006; Wandra et al. Reference Wandra, Depary, Sutisna, Margono, Suroso, Okamoto, Craig and Ito2006). A putative species-specific T. solium coproantigen ELISA has been reported which incorporated an anti-T. solium somatic capture antibody and an anti-T. solium ES detection conjugate; it showed no cross-reactivity with T. saginata infections, and had a sensitivity of 96·4% for T. solium carriers in Peru (Guezala et al. Reference Guezala, Rodriguez, Zamora, Garcia, Gonzalez, Tembo, Allan and Craig2009).

Taenia and Echinococcus coproantigens are resistant to fixation in 5–10% formal saline, an advantage for community studies when stool samples cannot be frozen (Allan and Craig, Reference Allan and Craig2006). Immunobiochemical analysis of Echinococcus coproantigens indicates they are highly glycosylated, with O-linked saccharides and N-linked glycans predominant being present on the surface glycocalyx of the tapeworm and shed in host faeces (Elayoubi and Craig, Reference Elayoubi and Craig2004; Hulsmeier et al. Reference Hulsmeier, Deplazes, Naem, Nonaka, Hennet and Kohler2010). Interestingly, experimental Taenia spp. infections indicated detectable coproantigen levels by approximately 18–21 days post-infection (dpi) in dogs (Deplazes et al. Reference Deplazes, Gottstein, Stingelin and Eckert1990; Allan et al, Reference Allan, Craig, Garcia Noval, Mencos, Liu, Wang, Wen, Zhou, Stringer, Rogan and Zeyhle1992) and by 5 dpi in T. solium infected hamsters (Allan et al. Reference Allan, Avila, Garcia Noval, Flisser and Craig1990). However, in 5 T. saginata voluntary self-infections, coproantigens were detectable much later post-infection i.e. from 73 to 149 dpi (Tembo, Reference Tembo2010). Although it is most important to identify T. solium carriers, this assay would miss cases of T. saginata or T. asiatica in co-endemic areas, which might be a disadvantage for broader epidemiological studies on human taeniases.

Coproantigen detection and monitoring has proved very useful in epidemiological studies (reviewed by Allan and Craig, Reference Allan and Craig2006), in evaluating the effects of health education (Sarti et al. Reference Sarti, Flisser, Schantz, Gleizer, Loya, Plancarte, Avila, Allan, Craig, Nijeyaratne and Bronfman1997) and mass treatment (Sarti et al. Reference Sarti, Schantz, Avila, Ambrosio, Medina-Santillan and Flisser2000) on human taeniasis, but also at the clinical level, for example in detection of anthelmintic treatment failures for taeniasis in community intervention studies in Peru (Bustos et al. Reference Bustos, Rodriguez, Jiminez, Moyano, Castillo, Ayvar, Allan, Craig, Gonzalez, Gilman, Tsang and Garcia2012). In addition, coproantigen ELISA has proved an important surveillance tool for post-intervention studies (Allan et al. Reference Allan, Craig, Pawlowski, Singh and Prabhakar2002) and in geospatial analysis of porcine cysticercosis and taeniasis risk (O`Neal et al. Reference O`Neal, Moyano, Ayvar, Gonzalvez, Diaz, Rodriguez, Wilkins, Tsang, Gilman, Garcia and Gonzalez2012).

Copro-DNA detection

Reliable epidemiological information on the prevalence of taeniasis, especially T. solium taeniasis, depends on the accurate identification of the causative parasite. The diagnostic methods based on the microscopic examination are inadequate. Because proglottids of Taenia species are morphologically similar to each other, technical skills are needed to identify species. In addition, there is no difference in the morphology of eggs among taeniid parasites. Furthermore, the sensitivity of this method is far from satisfactory.

To obtain precise information on Taenia infections in a definitive host, tools using molecular techniques including conventional PCR, PCR-restriction fragment length polymorphism (RFLP), multiplex-PCR and Loop-mediated isothermal amplification (LAMP) have been developed. PCR is a highly specific and sensitive method for amplification of a specific DNA target from a few copies to many. Mayta et al. (Reference Mayta, Gilman, Prendergast, Castillo, Tinoco, Garcia, Gonzalez and Sterling2008) have developed T. solium-specific nested-PCR assay targeted to Tso31, oncosphere-specific protein, gene. With faeces, the specificity and sensitivity of this assay were 100 and 97%, respectively, and the lower detection limit was 10 eggs per 250 mg of faeces. This assay cannot detect T. saginata and T. asiatica, which may be a disadvantage for epidemiological studies of the different taeniases. For differential identification of human Taenia parasites, RFLP analysis of PCR products is often carried out because this method is simple and gives unambiguous results (Mayta et al. Reference Mayta, Talley, Gilman, Jimenez, Verastegui, Ruiz, Garcia and Gonzalez2000; Nunes et al. Reference Nunes, Dias, Dias, Aoki, de Paula, Lima and Garcia2005). The PCR-RFLP for taeniasis can now differentiate T. solium from T. saginata, but whether these assays can differentiate T. asiatica from other two species is not clear. In addition, PCR plus restriction enzyme treatment is relatively time-consuming. To overcome this problem, differential identification by PCR assay only without restriction enzyme treatments of PCR products has led to the use of a multiplex-PCR which can amplify several target genes by using several primer sets in a single reaction (Yamasaki et al. Reference Yamasaki, Allan, Sato, Nakao, Sako, Nakaya, Qiu, Mamuti, Craig and Ito2004a, Reference Yamasaki, Matsunaga, Yamamura, Chang, Kawamura, Sako, Nakao, Nakaya and Itob; Gonzalez et al. Reference Gonzalez, Montero, Morakote, Puente, Diaz De Tuesta, Serra, Lopez-Velez, McManus, Harrison, Parkhouse and Garate2004, Reference Gonzalez, Bailo, Ferrer, Garcia, Harrison, Parkhouse, McManus and Garate2010). Gonzalez et al. (Reference Gonzalez, Montero, Morakote, Puente, Diaz De Tuesta, Serra, Lopez-Velez, McManus, Harrison, Parkhouse and Garate2004) have demonstrated the usefulness of the multiplex-PCR targeted HDP2, non-coding repetitive DNA fragments in nuclear DNA, for differential identification of T. saginata and T. solium, but this assay lacked the ability to differentiate T. asiatica from T. saginata. Recently, these authors have developed a novel multiplex-PCR assay targeted to the HDP2 DNA fragment which can differentiate three Taenia parasites (Gonzalez et al. Reference Gonzalez, Bailo, Ferrer, Garcia, Harrison, Parkhouse, McManus and Garate2010). The sensitivity limit and the capability for detection of parasites in faeces with this novel assay are still unknown. Yamasaki et al. (Reference Yamasaki, Allan, Sato, Nakao, Sako, Nakaya, Qiu, Mamuti, Craig and Ito2004a, Reference Yamasaki, Matsunaga, Yamamura, Chang, Kawamura, Sako, Nakao, Nakaya and Itob) have established the multiplex-PCR for a comprehensive identification of human taeniid cestodes based on cox1 genes. Unlike HDP2-multiplex-PCR assay, this multiplex PCR method can differentiate not only three human taeniid parasites but two genotypes of T. solium. It has been demonstrated that this multiplex PCR method could provide reliable results if more than 50 eggs were present in 1 g of faeces. In this way, with the increase of genetic information available for the different Taenia species, higher specific and sensitive PCR assays should emerge.

It is not easy to exploit PCR techniques in the laboratories of developing countries because they require sophisticated equipment such as a thermal cycler. Furthermore, Taq DNA polymerase used in PCR is often inactivated by inhibitors present in biological material such as faeces, sometimes causing sensitivity and reproducibility problems. The recently developed LAMP method amplifies DNA with high specificity, sensitivity and rapidity under isothermal conditions (Notomi et al. Reference Notomi, Okayama, Masubuchi, Yonekawa, Watanabe, Amino and Hase2000). This method requires a Bst DNA polymerase with strand displacement activity, four primers recognizing six regions on the target DNA and simple incubators such as water bath, block heater or thermos bottle. Unlike the Taq DNA polymerase, the Bst DNA polymerase resists many enzyme inhibitors in biological material, which means it is suitable for use with clinical samples (Mori et al. Reference Mori, Nagamine, Tomita and Notomi2001; Mori and Notomi, Reference Mori and Notomi2009). For a differential detection and identification of Taenia species, Nkouawa et al. (Reference Nkouawa, Sako, Nakao, Nakaya and Ito2009) have developed the LAMP method targeting cox1 and cathepsin L-like cysteine peptidase (clp) genes. This method is highly sensitive and specific for differential detection of Taenia species by using DNA prepared from proglottids, cysticerci and faeces of taeniasis patients in Indonesia and Sichuan (China). Evaluation using faeces from taeniasis patients has revealed higher sensitivity of the LAMP method than that of PCR (88·4 vs 37·2%, Nkouawa et al. Reference Nkouawa, Sako, Li, Chen, Wandra, Swastika, Nakao, Yanagida, Nakaya, Qiu and Ito2010). Furthermore, Nkouawa et al. (Reference Nkouawa, Sako, Li, Chen, Nakao, Yanagida, Okamoto, Giraudoux, Raoul, Nakaya, Xiao, Qiu, Qiu, Craig and Ito2012) have demonstrated that differential identification of Taenia species by the LAMP method could be carried out successfully using a thermos bottle and hot water instead of an incubator requiring electricity if the reaction temperature could be maintained, indicating the usefulness of LAMP in the field. The fact that LAMP-positivity can be judged by the appearance of a large amount of white precipitate of magnesium pyrophosphate produced during DNA amplification visible to the naked eye is a great advantage in field (Mori et al. Reference Mori, Nagamine, Tomita and Notomi2001), in contrast to PCR which depends on electrophoresis to detect the specific amplicon. Unfortunately, the multiplex-LAMP with several primer sets in a single tube is far from practical at present when compared with multiplex-PCR (Yamasaki et al. Reference Yamasaki, Allan, Sato, Nakao, Sako, Nakaya, Qiu, Mamuti, Craig and Ito2004a, Reference Yamasaki, Matsunaga, Yamamura, Chang, Kawamura, Sako, Nakao, Nakaya and Itob). Therefore, one primer set needs one reaction mixture. In the case of Taenia-LAMP, three reaction mixtures containing T. solium-, T. saginata- and T. asiatica-primers must be set up separately if the target gene is cox1 gene. For future use, such complicated test methods should be simplified.

Molecular identification of the parasite

Several molecular tools have been developed and are now widely used for the species identification of human tapeworms. The molecular identification of Taenia spp. targets mainly the mitochondrial cox1 gene, as it is variable enough to differentiate three human Taenia species. The above-mentioned multiplex PCR and LAMP methods are available for all the parasite stages (adult worm, metacestodes and eggs) in fresh, frozen and ethanol-fixed specimens (Yamasaki et al. Reference Yamasaki, Allan, Sato, Nakao, Sako, Nakaya, Qiu, Mamuti, Craig and Ito2004a; Nkouawa et al. Reference Nkouawa, Sako, Nakao, Nakaya and Ito2009, Reference Nkouawa, Sako, Li, Chen, Wandra, Swastika, Nakao, Yanagida, Nakaya, Qiu and Ito2010). These methods are highly sensitive and reliable, and the LAMP method is even possible for the identification of the adult worm without sophisticated equipment in field surveys (Nkouawa et al. Reference Nkouawa, Sako, Li, Chen, Nakao, Yanagida, Okamoto, Giraudoux, Raoul, Nakaya, Xiao, Qiu, Qiu, Craig and Ito2012). DNA sequencing of mtDNA has also been used for the species identification. Although it requires more time, cost and labour, compared to the multiplex PCR and LAMP methods, DNA sequencing has two advantages. First, it is applicable to the formalin-fixed and paraffin-embedded (FFPE) histopathological specimens (Yamasaki et al. Reference Yamasaki, Matsunaga, Yamamura, Chang, Kawamura, Sako, Nakao, Nakaya and Ito2004b, Reference Yamasaki, Nagase, Kiyoshige, Suzuki, Nakaya, Itoh, Sako, Nakao and Ito2006; Yanagida et al. Reference Yanagida, Yuzawa, Joshi, Sako, Nakao, Nakaya, Kawano, Oka, Fujii and Ito2010; Jongwutiwes et al. Reference Jongwutiwes, Yanagida, Ito and Kline2011; Swastika et al. Reference Swastika, Dewiyani, Yanagida, Sako, Sudarmaja, Sutisna, Wandra, Dharmawan, Nakaya, Okamoto and Ito2012). In the case of cysticercosis, FFPE histopathological specimens are often the only available material for molecular identification. It is generally not suitable for molecular analyses because of the degradation of DNA in formalin-fixed materials. Therefore, short fragments of cox1 gene are amplified and sequenced for species identification (Yanagida et al. Reference Yanagida, Yuzawa, Joshi, Sako, Nakao, Nakaya, Kawano, Oka, Fujii and Ito2010; Jongwutiwes et al. Reference Jongwutiwes, Yanagida, Ito and Kline2011; Swastika et al. Reference Swastika, Dewiyani, Yanagida, Sako, Sudarmaja, Sutisna, Wandra, Dharmawan, Nakaya, Okamoto and Ito2012). It is even possible to use the specimens after long-term preservation in formalin (Jeon et al. Reference Jeon, Kim and Eom2011). Secondly, DNA sequencing provides a clue to trace back the origin of the infection of T. solium. It is known that there are two mitochondrial genotypes which are geographically separated into Asian and African/Latin American countries (Nakao et al. Reference Nakao, Okamoto, Sako, Yamasaki, Nakaya and Ito2002; Martinez-Hernandez et al. 2009). As T. solium generally has the unique mitochondrial haplotypes in each Asian country, it is possible to assess where the patient became infected (Yanagida et al. Reference Yanagida, Yuzawa, Joshi, Sako, Nakao, Nakaya, Kawano, Oka, Fujii and Ito2010, Reference Yanagida, Sako, Nakao, Nakaya and Ito2012). On the other hand, the African and Latin American haplotypes are often indistinguishable because of the recent introduction of the parasite from Europe (Nakao et al. Reference Nakao, Okamoto, Sako, Yamasaki, Nakaya and Ito2002). Recently, sympatric distribution of both mitochondrial genotypes was demonstrated in Madagascar (Michelet et al. Reference Michelet, Carod, Rakontondrazaka, Ma, Gay and Dauga2010; Michelet and Dauga, Reference Michelet and Dauga2012).

The species status of T. saginata and T. asiatica is still a hot topic. The two species are genetically closely related and morphologically very similar, but their intermediate hosts are different. While metacestodes of T. saginata develop in the skeletal muscle of cattle, those of T. asiatica develop in pig viscera. Recently, evidence of hybridization between the two species has been reported from China and Thailand (Okamoto et al. Reference Okamoto, Nakao, Blair, Anantaphruti, Waikagul and Ito2010; Yamane et al. Reference Yamane, Suzuki, Tachi, Li, Chen, Nakao, Nkouawa, Yanagida, Sako, Ito, Sato and Okamoto2012). In the endemic regions of these two countries, several adult worms showed nuclear-mitochondrial discordance (e.g. T. saginata-type mtDNA with T. asiatica-type nuclear DNA). Thus, when both species are sympatric, we cannot exclude the possibility of hybrid worms by using mtDNA markers alone. In such cases, several nuclear genes can be used for confirmation of hybrid worms (Nkouawa et al. Reference Nkouawa, Sako, Nakao, Nakaya and Ito2009; Yamane et al. Reference Yamane, Suzuki, Tachi, Li, Chen, Nakao, Nkouawa, Yanagida, Sako, Ito, Sato and Okamoto2012). However, it is not always possible to detect hybrids because the number of nuclear target genes is not sufficient. Apart from the debate whether T. saginata and T. asiatica are the same species or not, it is important to differentiate beef worms from pork worms. The intermediate host range of the hybrid worms is still not known. Further study is needed to clarify the host specificity of hybrid worms, by examining metacestodes from cattle and pigs in areas co-endemic for the two species using both mitochondrial and nuclear gene markers.

Spatial analysis of human and animal host infection distribution

Key questions in spatial eco-epidemiology are (1) is the distribution of cases in the geographical area surveyed similar to the one of non-cases (control) or (2) is there an abnormal concentration of cases in some locations? Location refers to household, village or any spatial unit at a given scale. This question implicitly involves considering the population of non-cases along with the population of cases in the framework of data analysis. Surprisingly, only very few studies dealing with Taenia spp. transmission have included a spatial approach to detect spatial patterns of infection. Two methods were developed: (1) the detection of spatial clusters of human and animal cases using Kulldorff spatial scan statistics, Ripley's K functions, or variance-to-mean ratio (VMR) and nearest neighbour index (NNI); (2) statistical modelling of seroprevalence or neurocysticercosis using the distance to the nearest tapeworm carrier as a covariate. Spatial scan statistics are used to detect clusters of cases by gradually scanning a circular window (centred on each individual, household and village) across space and/or time, computing the number of observed and expected observations inside and outside the window at each location (Kulldorff, Reference Kulldorff1997). Scan statistics use various probability models (e.g. Bernoulli, Poisson) to predict expected observations depending on the nature of the data. For each window, a likelihood ratio statistic is computed based on the number of observed and expected cases within and outside the window and Monte Carlo hypothesis testing provides a P value. Ripleys’ K function is based on the concept that the expected number of events (cases, controls) in a circle radius t is some function of t that depends on the pattern of the events (Fortin and Dale, Reference Fortin and Dale2005). In the absence of clustering it is expected that K function for cases across a range of distance is not different from K function for controls.

Lescano et al. (Reference Lescano, Garcia, Gilman, Guezala, Tsang, Gavidia, Rodriguez, Moulton, Green and Gonzalez2007, Reference Lescano, Garcia, Gilman, Gavidia, Tsang, Rodriguez, Moulton, Villaran, Montano and Gonzalez2009) have undertaken a comprehensive investigation of the epidemiological position of T. solium in both humans and pigs in seven low-income rural villages of the northern coast of Peru (212 households comprising 898 permanent residents). Mass treatment of human taeniasis identified 11 adult worm carriers, and epidemiological screening revealed seroprevalences of human and pig cysticercosis of 24% (196/803) and 30·8% (280/908), respectively. Taking a household as the statistical unit and using distance to the nearest carrier (from 0 to 2000 m) as covariate, they (1) failed to show any relationship with prevalence of neurocysticercosis-related seizure, (2) evidenced a weak but significant relationship with human seroprevalence (21% >50 m from a carrier and 32% from 2–50 m), and (3) showed a strong relationship with pig seroprevalence (18% >500 m, 36% between 500 and 51 m, 69% within 50 m). Morales et al. (Reference Morales, Martinez, Rosetti, Fleury, Maza, Hernandez, Villalobos, Fragoso, De Aluja, Larralde and Sciutto2008) showed that prevalence of porcine cysticercosis, as assessed by tongue examination, (13·3% out of 562 pigs) significantly varied among 13 villages of a rural area in Mexico. They investigated clustering patterns within each of the 13 villages comparing VMR and NNI of all pig-rearing farms, farms with healthy pigs and farms with infection-positive pigs. VMR indicated clustering of infected pigs in 3 villages only but the values were similar to VMR obtained for all farms and farms with healthy pigs, suggesting that clustering of pigs was due to clustering of farms and not to local determinants fostering transmission. Clustering of several indices of T. solium and T. saginata infection was determined using a spatial scan statistic in South India, North Tanzania and Eastern Spain, yielding contrasting outcomes. In Catalonia (Spain), Allepuz et al. (Reference Allepuz, Napp, Picado, Alba, Panades, Domingo and Casal2009) disclosed two clusters of farms with bovine infection in an epidemiological investigation aiming at understanding the causes of a T. saginata outbreak. In their study area of approximately 200 km² in India, Raghava et al. (Reference Raghava, Prabhakaran, Jayaraman, Muliyil, Oommen, Dorny, Vercruysse and Rajshekhar2010) detected clusters of human seropositivity to T. solium cysticercal antibodies, of positivity to circulating cysticercal antigens and of taeniasis, but failed to detect clusters of neurocysticercosis. In a Tanzanian study area of approximately 4000 km², Ngowi et al. (Reference Ngowi, Kassuku, Carabin, Mlangwa, Mlozi, Mbilinyi and Willingham2010) assessed baseline prevalence of T. solium cysticercosis in 784 pigs (lingual examination) and incidence of infection in 295 sentinel piglets left on average for 4 months in households (lingual examination and Ag-ELISA). Local clusters of households were detected for prevalence (n = 1), seroincidence (n = 1) and incidence of lingual infection (n = 2), and all these clusters geographically overlapped partly, suggesting one large hotspot of transmission in the north-east part of the area. Spatial pattern analysis using Ripley's K functions supported the general clustering pattern for seroincidence and incidence of lingual infection but did not evidence clustering for seroprevalence. No evidence on the processes explaining patterns and differences between studies was provided. The authors hypothesized that several factors were involved including the mobility of people who acquired infection, the loss of adult worms, the possible long delay between contact with a tapeworm carrier and seizure appearance, the under-detection of cases, differences in defaecation practices, pig rearing systems, and food and drinking habits. Other sources of variation should also be considered. The spatial range of investigations may be a key issue. For instance, spatial clusters may not be detected if the range of the whole study area is included within a cluster within which random or regular distribution might be detected. Nevertheless, these results can have direct applications on a given scale. For instance, in Tanzania they served as baseline information to target health education efforts (Ngowi et al. Reference Ngowi, Kassuku, Carabin, Mlangwa, Mlozi, Mbilinyi and Willingham2010).

Monitoring pig behaviour and activity range

Several studies have identified free-roaming pig behaviour as a risk factor associated with increased pig cysticercosis (Pouedet et al. Reference Pouedet, Zoli, Nguekam, Vondou, Assana, Speybroeck, Berkvens, Dorny, Brandt and Geerts2002; Sikasunge et al. Reference Sikasunge, Phiri, Phiri, Dorny, Siziya and Willingham2007; Pondja et al. Reference Pondja, Neves, Mlangwa, Afonso, Fafetine, Willingham, Thamsborg and Johansen2010), but some others have not (Morales et al. Reference Morales, Martinez, Rosetti, Fleury, Maza, Hernandez, Villalobos, Fragoso, De Aluja, Larralde and Sciutto2008; Jayashi et al. Reference Jayashi, Arroyo, Lightowlers, Garcia, Rodriguez and Gonzalez2012). This apparent discrepancy may lie within differences in human defaecation facilities (e.g. open-air defaecation in a household might lead to infection of a pig even if it is retained) and behaviour (defaecation outside a household) or pig confinement efficiency. Very few data are available on the pig activity ranges and behaviour within villages or settlements to evaluate, for instance, how far from their owner's house pigs can potentially become infected. Copado et al. (Reference Copado, De Aluja, Mayagoitia and Galindo2004) quantified the behaviour of free-ranging pigs, including the consumption of human faeces, in a rural setting with a population of 900 people living in 182 houses, located in the tropical area of Mexico (a place known to be endemic for T. solium transmission and where the frequency of outdoor defaecation by people is high). Their main findings were: (1) pigs spent more time feeding and moving around during the rainy season (1 h, 26 mn and 3 h, 01 mn, respectively) than during the dry season (43 mn and 1 h, 38 mn, respectively); (2) pigs walked 1023±493 m per day during the dry season vs 2775±1429 m during the rainy season; (3) the frequency of human faeces consumption was higher during the dry season (0·95 events/h vs 0·48 events/h); and (4) in both seasons adult females consumed human faeces more frequently (0·32/h during the dry season and 0·19/h during the rainy season) than pigs in the other age groups, and adult males consumed faeces more frequently than juveniles males. In an area of western Sichuan (China) where T. solium is suspected to be highly endemic in Tibetan communities (Li, unpublished data) we obtained original data on pig activity ranges during the course of an epidemiological investigation. Marihe village (Yajiang prefecture) comprises 21 households spread over 530 m along a banana-shaped valley at an altitude of 2550 m. In October 2011, a total of 22 pigs belonging to 16 households were individually marked using a paint code on both sides of the neck, and the geographical coordinate of the owner house was taken using hand-held GPS (Garmin GPSMAP 62). We then walked systematically in the village for 2 days and noted the geographical coordinates of each pig. Among the 22 marked pigs only 14 were observed outside their houses. The maximum Euclidian distance between the release point and the most distant observation ranged between 65 and 475 m with an average of 276 m (Fig. 1). This suggests that most pigs, at that time of the year (October), can occasionally walk across almost the entire area of Marihe village. Such behavioural data can be useful to understand the transmission system on a local scale in terms of seasonal patterns of pig infection and of the spatial scale at which pigs can become infected.

Fig. 1. Maps of observation locations of 14 colour-marked pigs in Marihe village (Sichuan, China) in 2011. White circle: village houses, White star: owner house where known, Red circle: observation location. Maximum Euclidian distance between the release point (owner house) and the most distant observation is given (‘max. dist.’), except for pig 31 for which maximum Euclidian distance is calculated between the capture point and the most distant observation. Many pigs occasionally walk across the entire village.